TWI457564B - Nucleic acid molecule, diagnostic kit, biochip and method for the detection of food pathogenic bacteria - Google Patents

Description

Nucleic acid molecules, test kits, biochips and methods for detecting food pathogens

The present invention relates to nucleic acid molecules for detecting food pathogens. The invention also relates to diagnostic kits, biochips, and methods for detecting pathogenic bacteria present in a food sample using such nucleic acid molecules.

According to the statistics of the Department of Health, the total number of food poisoning cases occurred between 70 and 97 in the Republic of China, and the most common types of foods that cause microbial contamination include animal foods (such as seafood, poultry meat and their Processed products), fresh eggs, dairy products, and plant foods with high protein content (such as bean stuffing or bean products).

Pathogenic microorganisms that can cause food poisoning by environmental pollution include Moraxella catarrhalis , Pasteurella multocida , Staphylococcus aureus , Escherichia coli O157:H7 ( Escherichia coli O157:H7), Vibrio parahaemolyticus , Pseudomonas fluorescens , Salmonella spp., Aeromonas spp .) and Enterobacter spp. [such as Enterobacter sakazakii ]. Patients with food poisoning are often accompanied by symptoms such as fever, nausea, vomiting, diarrhea, and abdominal pain. In severe cases, they may even die. In addition, many food pathogenic bacteria cause epidemic infections between humans or animals via routes such as the mouth, nose or skin mucosa. Therefore, providing a method for detecting food pathogens early and quickly becomes an extremely important research and development subject.

In order to overcome the traditional strain culture and bacteriological characterization [eg gram staining, API identification system, physiological or biochemical characterization, antibiotic tolerance test, etc.], the operation process is more complicated and time consuming. Disadvantages, many molecular biological methods have been applied to the detection of food pathogens, including: polymerase chain reaction (PCR), multiplex polymerase chain reaction (Multiplex polymerase chain reaction), immunoassay (Immunoassay Method), DNA probe method, Immunomagnetic separation (IMS), biochip, and the like.

In recent years, the development of biochip technology has gradually gained attention and has been widely used in the detection of pathogenic bacteria, the classification of cancer, the development of new drugs, and the sequencing and identification of genes. Advantages of biochips include: small size, high confidence and accuracy of analytical results, fast analysis, and versatile experimental data with fewer samples and reagents. Biochips can be classified into the following four categories according to their functionality: (1) gene chip, also known as microarray chip; (2) microfluidic chip; (3) Protein chip; and (4) Lab-on-a-chip.

In general, DNA sequences in bacteria that contain highly conserved and highly variable DNA sequences are available for genus-specific or species-specific ( Detection or identification of species-specific).

In V. Spaniol et al . (2008), Microbes and Infection, 10:3-11, V. Spaniol et al . found a ubiquitous surface protein (UspA1) of Moraxella catarrhalis . [It is an outer membrane protein (OMP)] that gives this strain a unique ability to invade into epithelial cells. Therefore, the gene encoding UspA1 (i.e., uspA1 ) is expected to be the target gene for species-specific detection.

Heat shock protein (HSP) is a protective protein produced by cells in response to environmental stress. It is widely present in eukaryotic and prokaryotic cells and can be distinguished into the following six families depending on the molecular weight: HSP10 , HSP40, HSP60, HSP70, HSP90 and HSP100. The heat shock protein gene (also known as hsp ) is a highly conserved house-keeping gene that is often used to design specific primers for the detection or identification of bacterial species. Although the hsp gene sequence has been well studied, as far as the Applicant is aware, there is no literature or patent preamble that has revealed that the hsp10 gene can be used as Enterobacter sakazakii or Pasteurella. Multocida ) is the target gene for specific detection.

16S rDNA is fairly stable in evolution and it is a single-copy gene in the genomic DNA of many bacterial species. Therefore, 16S rDNA is often used as a target for genus-specific or species-specific detection. gene. For example, the 16S rRNA gene has been applied to the detection of Pasteurella multocida , Enterobacter spp., and Enterobacter sakazakii (PG Mbuthia et al . (2001). ), Journal of Clinical Microbiology , 39: 2627-2633; C. Dauga (2002), International Journal of Systematic and Evolutionary Microbiology , 52: 531-547; A. Lehner et al . (2004), BMC Microbiology , 4: 43 ).

In addition, in AJ Martinez-Murcia et al . (2005), International Microbiology , 8: 259-269, AJ Martinez-Murcia et al . found that 16S rDNA sequencing or 16S-23S rDNA intergenic spacer regions (intergenic spacer region) , ISR) restriction fragment length polymorphism (RFLP) is incapable of distinguishing strains of Aeromonas , and must use a house-keeping gene with high conservation ( For example, gyrB or rpoD ) can identify different Aeromonas species. Based on this finding, Applicants believe that the 16S-23S rDNA intergenic spacer region has the potential to supply genus-specific detection for Aeromonas.

In CY Lee et al . (1995), Applied and Environmental Microbiology , 61(4): 1311-1317, CY Lee et al . selected for colony from chromosomal DNA of Vibrio parahaemolyticus . The pR72H DNA fragment (which is conserved among all Vibrio cholerae strains) was designed with a set of primer pairs for VP32 and VP33. This primer pair can be used for PCR analysis to quickly detect Vibrio cholerae from contaminated shellfish or fish samples.

Dr. Jiang Yuxi, Ph.D., in the Department of Food and Applied Biotechnology, National Chung Hsing University [Name: "The development of biofilms for the detection of food pathogens and Staphylococcus aureus enterotoxin and the molecular classification and immediate polymerase chain reaction of Staphylococcus aureus (Development of Microarrays for Food Bacterial Pathogens and Staphylococcus aureus Enterotoxin Genes, as well as Molecular Typing and Real-Time PCR for Staphylococcus aureus )"] Escherichia coli O157:H7 ( Escherichia coli O157:H7) against different serotypes A randomly amplified random amplified polymorphic DNA (RAPD) fragment (referred to as Q3H1 having a size of about 456 bp) is designed to have a set of primer pairs for HAF1 and HAR1. In addition, the doctoral thesis also designed a set of primer pairs Sspp1S and Sspp1A for a different RAPD fragment shared by different Salmonella spp . (ie, a 1.8 kb Hin dIII DNA fragment). . The experimental results by PCR confirmed that the primers were highly specific for the detection of HAF1 and HAR1 and the primer pairs Sspp1S and Sspp1A for E. coli O157:H7 strain and Salmonella species, respectively.

Master's thesis by Zhang Yuxin, Institute of Biotechnology, Hongkong University of Science and Technology [Name: "Using the heat shock protein gene and its interval sequence design S. aureus , S. saprophyticus , S. epidermidis , S. haemolyticus , Streptococcus agalactiae , Strep. Uberis , Strep. bovis specific PCR primers and microarrays for the detection of S. aureus , S. saprophyticcus , S. epidermidis , S. haemolyticus, Strep. agalactiae, Strep. Uberis and Strep. bovis using heat shock protein gene)"] The nucleotide sequence of the complete genome of Staphylococcus aureus using Primer Premier 5.0 software [NCBI accession number: BA000018 A set of specific primer pairs SAU3 and SAU4 were designed, wherein SAU3 corresponds to the nucleotide residue positions 1771358 to 1771375 shown in NCBI Accession No. BA000018, and SAU4 corresponds to the NCBI registration number BA000018. Nucleotide residues are located at positions 1771763 to 1771746. It has been experimentally confirmed that this primer pair is highly specific for S. aureus and is available for species-specific detection.

In D. Dufour et al. (2008), International Journal of Food Microbiology , 125: 188-196, D. Dufour et al. found that many strains of Pseudomonas fluorescens produce an extracellular casein breakdown. Extracellular caseinolytic metalloprotease (abbreviated as AprX), its coding gene aprX is available for rapid detection of P. fluorescens strains present in raw milk.

Although the above literature has been reported, there is still a need in the art to screen out nucleic acid molecules that are highly specific and sensitive to food pathogens for rapid detection or identification.

Summary of invention

Thus, in a first aspect, the present invention provides a nucleic acid molecule reagent for detecting a food pathogen comprising at least one of the following:

(a) a nucleic acid molecule for detecting M. catarrhalis having a nucleotide sequence selected from the group consisting of: sequence identification number: 1 and sequence identification number: 2;

(b) a nucleic acid molecule for detecting Pasteurella septicum having a nucleotide sequence selected from the group consisting of: Sequence ID: 3 and Sequence ID: 4.

(c) a nucleic acid molecule for detecting a species of Aeromonas, having a nucleotide sequence selected from the group consisting of: Sequence ID: 5 and Sequence ID:

(d) a nucleic acid molecule for detecting an Enterobacter species having a nucleotide sequence selected from the group consisting of: Sequence ID: 7 and Sequence ID: 8.

(e) a nucleic acid molecule for detecting Enterobacter sakazakii having a nucleotide sequence selected from the group consisting of: Sequence ID: 9, Sequence ID: 10, and Sequence ID: 21.

(f) a nucleic acid molecule for detecting Pseudomonas fluorescens having a nucleotide sequence selected from the group consisting of: sequence identification number: 11 and sequence identification number: 12;

(g) a nucleic acid molecule for detecting S. aureus having a nucleotide sequence as shown in SEQ ID NO:22;

(h) a nucleic acid molecule for detecting E. coli O157:H7 having a nucleotide sequence as shown in SEQ ID NO: 23;

(i) a nucleic acid molecule for detecting Vibrio cholerae having a nucleotide sequence as shown in SEQ ID NO: 24;

(j) A nucleic acid molecule for detecting a Salmonella species having a nucleotide sequence as shown in SEQ ID NO: 25.

The nucleic acid molecules described above have a high degree of specificity and sensitivity for the target species to be detected, and thus are useful for detecting pathogenic bacteria present in a food sample. Accordingly, in a second aspect, the present invention provides a method for detecting the presence or absence of a pathogen in a food sample, comprising:

The food sample is subjected to a DNA amplification reaction using at least one set of primer pairs, wherein the at least one set of primer pairs is selected from the group consisting of:

(a) A primer pair for detecting M. catarrhalis comprising a nucleotide sequence as shown in sequence identification number: 1 and a primer having a sequence number: 2 a reverse primer of the indicated nucleotide sequence;

(b) a primer pair for detecting Pasteurella septicum comprising a nucleotide sequence as shown in sequence identification number: 3, and a sequence having a sequence identification number: 4 a reverse primer for the nucleotide sequence;

(c) a primer pair for detecting a species of Aeromonas, comprising a nucleotide sequence as shown in sequence identification number: 5, and a sequence having a sequence number: 6 a reverse primer of the nucleotide sequence shown;

(d) a primer pair for detecting an Enterobacter species comprising a nucleotide sequence as shown in sequence identification number: 7 and a primer having a sequence number: 8 a reverse primer of a nucleotide sequence;

(e) a primer pair for detecting Enterobacter sakazakii, comprising a nucleotide sequence forward primer as shown in sequence identification number: 9 and a sequence number as indicated by sequence number: 10. a reverse primer for the nucleotide sequence;

(f) a primer pair for detecting Pseudomonas fluorescens comprising a nucleotide sequence as shown in sequence identification number: 11 and a sequence having a sequence number: 12 a reverse primer of the indicated nucleotide sequence;

Detecting whether there is a DNA fragment amplified by using the at least one set of primer pairs, wherein the presence of the DNA fragment indicates the presence of a pathogen corresponding to the at least one set of primer pairs.

Nucleic acid molecules according to the invention are also contemplated as probes. Accordingly, in a third aspect, the present invention provides a method for detecting the presence or absence of a pathogen in a food sample, comprising: subjecting the food sample to a hybridization reaction using at least one probe, wherein the at least one probe The nucleotide sequence and the corresponding pathogen are as defined above for the nucleic acid molecules; and detecting whether there is a hybrid formed by performing hybridization reaction using the at least one probe, wherein the hybridization The presence of the object indicates the presence of a pathogen corresponding to the at least one probe.

In a fourth aspect, the invention provides a test kit for detecting a food pathogen comprising at least one nucleic acid molecule reagent as described above.

In a fifth aspect, the invention provides a biochip for detecting a food pathogen comprising at least one nucleic acid molecule reagent as described above.

The above and other objects, features and advantages of the present invention will become apparent from

Detailed description of the invention

It is to be understood that if any of the previous publications is quoted here, the prior publication does not constitute an acknowledgement that in Taiwan or any other country, the pre-existing publication forms a common general knowledge in the art. Part of it.

For the purposes of this specification, it will be clearly understood that the term "comprising" means "including but not limited to" and the term "comprises" has a corresponding meaning.

All technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention pertains, unless otherwise defined. A person skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which can be used to practice the invention. Of course, the invention is in no way limited by the methods and materials described. For clarity, the following definitions are used herein.

As used herein, the terms "nucleic acid", "nucleic acid sequence" or "nucleotide sequence" and the like mean a deoxyribonucleotide sequence or a ribonucleotide sequence in the form of a single strand or a double strand, and includes There are known naturally occurring nucleotides or artificial chemical mimics. As used herein, the term "nucleic acid" is used interchangeably with "gene," "DNA," "cDNA," "mRNA," "oligonucleotide," and "polynucleotide."

As used herein, the terms "nucleic acid fragment" and "DNA fragment" are used interchangeably and mean a DNA polymer in the form of a separate segment. Or as a component of a larger DNA construct, which may be derived from isolated DNA or chemically prepared by methods well known in the art. Or enzyme synthesis.

Unless otherwise indicated, a nucleic acid sequence encompasses complementary sequences, as well as their conservative analogs, associated naturally occurring structural variants, and in addition to the specific sequences disclosed herein. / or synthetic non-naturally occurring analogs.

As used herein, terms such as "hybridization" and "annealing" are used interchangeably and mean two single nucleotide sequences by virtue of the Watson-Crick rule ( Watson-Crick rules) are specifically sequence-coupled to each other via hydrogen bonding of their purine and/or pyrimidine bases, thereby forming a hybrid. The hybrid is a duplex nucleic acid complex having a stable nucleic acid structure comprising RNA: RNA, RNA: DNA or DNA: DNA double helix molecules.

Traditionally, strain culture and bacteriological characterization have been used to detect specific pathogens that cause food poisoning, but these assays are cumbersome, time consuming, and inaccurate in their operation. Therefore, how to quickly and accurately detect pathogens present in foods is particularly important. Molecular biology methods [such as polymerase chain reaction (PCR) and biochip technology] have the advantages of high speed, high reliability and accuracy of analysis results, so the applicant is committed to developing rapid detection methods in this area. .

In general, highly conserved and highly variable DNA sequences are included in the genomic DNA sequence of bacteria, which are useful for the detection or identification of genus-specific or species-specific. Therefore, the applicant tried to extract M. catarrhalis, Pasteurella septicum, Staphylococcus aureus, Escherichia coli O157:H7, Vibrio cholerae, Pseudomonas fluorescens, Salmonella species, gas producing unit cells Nucleic acid molecules with high specificity and sensitivity for these food pathogens are designed for rapid detection or identification, respectively, in the genomic DNA sequences of the genus Species and Enterobacter species (such as Enterobacter sakazakii).

In the present invention, the applicant is based on the uspA1 gene in the NCBI accession number CP002005 [complete genome of M. catarrhalis RH4], NCBI accession number PMU30165 [the groESL heat shock control group of Pasteurella septicum ( heat shock operon), groES entire coding sequence of groEL gene (complete coding sequence) groES gene] among, NCBI accession number CP000462 [addicted aquatic Aeromonas hydrophila subsp ATCC 7966 (Aeromonas hydrophila subsp. hydrophila ATCC 7966) complete 16S-23S rRNA intergenic spacer region (ISR) in the genome] 16S rRNA gene in NCBI accession number CP000653 (complete genome of Enterobacter 638), NCBI accession number CP000783 (Enterobacter sakazakii ATCC BAA) The hsp10 gene in the complete genome of -894 and the nucleotide sequence shown in NCBI Accession No. EU360312 (the complete coding sequence for the AprX gene of Pseudomonas fluorescens ATCC 17400) were designed for mucositis Mora Bacillus, Pasteurella septicum, Aeromonas species, Enterobacter species, Enterobacter sakazakii, and Pseudomonas fluorescens A pair of primers McF / McR, PmF / PmR, AeGF / AeGR, EnGF / EnGR, Esa1 / Esa2 and PFF / PFR (which has the nucleotide sequence are aggregated in Table 1).

In addition to designing primer pairs for the detection of food pathogens, the applicant also relied on the hsp10 gene in NCBI Accession No. CP000783 (the complete genome of Enterobacter sakazakii ATCC BAA-894), NCBI Accession No. BA000018 [Staphylococcus aureus The nucleotide sequence shown in the complete genome of N315 ( Staphylococcus aureus subsp. aureus N315 ), shown in NCBI Accession No. AE005174 [E. coli O157:H7 EDL933 ( Escherichia coli O157:H7 EDL933 ) complete genome] nucleotide sequence, NCBI accession number L30116 (Vibrio parahaemolyticus pR72H DNA fragment) from among the nucleotide sequence shown and NCBI Accession No. AM933172 [Salmonella typhimurium subsp P125109 (Salmonella enterica subsp. enterica serovar enteritidis str. P125109) Specific nucleotides shown in the complete genome], and designed specific probes for Enterobacter sakazakii, Staphylococcus aureus, Escherichia coli O157:H7, Vibrio cholerae, and Salmonella species (they The nucleotide sequences possessed are summarized in Table 4). In addition, according to the above five primers, including McF, PmR, AeGR, EnGF and PFF, they are also used as species against M. catarrhalis, Pasteurella septicum, Aeromonas, A specific probe for Enterobacter species and Pseudomonas fluorescens.

In order to evaluate the detection specificity and sensitivity of the primers and/or probes according to the present invention to food pathogens, Applicants used genomic DNA of bacterial strains obtained from different food samples as templates, and respectively used the 6 sets of primers of the present invention. PCR reaction is performed on McF/McR, PmF/PmR, AeGF/AeGR, EnGF/EnGR, Esa1/Esa2, and PFF/PFR, or a combination of primers is used for multiplex PCR, wherein the primer combination is At least one set of primers in accordance with the present invention is constructed with at least one set of conventionally disclosed primer pairs (as shown in Table 4). The PCR amplification product obtained by PCR or multiplex PCR is analyzed by agarose gel electrophoresis and found that the primer pair according to the present invention has high specificity and sensitivity for the target species to be detected. (sensitivity).

Further, the applicant further takes a hybridization reaction based on the PCR amplification product obtained above and a biochip spotted with the probe according to the present invention. The map after hybridization shows that the probe according to the invention is also highly specific and sensitive to the target species to be detected.

Based on the above, the primer pair and probe according to the present invention are expected to be available for rapid detection of pathogenic bacteria present in a food sample. Accordingly, the present invention provides a nucleic acid molecule reagent for detecting a food pathogen comprising at least one of the following:

(a) a nucleic acid molecule for detecting M. catarrhalis having a nucleotide sequence selected from the group consisting of: sequence identification number: 1 and sequence identification number: 2;

(b) a nucleic acid molecule for detecting Pasteurella septicum having a nucleotide sequence selected from the group consisting of: Sequence ID: 3 and Sequence ID: 4.

(c) a nucleic acid molecule for detecting a species of Aeromonas, having a nucleotide sequence selected from the group consisting of: Sequence ID: 5 and Sequence ID:

(d) a nucleic acid molecule for detecting an Enterobacter species having a nucleotide sequence selected from the group consisting of: Sequence ID: 7 and Sequence ID: 8.

(e) a nucleic acid molecule for detecting Enterobacter sakazakii having a nucleotide sequence selected from the group consisting of: Sequence ID: 9, Sequence ID: 10, and Sequence ID: 21.

(f) a nucleic acid molecule for detecting Pseudomonas fluorescens having a nucleotide sequence selected from the group consisting of: sequence identification number: 11 and sequence identification number: 12;

(g) a nucleic acid molecule for detecting S. aureus having a nucleotide sequence as shown in SEQ ID NO:22;

(h) a nucleic acid molecule for detecting E. coli O157:H7 having a nucleotide sequence as shown in SEQ ID NO: 23;

(i) a nucleic acid molecule for detecting Vibrio cholerae having a nucleotide sequence as shown in SEQ ID NO: 24;

(j) A nucleic acid molecule for detecting a Salmonella species having a nucleotide sequence as shown in SEQ ID NO: 25.

Nucleic acid molecules according to the present invention may be further attached or conjugated to a detectable label using standard techniques well known to those of ordinary skill in the art to permit detection or quantification of such pathogenic bacteria. Detectable labels suitable for use in the present invention include, but are not limited to, hapten labels [eg, biotin and digoxigenin (Dig)], chemiluminescent labels. , fluorescent markers (fluorescent labels) [e.g., fluorescein (Fluorescein), rose Bengal (rhodamine), NH ₂ - glycoside cyanine _{5 (NH 2 -cyanin 5, Cy5} ) and NH ₂ - 3 cyanine glycosides (NH ₂ -cyanin 3, Cy3)], enzyme labeling, radioactive labeling (eg, ³² P), particle labels [eg, gold colloids], and colorimetric labels [eg, Dye and colored glass or plastic beads].

According to the present invention, when the nucleic acid molecules are used as probes, at least one nucleotide residue may be added or deleted at their 5' and/or 3' ends, so that the length thereof is Within the range of about 15 to 30 nucleotide residues. Furthermore, at least one thymidine may be added to the 5' end and/or the 3' end of the nucleic acid molecules such that they may have better immobilization when spotted on the surface of the biochip. Immobilization, and enhance the detection signals they obtain when performing hybridization reactions. These are techniques that are familiar and familiar to those skilled in the art. Preferably, the number of thymidines added at the 5' and/or 3' ends of the nucleic acid molecules falls within the range of about 10 to 35.

The invention also provides a method for detecting the presence or absence of a pathogen in a food sample, comprising: subjecting the food sample to a DNA amplification reaction using at least one set of primer pairs, wherein the at least one set of primer pairs is selected from the group consisting of In the group consisting of: (a) a primer pair for detecting M. catarrhalis comprising a nucleotide sequence as shown in sequence identification number: 1 and a primer a reverse primer having a nucleotide sequence as shown in sequence identification number: 2; (b) a primer pair for detecting Pasteurella septicum comprising a sequence as shown in sequence identification number: The nucleotide sequence is preceded by a primer, and a primer having a nucleotide sequence as shown in SEQ ID NO: 4; (c) a primer pair for detecting a species of Aeromonas, which comprises a forward sequence of a nucleotide sequence as shown in sequence identification number: 5, and a reverse primer having a nucleotide sequence as shown in sequence identification number: 6; (d) one for detecting the intestine a pair of primers of the genus Bacillus, which contains A nucleotide sequence as shown in sequence identification number: 7 is preceded by a primer, and a reverse primer having a nucleotide sequence as shown in sequence identification number: 8; (e) one for detecting sakisaki a pair of primers of Enterobacter, which contains one For example, the nucleotide sequence shown in sequence identification number: 9 is forward-introduced, and a reverse primer having a nucleotide sequence as shown in sequence identification number: 10; and (f) one is used to detect fluorescent false A primer pair of a bacterium comprising a forward sequence of a nucleotide sequence as shown in SEQ ID NO: 11 and a reverse primer having a nucleotide sequence as shown in SEQ ID NO: 12. And detecting whether there is a DNA fragment amplified by using the at least one set of primer pairs, wherein the presence of the DNA fragment indicates the presence of a pathogen corresponding to the at least one set of primer pairs.

According to the present invention, total genomic DNA is extracted from the food sample as a template before the DNA amplification reaction is carried out.

According to the present invention, the DNA amplification reaction can be carried out by using at least one of the following methods: polymerase chain reaction (PCR), reverse transcriptase polymerase chain reaction (RT-PCR), instant Real time quantitative PCR, nested PCR, hot-start PCR, in-situ PCR, degenerate oligonucleotide primer PCR Degenerate oligonucleotide primer PCR, DOP PCR, micro PCR, multiplex polymerase chain reaction, restriction fragment length polymorphism nucleic acid Sequence-based amplification (NASBA), transcription-mediated amplification (TMA), loop-mediated isothermal amplification (LAMP), and rolling circle amplification , RCA).

According to the present invention, the DNA amplification reaction can be carried out on a biochip selected from the group consisting of microfluidic wafers (for example, sample pretreatment wafers, reactive wafers, and analytical wafers) and Wafer Lab.

According to the present invention, the detection of the DNA fragment can be carried out by a hybridization reaction using a probe. Preferably, the probe has a nucleotide sequence selected from the group consisting of: sequence identification number: 1. sequence identification number: 2. sequence identification number: 3. sequence identification number: 4. Sequence identification number: 5, sequence identification number: 6, sequence identification number: 7, sequence identification number: 8, sequence identification number: 9, sequence identification number: 10, sequence identification number: 21, sequence identification number: 11 and sequence identification Number: 12.

In a preferred embodiment of the present invention, the probe has a nucleotide sequence selected from the group consisting of: sequence identification number: 1, sequence identification number: 4, sequence identification number: 6 , sequence identification number: 7, sequence identification number: 21 and sequence identification number: 11.

According to the present invention, the hybridization reaction can be carried out on a biochip selected from the group consisting of a gene wafer, a microfluidic wafer (for example, a sample pretreatment wafer, a reactive wafer, and an analytical wafer). And the wafer lab. In a preferred embodiment of the invention, the hybridization reaction is carried out on a gene wafer.

According to the invention, the food sample may be, but is not limited to, fluid milk products such as milk and goat milk; beverages; meat products such as chicken, beef, lamb and pork; sea foods ), such as fish and shellfish; animal feeds; water; dairy products such as yogurt, ice cream, and cream cheeses; vegetables ; fruit; and canned foods. In a preferred embodiment of the invention, the food sample is milk. In another preferred embodiment of the invention, the food sample is chicken.

Nucleic acid molecules according to the present invention can be used as probes and directly subjected to hybridization reactions with food samples, as is well known and commonly employed by those skilled in the art.

Accordingly, the present invention also provides a method for detecting the presence or absence of a pathogen in a food sample, comprising: subjecting the food sample to a hybridization reaction using at least one probe, wherein the at least one probe has a nucleoside The acid sequence and the corresponding pathogen are as defined above for the nucleic acid molecules; and detecting whether there is a hybrid formed by performing hybridization reaction using the at least one probe, wherein the presence of the hybrid indicates The presence of a pathogen corresponding to the at least one probe.

In a preferred embodiment of the present invention, the at least one probe has a nucleotide sequence selected from the group consisting of: sequence identification number: 1, sequence identification number: 4, sequence identification number : 6, sequence identification number: 7, sequence identification number: 21, sequence identification number: 11, sequence identification number: 22, sequence identification number: 23, sequence identification number: 24 and sequence identification number: 25.

According to the present invention, the hybridization reaction can be carried out on a biochip selected from the group consisting of a gene wafer, a microfluidic wafer (for example, a sample pretreatment wafer, a reactive wafer, and an analytical wafer). And the wafer lab. In a preferred embodiment of the invention, the hybridization reaction is carried out on a gene wafer.

The nucleic acid molecule according to the invention can be used to prepare a test kit for detecting a food pathogen. In addition to the nucleic acid molecules, the test kit can further comprise a nucleic acid dye for monitoring DNA amplification products. Preferably, the nucleic acid dye is selected from the group consisting of: ethidium bromide (EtBr), SYBR GREEN I, SYBR GREEN II, SYBR Orange, SYBR Gold, Phopidium Iodide, SYTOX Blue, SYPRO Ruby and Coomassie Blue.

Nucleic acid molecules according to the present invention are also contemplated for use in biochips to rapidly detect pathogenic bacteria present in a food sample.

Detailed description of the preferred embodiment

The invention is further described in the following examples, but it should be understood that these examples are for illustrative purposes only and are not to be construed as limiting.

Example Experimental Materials:

1. The bacterial strains used in the following examples are Biosource Collection and Research Center purchased from the Food Industry Research and Development Institute (FIRDI) in Taiwan. , BCRC) (300 Food Road, Hsinchu City, Taiwan), National Ping Tung University of Science and Technology (PT), American Type Culture Collection (ATCC), New York, USA The City of New York Department of Health, USA (US), and the United State Department of Agriculture (USDA), where these strains include:

(1) Moraxella spp., including Moraxella catarrhalis BCRC 10629 and 10628, Moraxella osloensis BCRC 10705, and Moraxella Bovis ) BCRC 11229;

(2) Pasteurella multocida BCRC 16079, 13850, 13851 and 15427;

(3) Aeromonas spp., including Aeromonas sp. A. hydrophila BCRC 13018 and 16704, Aeromonas aeruginosa ( A. caviae ) BCRC 13933, mineral-free springs Aeromonas ( A. eucrenophila ) BCRC 14134 and Aeromonas aeruginosa ( A. sobria ) BCRC 13934;

(4) Enterobacter species (Enterobacter spp.), Including Enterobacter sakazakii (E. Sakazakii) BCRC 14122,13988 and 14153, Enterobacter cloacae (E. Cloacae) BCRC 11507,10401 and 12831, Enterobacter aerogenes ( E. aerogenes ) BCRC 17147 and 15630, E. amnigenus BCRC 13987, E. gergoviae BCRC 17150, Escherichia coli ( E. hormaechei ) BCRC 17676, Enterobacter cloacae ( E. asburiae ) BCRC 17675, E. ludwigii BCRC 17716 and E. intermedius BCRC 14808;

(5) Pseudomonas spp., including Pseudomonas fluorescens BCRC 16016, 13902, 11028, 13904 and 10304, P. synxantha BCRC 11022 P. putida BCRC 12857, 10459 and 11016, P. aeruginosa BCRC 10944 and P. mendocina BCRC 10458;

(6) Acinetobacter spp., including Acinetobacter sp. strains BCRC 15638, 15417, 15420 and 17454, A. calcoaceticus BCRC 11562, A. baumannii BCRC 15556 and 15318, A. radioresistens BCRC 15425, A. lwoffii BCRC 14855, A. junii BCRC 14854, A. haemolyticus BCRC 14852 And A. johnsonii BCRC 14853;

(7) Listeria spp., including Listeria monocytogenes BCRC 14845, 14846, 14847, 14848, 14930, 15327, 15330, 15331, 15332, 15333, 15334, 15335, 15336, 15337 and 15338, Listeria seeligeri BCRC 15361, Listeria ivanovii BCRC 15383 and Listeria welshimeri BCRC 15362;

(8) Staphylococcus aureus ATCC 700699, BCRC 10451, 10780, 10781, 10782, 10823, 10908, 11863, 12154, 12652, 12653, 12654, 12656, 12657, 13008, 13824, 13825, 13826, 13827 , 13829, 13830, 13831, 13958, 14961, 14963, 14972, 14987, 15205 and 15309;

(9) Salmonella spp., including S. Typhimurium BCRC 10747 and 12947, S. hadar PT 677, S. infantis USDA 1117B With US, S. salamae BCRC 15450, S. barley ( S. bareilly ) USDA 1 and US 1, California Salmonella ( S. california ) US 2, S. cerevisiae ( S. Cerro ) USDA 671D, USDA 2 and US 3, S. choleraesuis PT and BCRC 10743, S. derby PT 601, S. dublin US and USDA 1146B1 S. enteritidis ATCC 13076 and US, S. florida US, S. hadar PT 677 and USDA 1362E, S. indiana US, Kentucky sand door S. kentucky USDA 1073E, S. minnesota US, S. miami USDA, S. newington USDA, S. newport PT With US, A type Cold bacillus (S. paratyphi A) PT 398, Tennessee Salmonella (S. tennessee) USDA 1258 and Thompson Salmonella (S. thompson) USDA 1363E;

(10) Streptococcus spp., including Streptococcus agalactiae BCRC 10787, 5 strains of Streptococcus agalactiae isolates HK 88001 to 88005, and Streptococcus pneumoniae BCRC 10794;

(11) Vibrio spp., including Vibrio parahaemolyticus BCRC 12863, 12963, 13023 and 13025, Vibrio mimicus BCRC 14690, Vibrio vulnificus BCRC 12905, Vibrio fluvialis BCRC 12830 and Vibrio furnissii BCRC 15889;

(12) Escherichia coli BCRC 14824 and 14825, Escherichia coli O157:H7 ( Eschrichia coli O157:H7) BCRC 13084, 13086, 13094, 13095, 13096, ATCC 35150 and 43890;

(13) Yersinia spp., including Yersinia enterocolitica BCRC 10807, 12986 and 13999, and Yersinia ruckeri BCRC 14124;

(14) Clostridium spp., including Clostridium perfringens BCRC 16152, Clostridium butyricum BCRC 11665, Clostridium celatum BCRC 14521, tying Clostridium nexile BCRC 14523 and Clostridium haemolyticum BCRC 10643;

(15) Corynebacterium spp., including Corynebacterium minutissimum BCRC 14870, Corynebacterium cystitidis BCRC 12320, Corynebacterium pseudotuberculosis BCRC 14872, Corynebacterium renale BCRC 10657, Corynebacterium matruchotii BCRC 14869, Corynebacterium variabile BCRC 14862, Corynebacterium flavescens BCRC 12131, and glutamate stick Corynebacterium glutamicum BCRC 10027;

(16) Kurthia spp., including Kurthia gibsonii BCRC 17033 and Kocuria rosea BCRC 11577;

(17) Neisseria spp., including Neisseria meningitides BCRC 10714 and Neisseria mucosa BCRC 10716;

(18) Plesiomonas shigelloides BCRC 14120;

(19) Serratia spp., including Serratia marcescens BCRC 11576, 12833 and 12832, Serratia entomophila BCRC 15867, liquefied Serra Serratia liquefaciens BCRC 19989 and Serratia rubidaea BCRC 13990;

(20) Alcaligenes faecalis BCRC 10828;

(21) Brevibacterium linens ATCC 19391;

(22) Bacillus cereus BCRC 10250, 11837 and 14195;

(23) Citrobacter freundii BCRC 12291;

(24) Hafnia alvei BCRC 10906;

(25) Kluyvera ascorbata BCRC 11645;

(26) Klebsiella pneumonia BCRC 10692;

(27) Lactobacillus spp., including Lactobacillus acidophilus BCRC 14079, Lactobacillus casei BCRC 14084, and Lactobacillus subsp. lactis ( L. delbrueckii subsp. Lactis BCRC 12256;

(28) Shigella spp., including Shigella flexneri BCRC 10772, Shigella sonnei BCRC 10773 and 10774, and Shigella boydii BCRC 15959;

(29) Proteus vulgaris ATCC 8427.

2. Tryptic Soy Broth (TSB) and Plate Count Agar (PCA) used in the following examples were purchased from Difco (Difco Uboratories, Detroit, Michigan U.S.A.).

3. Biotin-labeled primer pairs and probes used in the following examples were synthesized by Biotech (Taipei, Taiwan).

General experimental method: 1. Extraction of genomic DNA:

The extraction of the genomic DNA of the bacterial strain was carried out using the Blood and Tissue Genomic DNA Miniprep System (VIOGENE). First, 1 mL of the bacterial culture or food sample was placed in a microcentrifuge tube, followed by centrifugation at 10,000 rpm for 5 minutes. After the supernatant was removed, the pellets were washed twice with 1 mL of sterile water. Thereafter, 200 μL of sterile water was added to fully suspend the cells, followed by the addition of 20 μL of lysozyme (2 mg/mL) and 20 μL of RNase A (2 mg/mL) and reacted at 37 ° C for 3 hours. Next, 200 μL of Ex buffer and 15 μL of proteinase K (10 mg/mL) were added and shake-mixed, whereby the resulting mixture was placed in a 60 ° C water bath for overnight reaction. Thereafter, the mixture was placed in a 70 ° C water bath for 30 minutes to deactivate the proteinase K, followed by the addition of 250 μL of absolute alcohol and a slight oscillating mixture for a few seconds. Next, a B/T Genomic DNA Mini Column was placed in a collection tube, and all the mixture was transferred to the column, followed by 8,000 rpm. The centrifugation lasted 2 minutes. Thereafter, the column was placed in a new collection tube, and the precipitate in the column was washed twice with 500 μL of WS buffer, followed by centrifugation at 8,000 rpm for 2 minutes. After the eluate was removed, the column was placed back into the collection tube and centrifuged at 12,000 rpm for 3 minutes. Finally, the column was placed in a new microcentrifuge tube, and then 100 μL of sterile water (preheated to 70 ° C) was added to the center of the column and allowed to stand for 5 minutes, then at 13,000 rpm. The centrifugation was carried out for 30 seconds to elute the genomic DNA of the bacterial strain. The genomic DNA thus obtained was stored at -20 ° C for use.

Example 1. Design of primer pairs for detecting food pathogenic bacteria

Based on the NCBI accession number CP002005 [complete genome of M. catarrhalis RH4], the uspA1 gene, NCBI accession number PMU30165 [the groESL heat shock operon of septicum bacillus , groES and the complete coding sequence of groEL gene (complete coding sequence) groES gene] among, NCBI accession number CP000462 [tropic 16S-23S aquatic Aeromonas hydrophila subsp complete genome ATCC 7966 (Aeromonas hydrophila subsp. hydrophila ATCC 7966) ] a among 16S rRNA gene in the rRNA intergenic spacer region (ISR), NCBI accession number CP000653 (complete genome of Enterobacter 638), NCBI accession number CP000783 (complete genome of Enterobacter sakazakii ATCC BAA-894) The nucleotide sequence shown in the hsp10 gene and the NCBI accession number EU360312 (the complete coding sequence of the AprX gene of Pseudomonas fluorescens ATCC 17400), and against M. catarrhalis, Pasteurella septicum , respectively Aeromonas species, Enterobacter species, Enterobacter sakazakii, and Pseudomonas fluorescens were used to design multiple sets of primer pairs.

These primers then use the Gene Blast software provided on the NCBI website to separately access the bacterial gene database of the NCBI website (http://www.ncbi.nlm.nih.gov/genomes/genlist.cgi?taxid=2&type= Alignment analysis of all gene sequences in 0&name=Complete%20Bacteria), thereby screening for M. catarrhalis, Pasteurella septicum, Aeromonas species, Enterobacter species, and Sakae Specific primers for Enterobacter sakazakii and Pseudomonas fluorescens vs. McF/McR, PmF/PmR, AeGF/AeGR, EnGF/EnGR, Esa1/Esa2, and PFF/PFR (see Table 1), and then the pair of primers Biotin was later calibrated for subsequent experiments.

Example 2. Analysis of the specificity and sensitivity of the primer of the present invention on the detection of food pathogens

In order to evaluate the specificity and sensitivity of the six sets of biotin-labeled primer pairs designed in Example 1 above, the following experiments were carried out.

A. The specificity test of the pair of primers:

First, 198 bacterial strains described in the above item 1 of the "Experimental Materials" were inoculated separately into TSB broth medium, and cultured overnight in a constant temperature shaking incubator (26 to 37 ° C, 150 rpm). Next, 1 mL of the bacterial culture was taken and the genomic DNA was extracted according to the method described in the first item "Extraction of genomic DNA" in "General Experimental Methods" above. Then, the obtained genomic DNA of each bacterial strain was used as a template, and the biotin-labeled primer pairs designed in the above Example 1 were respectively used, McF/McR, PmF/PmR, AeGF/AeGR, EnGF/EnGR, Esa1/Esa2, and PFF/PFR were used for polymerase chain reaction (PCR), and the reaction conditions of PCR are shown in Table 2 below.

After the completion of the PCR, it was confirmed by 2% agarose gel electrophoresis whether or not a PCR product of a predetermined size as shown in Table 1 was obtained.

B. Sensitivity test of the pair of primers:

To understand the sensitivity of the six sets of primers disclosed in the present invention to the detection of food pathogens, Moraxella catarrhalis BCRC 10629, Pasteurella septicum BCRC 13850, Aeromonas hydrophila BCRC 13018, Enterobacter aerogenes BCRC 15630, Enterobacter sakazakii BCRC 14122, and Pseudomonas fluorescens BCRC 11028 were used for the following experiments. First, these 6 strains were inoculated separately into TSB broth culture medium and cultured in a constant temperature shaking incubator (culture temperature: Moraxella catarrhalis BCRC 10629 and Pasteurella bacillus BCRC 13850 at 37 ° C, water-producing gas Monocytogenes BCRC 13018, Enterobacter aerogenes BCRC 15630 and Enterobacter sakazakii BCRC 14122 were 30 ° C, Pseudomonas fluorescens BCRC 11028 was 26 ° C; and shaking speed: all strains were 150 rpm). The resulting bacterial culture was adjusted to a concentration of 10 ⁹ CFU/mL (counted by plate count medium) in TSB broth medium, and used as a stock for 10-fold serial dilution ( 10-fold serial dilution), whereby diluted bacterial solutions having different concentrations (10 ⁶ , 10 ⁵ , 10 ⁴ , 10 ³ , 10 ² and 10 ¹ CFU/mL) were obtained. Thereafter, 1 mL of each of the diluted bacterial solutions containing different bacterial concentrations was taken, and genomic DNA was extracted according to the method described in the first item "Extraction of genomic DNA" in the "General Experimental Method" above.

In addition, a part of the diluted bacterial solution containing the different bacterial concentrations obtained according to the above manner is placed in a constant temperature shaking incubator (the culture temperature and the oscillation speed of each strain are as described above) for enrichment culturing for a duration of time. 10 hours. Thereafter, 1 mL of each of the diluted bacterial solutions containing different bacterial concentrations in the proliferation culture was taken, and genomic DNA was extracted according to the method described in the first item "Extraction of genomic DNA" in the "General Experimental Method" above.

Genomic DNA of each of the non-proliferation-cultured and culture-grown bacterial strains obtained above was used as a template, and PCR was carried out using biotin-labeled primer pairs as shown in Table 1 above, respectively, and PCR The reaction conditions are as shown in Table 2 above.

After the completion of the PCR, it was confirmed by 2% agarose gel electrophoresis whether or not a PCR product of a predetermined size as shown in Table 1 was obtained.

result: A. The specificity test of the pair of primers:

Agarose gel electrophoresis analysis revealed that only when the genomic DNA of each bacterial strain was used as a template and the biotin-labeled primer was used to perform PCR on McF/McR, only M. catarrhalis had a size of about 112 bp PCR amplification product.

In addition, when PCR was performed using PmF/PmR with biotin-labeled primers, only S. septicum had a PCR amplification product of approximately 243 bp in size; when biotin-labeled primers were used for AeGF/ When AeGR is used for PCR, only Aeromonas species (such as Aeromonas hydrophila, Aeromonas aeruginosa, Aeromonas aeruginosa, and Aeromonas aeruginosa) have a size of about Is a 438 bp PCR amplification product; when using the biotin-labeled primer pair for EnGF/EnGR for PCR, only Enterobacter species (such as Enterobacter sakazakii, Enterobacter cloacae, Enterobacter aerogenes, River sausage) Bacillus, Enterobacter jejuni, Enterobacter sinensis, Enterobacter cloacae, Enterobacter serovar and Enterobacter sinensis have a PCR amplification product of approximately 491 bp in size; when using biotin-labeled primers When PCR was performed on Esa1/Esa2, only A. sakazakii obtained a PCR amplification product of approximately 491 bp in size; and when PCR was performed using biotin-labeled primer pair PFF/PFR, only fluorescent false Monocytogenes has a PCR amplification product with a size of approximately 779 bp. .

The results of this experiment show that biotin-labeled primers for McF/McR, PmF/PmR, AeGF/AeGR, EnGF/EnGR, Esa1/Esa2, and PFF/PFR for M. catarrhalis, Pasteurella septicum, Aeromonas species, Enterobacter species, Enterobacter sakazakii, and Pseudomonas fluorescens have detection specificity.

B. Sensitivity test of the pair of primers:

Table 3 below shows the results of sensitivity tests of six sets of biotin-labeled primer pairs according to the present invention. It can be seen from Table 3 that the detection sensitivity of biotin-labeled primers to McF/McR, PmF/PmR, AeGF/AeGR, EnGF/EnGR, Esa1/Esa2, and PFF/PFR for non-proliferated food pathogens can reach 10 ³ CFU/mL, and the detection sensitivity of the proliferated cultured food pathogen can reach 10 ⁰ CFU/mL. The results of this experiment show that biotin-labeled primers have high sensitivity to McF/McR, PmF/PmR, AeGF/AeGR, EnGF/EnGR, Esa1/Esa2 and PFF/PFR and can be used to detect food pathogens.

Example 3. Design of probes for detecting food pathogens

According to NCBI accession number CP000783 (the complete genome of Enterobacter sakazaki ATCC BAA-894)Hsp10 Gene, NCBI accession number BA000018 [Staphylococcus aureus subspeciesN315 (Staphylococcus Aureus Subsp. aureus N315 The nucleotide sequence shown in the complete genome], NCBI accession number AE005174 [E. coli O157:H7EDL933 (Escherichia coli O157:H7EDL933 The nucleotide sequence shown in the complete genome], the nucleotide sequence shown in NCBI Accession No. L30116 (Vibrio cholerae pR72H DNA fragment), and the NCBI accession number AM933172 [S. typhimurium subspecies]P125109 (Salmonella Enterica subsp. enterica serovar Enteritidis str. P125109 The nucleotide sequences shown in the complete genome], and various probes were designed for Enterobacter sakazakii, Staphylococcus aureus, Escherichia coli O157:H7, Vibrio cholerae, and Salmonella species, respectively.

These probes then use the Gene Blast software provided on the NCBI website to separately access the bacterial gene database on the NCBI website (http://www.ncbi.nlm.nih.gov/genomes/genlist.cgi?taxid=2&type= Alignment analysis of all gene sequences in 0&name=Complete%20Bacteria), thereby screening for specificity against Enterobacter sakazakii, Staphylococcus aureus, Escherichia coli O157:H7, Vibrio cholerae, and Salmonella species Probe.

In addition, the five primers designed in the above Example 1, including McF, PmR, AeGR, EnGF, and PFF, were also used as the M. catarrhalis, Pasteurella septicum, and gas-producing cells, respectively. Specific probes for the genus Fungi, Enterobacter species, and Pseudomonas fluorescens.

For clarity, the primer pairs and probe related information (including: nucleotide sequence, size of the amplified PCR product, corresponding to the target gene or the location of the complete genomic DNA, etc.) according to the present invention have been integrated. In Table 4 below.

Example 4. Evaluation of the ability of a primer according to the present invention to detect pathogenic bacteria in foods

To assess the specificity of the six sets of biotin-labeled primer pairs according to the present invention and the conventionally disclosed four sets of biotin-labeled primer pairs (as shown in Table 4) for pathogens present in foods. With sensitivity, milk samples containing different pathogens and chicken samples were taken for PCR reaction.

A. Preparing food samples containing food pathogenic bacteria (1) Preparation of milk samples containing M. catarrhalis, Pasteurella septicum, Aeromonas hydrophila or Enterobacter aerogenes:

First, M. catarrhalis BCRC 10629, Pasteurella multocida BCRC 13850, Aeromonas hydrophila BCRC 13018, and Enterobacter aerogenes BCRC 15630 were inoculated into TSB broth medium and cultured at a constant temperature. Box (culture temperature: M. catarrhalis BCRC 10629 and S. septicum BCRC 13850 at 37 ° C, Aeromonas hydrophila BCRC 13018 and Enterobacter aerogenes BCRC 15630 at 30 ° C; Oscillation rate: all strains Incubate overnight at 150 rpm). The formed bacterial culture was adjusted to a concentration of 10 ⁹ CFU/mL (counted by the plate count medium) in TSB broth medium, and 10 times serial dilution was performed as a stock solution, thereby Diluted bacterial solutions with different concentrations (10 ⁹ ~ 10 ² CFU/mL) were obtained. Then, take 100 μL of each diluted liquid solution containing different bacterial concentrations, and then add 1 mL of milk and 8.9 mL of TSB broth medium and mix them evenly, thereby obtaining different bacterial concentrations (10 ⁷ ~ 10 ⁰ Milk sample of CFU/mL).

In addition, a part of the milk samples containing different bacterial concentrations obtained according to the above manner are placed in a constant temperature shaking incubator (the culture temperature and the oscillation speed of each strain are as described above), and the proliferation culture is carried out for 10 hours. This resulted in a proliferated culture of milk samples containing different bacterial concentrations.

(2) Preparation of milk samples containing Staphylococcus aureus, Enterobacter sakazakii, Escherichia coli O157:H7, Vibrio cholerae, Salmonella typhimurium or Pseudomonas fluorescens and chicken samples:

In this experiment, the applicant selected Staphylococcus aureus ATCC 700699, Enterobacter sakazakii BCRC 14122, Escherichia coli O157:H7 BCRC 14824, Vibrio cholerae BCRC 13025, Salmonella typhimurium ATCC 14028, and Pseudomonas fluorescens BCRC 11028 and prepare a milk sample containing the pathogens according to the method described in the above point (1), and the culture temperature of each strain is as follows: Staphylococcus aureus ATCC 700699, Escherichia coli O157: H7 BCRC 14824, Vibrio cholerae BCRC 13025 and Salmonella typhimurium ATCC 14028 were 37 ° C, Enterobacter sakazakii BCRC 14122 was 30 ° C, and Pseudomonas fluorescens BCRC 11028 was 26 °C. In addition, the preparation of chicken samples containing such pathogenic bacteria was generally carried out in the manner described above, except that the ground chicken was used instead of milk.

B. The specificity test of the pair of primers:

Milk samples containing Moraxella catarrhalis, Pasteurella septicum, Aeromonas hydrophila or Enterobacter aerogenes produced in the unproliferated culture prepared in point (1) of item A above (each The sample contained 1 mL of a bacterial concentration of 10 ⁷ CFU/mL, and the genomic DNA was extracted according to the method described in the first item "Extraction of genomic DNA" in "General Experimental Methods" above. Then, PCR was carried out using the obtained genomic DNA of each bacterial strain as a template and using biotin-labeled primers for McF/McR, PmF/PmR, AeGF/AeGR, and EnGF/EnGR, respectively, and the reaction conditions of PCR were as follows. The one shown in Table 2 above. After the completion of the PCR, each of the strains obtained four PCR amplification products, respectively, so that a total of 16 PCR amplification products were obtained, which were collectively referred to as the first group PCR product.

In addition, the unproliferated culture containing Staphylococcus aureus, Enterobacter sakazakii, Escherichia coli O157:H7, Vibrio cholerae, Salmonella typhimurium or fluorescein prepared in the above item (2) Take 1 mL of the Pseudomonas milk sample and the chicken sample (each sample contains a bacterial concentration of 10 ⁷ CFU/mL), and follow the item "Extraction of genomic DNA" in item 1 of the "General Experimental Methods" above. The method described is for the extraction of genomic DNA. Then, PCR was carried out using the obtained genomic DNA of each bacterial strain as a template and using biotin-labeled primer pairs for SAU3/SAU4, Esa1/Esa2, HAF1/HAR1, VP33/VP32, Sspp1S/Sspp1A, and PFF/PFR, respectively. And the reaction conditions of PCR are as shown in Table 2 above. According to the above, when PCR is carried out from bacterial genomic DNA obtained from a milk sample and a chicken sample, each of the strains obtains 12 PCR amplification products, respectively, so that a total of 72 PCR amplification products are obtained, which are collectively referred to as Group 2 PCR product.

The obtained first group and the second group PCR products were each subjected to 2% agarose gel electrophoresis analysis to confirm whether or not a PCR product of a predetermined size was obtained.

C. Sensitivity test of the pair of primers:

For the non-proliferation culture and the proliferation culture prepared in point (1) of item A above, containing different concentrations (10 ⁰ , 10 ¹ , 10 ² , 10 ³ , 10 ⁴ and 10 ⁵ CFU/mL) The milk samples of Moraxella catarrhalis were each taken 1 mL, and genomic DNA was extracted according to the method described in the first item "Extraction of genomic DNA" in "General Experimental Methods" above. Next, the obtained genomic DNA of M. catarrhalis which has not been subjected to proliferation culture and proliferation culture is used as a template, and PCR is carried out by using biotin-labeled primers for McF/McR, and the reaction conditions of PCR are as follows. The one shown in Table 2 above. After completion of the PCR, a total of 12 PCR amplification products were obtained, which are collectively referred to as the third group PCR product.

The sensitivity test for milk samples containing Pasteurella septicum, Aeromonas hydrophila or Enterobacter aerogenes is generally carried out in the manner described above for milk samples containing M. catarrhalis, different The reason is that when PCR is carried out using genomic DNA of Pasteurella septicum, Aeromonas hydrophila and Enterobacter aerogenes as a template, the selected pair of primers are biotin-labeled PmF/PmR and AeGF, respectively. /AeGR and EnGF/EnGR. After the completion of the PCR, 15 strains of PCR amplification products were obtained from Pasteurella septicum, Aeromonas hydrophila, and Enterobacter aerogenes, which were collectively referred to as Group 4-6 PCR products, respectively.

In addition, the non-proliferation culture and the proliferation culture prepared in point (2) of item A above contain different bacterial concentrations (10 ⁰ , 10 ¹ , 10 ² , 10 ³ , 10 ⁴ and 10 ⁵ CFU / The sensitivity test of the milk sample and the chicken sample of mL) is generally carried out in the manner described above for the milk sample containing M. catarrhalis, except that: Staphylococcus aureus, Sakasaki When the genomic DNA of Bacillus, Escherichia coli O157:H7, Vibrio cholerae, Salmonella typhimurium, and Pseudomonas fluorescens was used as a template for PCR, the selected pair of primers were biotin-labeled SAU3/SAU4 and Esa1, respectively. /Esa2, HAF1/HAR1, VP33/VP32, Sspp1S/Sspp1A, and PFF/PFR. According to the above, when performing PCR using bacterial genomic DNA obtained from milk samples, Staphylococcus aureus, Enterobacter sakazakii, Escherichia coli O157:H7, Vibrio cholerae, Salmonella typhimurium, and Pseudomonas fluorescens Each of them will receive 12 PCR amplification products, which are collectively referred to as the 7th to 12th cluster PCR products, and when PCR is performed on bacterial genomic DNA obtained from chicken samples, Staphylococcus aureus and Enterobacter sakazakii Escherichia coli O157:H7, Vibrio cholerae, Salmonella typhimurium and Pseudomonas fluorescens each obtained 12 PCR amplification products, which were collectively referred to as the 13th to 18th cluster PCR products.

Finally, the obtained 3rd to 18th cluster PCR products were each subjected to 2% agarose gel electrophoresis analysis to confirm whether a PCR product of a predetermined size as shown in Table 4 was obtained.

result: A. The specificity test of the pair of primers:

Analysis by agarose gel electrophoresis revealed that genomic DNA of M. catarrhalis, P. septicum, Aeromonas hydrophila or Enterobacter aerogenes obtained from milk samples was used as a template and used. When biotin-labeled primers were used for PCR on McF/McR, only M. catarrhalis obtained a PCR amplification product of approximately 112 bp in size; when using biotin-labeled primers for PmF/PmR At the time of PCR, only a 243 bp PCR amplification product was obtained from Pasteurella septicum; when PCR was performed on AeGF/AeGR using biotin-labeled primers, only the genus Aeromonas aeruginosa had a A PCR amplification product of approximately 438 bp in size; and when PCR was performed using EnGF/EnGR with a biotin-labeled primer, only Enterobacter aerogenes obtained a PCR amplification product of approximately 491 bp in size.

In addition, when using genomic DNA of Staphylococcus aureus, Enterobacter sakazakii, Escherichia coli O157:H7, Vibrio cholerae, Salmonella typhimurium or Pseudomonas fluorescens from milk samples and chicken samples, respectively When using a template and biotin-labeled primer pair for SAU3/SAU4 for PCR, only S. aureus can obtain a PCR amplification product of approximately 406 bp in size; when using biotin-labeled primer pair Esa1/Esa2 When performing PCR, only A. sakazakii obtained a PCR amplification product of about 491 bp in size; when using biotin-labeled primers for HAF1/HAR1 for PCR, only E. coli O157:H7 got a size. A PCR amplification product of approximately 316 bp; when PCR was performed using biotin-labeled primer pair VP33/VP32, only Vibrio cholerae obtained a PCR amplification product of approximately 387 bp; when biotin was used When the calibrated primer is used for PCR on Sspp1S/Sspp1A, only Salmonella typhimurium has a PCR amplification product of approximately 237 bp in size; and when PCR is performed using biotin-labeled primers for PFF/PFR, only firefly Pseudomonas phoeroides has a PCR amplification product of approximately 779 bp in size.

The results of this experiment show that biotin-labeled primer pair McF/McR, PmF/PmR, AeGF/AeGR, EnGF/EnGR, SAU3/SAU4, Esa1/Esa2, HAF1/HAR1, VP33/VP32, Sspp1S/Sspp1A, and PFF/ PFR can specifically detect M. catarrhalis, S. septicum, Aeromonas hydrophila, Enterobacter aerogenes, Staphylococcus aureus, Enterobacter sakazakii, large intestine present in food. Bacillus O157:H7, Vibrio cholerae, Salmonella typhimurium, and Pseudomonas fluorescens.

B. Sensitivity test of the pair of primers:

Table 5 below shows the detection sensitivity of six sets of biotin-labeled primer pairs according to the present invention and the conventionally disclosed four sets of biotin-labeled primer pairs for pathogens present in milk samples and chicken samples. As can be seen from Table 5, the sensitivity of biotin-labeled primers to McF/McR, PmF/PmR, AeGF/AeGR, and EnGF/EnGR for pathogens in non-proliferated milk samples can reach 10 ³ ~ 10 ⁴ CFU/ mL, and the detection sensitivity of pathogenic bacteria in the cultured milk samples can reach 10 ⁰ CFU/mL. In addition, the detection sensitivity of biotin-labeled primer pairs SAU3/SAU4, Esa1/Esa2, HAF1/HAR1, VP33/VP32, Sspp1S/Sspp1A, and PFF/PFR for pathogens in unproliferated milk samples or chicken samples It can reach 10 ² ~ 10 ⁴ CFU/mL, and the detection sensitivity of pathogenic bacteria in the cultured milk sample or chicken sample can reach 10 ⁰ CFU/mL.

The results of this experiment show that the six groups of biotin-labeled primers according to the present invention have McF/McR, PmF/PmR, AeGF/AeGR, EnGF/EnGR, Esa1/Esa2 and PFF/PFR for the target species to be detected. It has high sensitivity and can therefore be used to detect pathogenic bacteria in food.

Example 5. Detection of pathogenic bacteria in food by MU1tiplex polymerase chain reaction (mu1tiplex PCR)

In order to evaluate a primer set consisting of two sets of biotin-labeled primers of the present invention, Esa1/Esa2 and PFF/PFR, and four sets of biotin-labeled primer pairs (as shown in Table 4) disclosed by the prior art. In combination with the detection effect on the multiplex polymerase chain reaction, the unproliferated milk samples containing different pathogens prepared in the above-mentioned Example 4, point (2), and the chicken samples were taken for the following experiment.

experimental method: A, specificity test of multiplex polymerase chain reaction:

First, milk samples containing Staphylococcus aureus, Enterobacter sakazakii or Salmonella typhimurium prepared in the unproliferated culture prepared in point (2) of item A of Example 4 above, and chicken samples (each sample) One mL of each was used to contain a bacterial concentration of 10 ⁷ CFU/mL, and genomic DNA was extracted according to the method described in the first item "Extraction of genomic DNA" in "General Experimental Methods" above. Next, genomic DNA (concentration of 100 μg/μL) of each bacterial strain obtained from the milk sample was mixed according to the combination and the amount ratio shown in Table 6 below, thereby obtaining a genomic DNA mixture of the milk sample 1 ~7, and genomic DNA (concentration of 100 μg/μL) derived from each bacterial strain in the chicken sample was also mixed as described above, thereby obtaining a genomic DNA mixture 1 to 7 of the chicken sample. Then, using the obtained milk sample and the genomic DNA mixture 1-7 of the chicken sample as a template, and using a combination of primers composed of SAU3/SAU4, Esa1/Esa2, and Sspp1S/Sspp1A by biotin-labeled primers. The multiplex polymerase chain reaction was carried out, and the reaction conditions of the multiplex polymerase chain reaction were as shown in Table 7 below. After completion of multiplex PCR, a total of 14 PCR amplification products were obtained, of which 7 PCR amplification products derived from milk samples were collectively referred to as Group 19 PCR products, and 7 PCR amplifications derived from chicken samples. The products are collectively referred to as the 20th population PCR product.

In addition, a milk sample containing Vibrio cholerae, Escherichia coli O157:H7 or Pseudomonas fluorescens and a chicken sample prepared by the non-proliferation culture prepared in the item (2) of the above item A of Example 4 ( Each sample contained 1 mL of a bacterial concentration of 10 ⁷ CFU/mL, and the genomic DNA was extracted according to the method described in the first item "Extraction of genomic DNA" in "General Experimental Methods" above. Next, genomic DNA (concentration of 100 μg/μL) of each bacterial strain obtained from the milk sample was mixed according to the combination and the amount ratio shown in Table 8 below, thereby obtaining a genomic DNA mixture of the milk sample 8 ~14, and genomic DNA (concentration: 100 μg/μL) derived from each bacterial strain in the chicken sample was also mixed as described above, thereby obtaining a genomic DNA mixture 8-14 of the chicken sample. Then, using the obtained milk sample and the genomic DNA mixture 8-14 of the chicken sample as a template, and using a combination of primers composed of biotin-labeled primers for HAF1/HAR1, VP33/VP32, and PFF/PFR. The multiplex polymerase chain reaction was carried out, and the reaction conditions of the multiplex polymerase chain reaction were as shown in Table 9 below. After completion of multiplex PCR, a total of 14 PCR amplification products were obtained, of which 7 PCR amplification products derived from milk samples were collectively referred to as Group 21 PCR products, and 7 PCR amplifications derived from chicken samples. The products are collectively referred to as the 22nd group PCR product.

Finally, the obtained 19th to 22th PCR products were separately subjected to 2% agarose gel electrophoresis analysis to confirm whether a PCR product of a predetermined size was obtained.

B. Sensitivity test of multiplex polymerase chain reaction:

First, milk samples containing Staphylococcus aureus, Enterobacter sakazakii or Salmonella typhimurium prepared in the unproliferated culture prepared in point (2) of item A of Example 4 above, and chicken samples (each sample) One mL of each was used to contain a bacterial concentration of 10 ⁴ CFU/mL, and genomic DNA was extracted according to the method described in the first item "Extraction of genomic DNA" in "General Experimental Methods" above. Then, multiplex PCR was performed using the genomic DNA of each of the obtained bacterial strains as a template, and a combination of primers consisting of SAU3/SAU4, Esa1/Esa2, and Sspp1S/Sspp1A by biotin-labeled primers was used for multiplex PCR. The reaction conditions of the PCR were as shown in Table 7 above.

In addition, a milk sample containing Vibrio cholerae, Escherichia coli O157:H7 or Pseudomonas fluorescens and a chicken sample prepared by the non-proliferation culture prepared in the item (2) of the above item A of Example 4 ( Each sample contained 1 mL of a bacterial concentration of 10 ⁴ CFU/mL, and the genomic DNA was extracted according to the method described in the first item "Extraction of genomic DNA" in "General Experimental Methods" above. Then, multiplex PCR was performed using the genomic DNA of each of the obtained bacterial strains as a template, and a combination of primers composed of HAF1/HAR1, VP33/VP32, and PFF/PFR, which were labeled with biotin, was used to perform multiplex PCR. The reaction conditions of the PCR were as shown in Table 9 above.

After completion of multiplex PCR, the obtained PCR products were separately subjected to 2% agarose gel electrophoresis analysis to confirm whether a PCR product of a predetermined size was available.

result: A, specificity test of multiplex polymerase chain reaction:

Figure 1 shows the multiplex PCR when a genomic DNA mixture 1-7 derived from a milk sample is used as a template and a combination of primers consisting of SAU3/SAU4, Esa1/Esa2 and Sspp1S/Sspp1A is performed using a biotin-labeled primer. The result of agarose gel electrophoresis analysis of the obtained PCR product, wherein the diameter M represents a DNA ladder marker, and the diameters 1 to 7 represent genomic DNA mixtures 1 to 7, respectively. As can be seen from Fig. 1, a combination of primers composed of SAU3/SAU4, Esa1/Esa2 and Sspp1S/Sspp1A, which are biotin-labeled primers, can simultaneously detect the Staphylococcus aureus present in the milk sample. Enterobacter sakazakii and Salmonella typhimurium. In addition, the same experimental results were obtained when multiplex PCR was carried out using this primer combination and using genomic DNA mixtures 1 to 7 derived from chicken samples as templates (data not shown).

Figure 2 shows the multiplex PCR when a genomic DNA mixture 8-14 derived from a milk sample is used as a template and a combination of primers consisting of HAF1/HAR1, VP33/VP32 and PFF/PFR by biotin-labeled primers is used. The result of agarose gel electrophoresis analysis of the obtained PCR product, wherein the diameter M represents a DNA ladder marker, and the diameters 1 to 7 represent genomic DNA mixtures 8 to 14, respectively. As can be seen from Fig. 2, a combination of primers composed of HAF1/HAR1, VP33/VP32 and PFF/PFR, which are labeled with biotin, can simultaneously detect the Vibrio cholerae and the large intestine present in the milk sample. Bacillus O157:H7 and Pseudomonas fluorescens. In addition, the same experimental results were obtained when multiplex PCR was carried out using this primer combination and using genomic DNA mixtures 8 to 14 derived from chicken samples as templates (data not shown).

The above experimental results confirmed that: a pair of biotin-labeled primer pairs (i.e., Esa1/Esa2 and PFF/PFR) of the present invention and four sets of biotin-labeled primer pairs disclosed by the prior art (i.e., SAU3/) A primer combination consisting of SAU4, Sspp1S/Sspp1A, HAF1/HAR1, and VP33/VP32) can be used for multiplex PCR, thereby simultaneously detecting the E. sakazakii, fluorescent pseudomons present in food. Bacteria, Staphylococcus aureus, Salmonella typhimurium, Escherichia coli O157:H7, and Vibrio cholerae.

B. Sensitivity test of multiplex polymerase chain reaction:

Analysis by agarose gel electrophoresis revealed that a combination of primers consisting of SAU3/SAU4, Esa1/Esa2, and Sspp1S/Sspp1A was performed using biotin-labeled primers, and was obtained from milk samples or chicken samples. When genomic DNA of S. aureus is used as a template for multiplex PCR, a PCR amplification product of about 406 bp in size can be obtained; when genomic DNA of Salmonella typhimurium obtained from milk samples or chicken samples is used as a template When performing multiplex PCR, a PCR amplification product having a size of about 237 bp can be obtained; and when multiplex PCR is performed using genomic DNA of Enterobacter sakazakii obtained from a milk sample or a chicken sample as a template, A PCR amplification product of approximately 491 bp in size.

In addition, when a combination of primers consisting of HAF1/HAR1, VP33/VP32, and PFF/PFR, which are biotin-labeled primers, is used, and genomic DNA of Vibrio cholerae obtained from milk samples or chicken samples is used as a template. When performing multiplex PCR, a PCR amplification product of about 387 bp in size can be obtained; when multiplex PCR is performed using genomic DNA of E. coli O157:H7 obtained from a milk sample or a chicken sample as a template, a PCR amplification product of approximately 316 bp in size; and when multiplex PCR is performed using genomic DNA of Pseudomonas fluorescens obtained from a milk sample or a chicken sample as a template, a size of approximately 779 bp is obtained PCR amplification products.

The above experimental results confirm that when multiplex PCR is performed using a combination of a biotin-labeled primer pair of the present invention and a conventional biotin-labeled primer pair to perform multiplex PCR, the food for non-proliferation culture is used. The detection sensitivity of pathogens can reach 10 ⁴ CFU/mL.

Example 6. Evaluation of the ability of a probe according to the present invention to detect pathogenic bacteria in food

In order to evaluate the detection specificity and sensitivity of the 10 probes (as shown in Table 4) designed in the above Example 3 for the pathogens present in the food, the results obtained in the above Examples 4 and 5 The first to 22th PCR products were taken for the following experiments.

A. Preparation of biochips:

First, in order to allow the 10 probes according to the present invention to have better immobilization when spotted on the surface of the biochip, the 5' or 3' end of the 10 probes The oligo (dT) [oligo (dT)] was modified, respectively, and the 40-mer probes thus obtained were shown in Table 10 below.

Thereafter, the synthesized 40-member probes 1 to 10 were each dissolved in sterile water to obtain a probe stock solution 1 to 10 having a concentration of 10 to 20 μM. Next, 25 μL of each of the probe stock solutions 1 to 10 was taken and mixed with 25 μL of a 2X probe solution (Dr. Chip Biotech., Taiwan) to form wafer probe solutions 1 to 10. The formed wafer probe solutions 1 to 10 were added to a 96-well culture dish, and the 40-member probe was then placed by an Automatic Arrayer (Ezspot SR-A300, Shuai Ran Precision, Taiwan). 1~10 is placed on plastic membrane (DR. Chip, Dr. Chip Biotech., Taiwan) in different combinations, and then UV cross-linker C1508 (UVItec, Cambridge, England) (254 nm/0.6-1.2 joules) These 40-member probes were immobitized on a plastic membrane to form 12 biochips with different probe combinations (see Table 11 below) . In addition, the biotin-labeled random sequence fragment and the 2X probe solution were used as a positive control and a negative control, respectively.

B. Specificity test of biochip:

In this experiment, the biochips 11 and 12 are respectively subjected to a specificity test, and the DNA probe spot position of the biochip 11 is shown in FIG. 3, wherein B2 represents the probe 1; B4 represents probe 3; D2 represents probe 2; D4 represents probe 4; A1, A5, C3, E1 and E5 all represent a positive control group; and D3 represents a negative control group. The position of the DNA probe spot on the biochip 12 is shown in Fig. 4, wherein A2 and A4 represent probes 7 and 5, respectively; B1 and B4 represent the positive control group; B2 and B3 both represent the negative control group; To C3 denotes probes 8 to 10, respectively; and C4 denotes probe 6.

First, 25 μL of the first group PCR product (total of 16 PCR amplification products) obtained in the "B, primer pair specificity test" of Example 4 above was placed in a 1.5 mL microcentrifuge tube. (microtube) and added 200 μL DR. Hyb ^TM buffer ^{(DR. Hyb TM buffer) (} Dr. Chip Biotech., Taiwan). Subsequently, denaturation was carried out at 99 ° C for 10 minutes, followed by standing on ice for 5 minutes, thereby obtaining 16 mixed solutions containing single-stranded DNA. Thereafter, the biochip 11 was placed in a hybridization box (DR. Chip, Dr. Chip Biotech., Taiwan) and the 16 mixed solutions obtained above were respectively added, and then the lid was capped and placed at a temperature. an oven set at 50 deg.] C ^{(DR. Mini TM oven, Dr} . Chip Biotech., Taiwan) for shaking the reaction for 1 hour. Thereafter, the solution in each hybridization cassette was removed and the biochips were washed once with 250 μL of Wash Buffer (Dr. Chip Biotech., Taiwan) at room temperature, followed by 250 μL. The wash buffer was washed once to remove non-specifically bound DNA-DNA hybrids (hybrids). Next, 200 μL of Blocking Reagent (Dr. Chip Biotech., Taiwan) was mixed with 0.2 μL of Streptomycin-AP (Nel751, Perkin Elmer Co., MA, USA). They were separately added to each hybridization box, and then placed in an oven set at 30 ° C for an oscillating reaction for 30 minutes. After removing the liquid from the hybridization cassettes, 500 μL of wash buffer was added and allowed to stand for 1 minute, followed by removal of the wash buffer. This washing-resting step was repeated 4 times. Thereafter, 196 μL of Detection Buffer (Dr. Chip Biotech., Taiwan) and 4 μL of BCIP/NBT substrate solution (NEL937, Perkin Elmer Co., MA, USA) Mix well and add to each hybridization box separately, then let stand for 15-30 minutes for chromogenic reaction. Thereafter, the cassette hybridisation oven ^{(DR. Mini TM Oven, Dr} . Chip Biotech., Taiwan) over 10-15 minutes of drying, and then to remove these biochips image analysis system (DR. AiM ^TM Reader, Dr. Chip Biotech., Taiwan) to scan.

Further, the second group PCR products (total of 72 PCR amplification products) obtained in the "B, primer pair specificity test" of Example 4 above were referred to the above-described manner for the first group PCR product. The denaturation reaction is carried out, whereby the obtained 72 kinds of mixed solutions containing single-stranded DNA are respectively subjected to hybridization reaction with the biochip 12, and the reaction conditions and operation steps relating to the hybridization reaction are as described above.

In addition, in order to further evaluate whether the biochip 12 can detect a plurality of pathogenic bacteria simultaneously present in a food, the applicant selects the "A, multiplex polymerase chain reaction specificity test" obtained in the above Example 5 19~22 group PCR products (7 PCR amplification products in each group, totaling 28 PCR amplification products) and denaturation reaction according to the method described above, thereby obtaining 28 kinds of single-stranded DNA The mixed solution is separately subjected to a hybridization reaction with the biochip 12, and the reaction conditions and operation steps relating to the hybridization reaction are as described above.

C. Sensitivity test of biochip:

The 3rd to 18th group PCR products obtained in the "C, primer pair sensitivity test" of the above Example 4 (12 PCR amplification products in each group, totaling 192 PCR amplification products) refer to the above B. The denaturation reaction was carried out in the manner described in the above section, whereby 192 kinds of mixed solutions containing single-stranded DNA were obtained. Thereafter, 12 kinds of mixed solutions containing single-stranded DNA obtained from the third group PCR product were respectively subjected to hybridization reaction with the biochip 1; 12 kinds of mixed solutions containing single-stranded DNA obtained from the fourth group PCR product Each of them is used for hybridization reaction with the biochip 2; 12 mixed solutions containing single-stranded DNA obtained from the 5th PCR product are respectively subjected to hybridization reaction with the biochip 3; and the 6th PCR product is obtained. 12 kinds of mixed solutions containing single-stranded DNA were respectively subjected to hybridization reaction with bio-disc 4; 24 kinds of mixed solutions containing single-stranded DNA obtained from the 8th and 14th-group PCR products were respectively taken with biochip 5 The hybridization reaction was carried out; 24 mixed solutions containing single-stranded DNA obtained from the 12th and 18th-group PCR products were respectively subjected to hybridization reaction with the biochip 6; 24 kinds of PCR products derived from the 7th and 13th group PCR products were contained. A mixed solution of single-stranded DNA is separately subjected to hybridization reaction with the bio-wafer 7; and 24 mixed solutions containing single-stranded DNA obtained from the 9th and 15th-group PCR products are respectively subjected to hybridization reaction with the bio-disc 8; 24 singles from the 10th and 16th cluster PCR products The mixed solution of DNA is separately subjected to hybridization reaction with the biochip 9; and 24 mixed solutions containing single strands of DNA derived from the 11th and 17th group PCR products are respectively subjected to hybridization reaction with the biochip 10, and The reaction conditions and procedures for the hybridization reaction are as described in item B above.

result: A, the specificity test of biochip:

In order to understand the specificity of detection of food pathogens when the DNA probe of the present invention is applied to a biochip, Moraxella catarrhalis, Pasteurella septicum, Aeromonas hydrophila obtained from milk samples Or the genomic DNA of Enterobacter aerogenes was used as a template and 16 PCR products obtained by PCR using biotin-labeled primers for McF/McR, PmF/PmR, AeGF/AeGR, and EnGF/EnGR, respectively, were used. A hybridization reaction is performed with the biochip 11. The map after hybridization shows that the PCR product obtained by performing PCR using McD./McR using the genomic DNA of M. catarrhalis as a template and biotin-labeled primer is taken with the biochip 11 In the hybridization reaction, a hybridization signal was observed at B2 (see Fig. 5A); when the PCR was performed using the genomic DNA of Pasteurella septicum as a template and using biotin-labeled primers for PmF/PmR. When the PCR product is brought into hybridization reaction with the biochip 11, a hybridization signal is observed at D2 (see Fig. 5B); when the genomic DNA of Aeromonas hydrophila is used as a template and biotin-labeled When the PCR product obtained by PCR after AeGF/AeGR is taken to hybridize with the biochip 11, a hybridization signal is observed at B4 (see FIG. 5C); and when Enterobacter aerogenes is produced When a genomic DNA was used as a template and a PCR product obtained by performing PCR on EnGF/EnGR using a biotin-labeled primer was subjected to hybridization reaction with the biochip 11, a hybridization signal was observed at D4 (see the figure). 5D).

Using genomic DNA from Staphylococcus aureus, Enterobacter sakazakii, Escherichia coli O157:H7, Vibrio cholerae, Salmonella typhimurium or Pseudomonas fluorescens from milk samples as template and using biotin separately The 36 primers obtained by performing PCR on SAU3/SAU4, Esa1/Esa2, HAF1/HAR1, VP33/VP32, Sspp1S/Sspp1A, and PFF/PFR were subjected to hybridization reaction with the biochip 12, respectively. The map after hybridization showed that the PCR product obtained after PCR using S. aureus genomic DNA as a template and using biotin-labeled primer pair SAU3/SAU4 was subjected to hybridization reaction with the biochip 12. At the time of A2, a hybridization signal was observed (see Fig. 6A); the PCR product obtained after PCR using the genomic DNA of Enterobacter sakazaki as a template and using biotin-labeled primer pair Esa1/Esa2 was When a hybridization reaction with the biochip 12 is carried out, a hybridization signal is observed at A4 (see Fig. 6B); when the genomic DNA of Escherichia coli O157:H7 is used as a template and the biotin-labeled primer is used for HAF1/ When the PCR product obtained by PCR after HAR1 is hybridized with the biochip 12, a hybridization signal is observed at C1 (see Fig. 6C); when the genomic DNA of Vibrio cholerae is used as a template and used When the PCR product obtained by performing PCR on VP33/VP32 by biotin-labeled primer is subjected to hybridization reaction with the biochip 12, a hybridization signal is observed at C2 (see Fig. 6D); Typhoid fever When the genomic DNA of the bacillus is used as a template and the PCR product obtained by PCR using the biotin-labeled primer pair Sspp1S/Sspp1A is subjected to hybridization reaction with the biochip 12, a hybridization signal is observed at C3. (See Fig. 6E); and the PCR product obtained after PCR using Pseudomonas fluorescens genomic DNA as a template and biotin-labeled primer pair PFF/PFR is taken with the biochip 12 At the time of the hybridization reaction, a hybridization signal was observed at C4 (see Fig. 6F).

In addition, when the genomic DNA of the pathogens obtained from the chicken samples was used as a template and the biotin-labeled primer pair SAU3/SAU4, Esa1/Esa2, HAF1/HAR1, VP33/VP32, Sspp1S/Sspp1A, and PFF were respectively used. The same experimental results (data not shown) were obtained when 36 kinds of PCR products obtained by PCR/PFR were hybridized with the biochip 12, respectively.

A mixture of 7 bacterial genomic DNAs containing different samples derived from milk (i.e., genomic DNA mixtures 1 to 7 listed in Table 6 above) was used as a template and a biotin-labeled primer pair SAU3 was used, respectively. The seven PCR products (i.e., the 19th group PCR products) obtained by performing multiplex PCR using the combination of primers composed of /SAU4, Esa1/Esa2, and Sspp1S/Sspp1A were respectively subjected to hybridization reaction with the biochip 12. The map after hybridization shows that when a multiplex PCR is performed using a mixture of genomic DNA of Staphylococcus aureus, Salmonella typhimurium and Enterobacter sakazaki (ie, genomic DNA mixture 1) as a template When the PCR product was subjected to hybridization reaction with the biochip 12, a hybridization signal was observed at A2, C3, and A4, respectively (see Fig. 7A); when the genomic DNA of Salmonella typhimurium (i.e., genomic DNA mixture) was used. 2) When the PCR product obtained by performing multiplex PCR as a template is subjected to hybridization reaction with the biochip 12, a hybridization signal is observed at C3 (see Fig. 7B); when the genome of Staphylococcus aureus is used When the PCR product obtained by performing multiplex PCR using DNA (ie, genomic DNA mixture 3) as a template is subjected to hybridization reaction with the biochip 12, a hybridization signal is observed at A2 (see FIG. 7C); When the PCR product obtained by performing multiplex PCR using genomic DNA of Enterobacter sakazaki (ie, genomic DNA mixture 4) as a template is subjected to hybridization reaction with the biochip 12, there is a view at A4. To a hybridization signal (see Figure 7D); PCR product obtained after multiplex PCR using a mixture of genomic DNA of Staphylococcus aureus and Salmonella typhimurium (i.e., genomic DNA mixture 5) as a template When a hybridization reaction with the biochip 12 is carried out, a hybridization signal is observed at A2 and C3, respectively (see Fig. 7E); when composed of genomic DNA of Salmonella typhimurium and Enterobacter sakazakii When the PCR product obtained by performing multiplex PCR using the mixture (i.e., genomic DNA mixture 6) as a template was subjected to hybridization reaction with the biochip 12, a hybridization signal was observed at C3 and A4, respectively (see Fig. 7F). And a PCR product obtained by performing multiplex PCR using a mixture of genomic DNA of Staphylococcus aureus and Enterobacter sakazaki (ie, genomic DNA mixture 7) as a template is taken with the biochip When a hybridization reaction was carried out, a hybridization signal was observed at A2 and A4, respectively (see Fig. 7G).

In addition, seven PCR products (i.e., the 20th PCR product) obtained after multiplex PCR were carried out using a mixture of bacterial genomic DNA containing different samples derived from chicken meat as a template and using primer combinations as described above, respectively. The same experimental results were obtained when the hybridization reaction with the biochip 12 was carried out, respectively (data not shown).

A mixture of 7 bacterial genomic DNAs containing different samples derived from milk (i.e., genomic DNA mixtures 8 to 14 listed in Table 8 above) was used as a template and a biotin-labeled primer pair HAF1 was used, respectively. The seven PCR products (i.e., the 21st group PCR products) obtained after multiplex PCR were combined with the primers composed of /HAR1, VP33/VP32, and PFF/PFR, respectively, were subjected to hybridization reaction with the biochip 12. The map after hybridization showed that multiplex PCR was performed using a mixture of genomic DNA of Escherichia coli O157:H7, Vibrio cholerae and Pseudomonas fluorescens (ie, genomic DNA mixture 8) as a template. When the obtained PCR product is subjected to hybridization reaction with the biochip 12, a hybridization signal is observed at C1, C2, and C4, respectively (see FIG. 8A); when genomic DNA of E. coli O157:H7 is used (ie, Genomic DNA Mixture 9) When a PCR product obtained by performing multiplex PCR as a template is subjected to hybridization reaction with the biochip 12, a hybridization signal is observed at C1 (see FIG. 8B); When the genomic DNA (i.e., genomic DNA mixture 10) is used as a template to perform multiplex PCR, the PCR product obtained by the multiplex PCR is hybridized with the biochip 12, and a hybridization signal is observed at C2 (see Fig. 8C). When the PCR product obtained by performing multiplex PCR using genomic DNA of Pseudomonas fluorescens (ie, genomic DNA mixture 11) as a template is subjected to hybridization reaction with the biochip 12, it is observed at C4. To a hybrid No. (see Fig. 8D); the PCR product obtained after multiplex PCR using a mixture of Escherichia coli O157:H7 and genomic DNA of Vibrio cholerae (i.e., genomic DNA mixture 12) was used as a template. When hybridizing with the biochip 12, a hybridization signal is observed at C1 and C2, respectively (see Fig. 8E); when consisting of a genomic DNA of Escherichia coli O157:H7 and Pseudomonas fluorescens When the PCR product obtained by performing multiplex PCR using the mixture (i.e., genomic DNA mixture 13) as a template was subjected to hybridization reaction with the biochip 12, a hybridization signal was observed at C1 and C4, respectively (see Fig. 8F). And the PCR product obtained after performing multiplex PCR using a mixture of genomic DNA of Vibrio cholerae and Pseudomonas fluorescens (ie, genomic DNA mixture 14) as a template is taken with the organism When the wafer 12 was subjected to the hybridization reaction, a hybridization signal was observed at C2 and C4, respectively (see Fig. 8G).

In addition, seven PCR products (i.e., the 22nd PCR product) obtained after multiplex PCR were carried out using a mixture of bacterial genomic DNA containing different chicken samples as a template and using the primer combinations as described above, respectively. The same experimental results were obtained when the hybridization reaction with the biochip 12 was carried out, respectively (data not shown).

The above experimental results confirmed that the probes 1 to 10 according to the present invention can specifically detect the presence of M. catarrhalis, S. septicum, Aeromonas hydrophila, and gas production in foods. Enterobacter, Enterobacter sakazakii, Pseudomonas fluorescens, Staphylococcus aureus, Escherichia coli O157:H7, Vibrio cholerae and Salmonella typhimurium. Therefore, the probes 1 to 10 according to the present invention can be applied to a gene chip for rapid detection of food pathogens.

B. Sensitivity test of biochip:

Table 12 shows the results of sensitivity tests obtained using biochips 1 to 10 to detect pathogens present in milk samples and chicken samples. As can be seen from Table 12, the detection sensitivity of the biochips 1 to 4 for the pathogens in the unproliferated milk samples can reach 10 ² to 10 ³ CFU/mL, and the detection sensitivity of the pathogenic bacteria in the proliferated milk samples can be Up to 10 ⁰ CFU/mL. In addition, the sensitivity of the biochips 5 to 10 for the unproliferated milk samples or the pathogenic bacteria in the chicken samples can reach 10 ² to 10 ³ CFU/mL, while for the probiotic cultured milk samples or the pathogens in the chicken samples. The detection sensitivity can reach 10 ⁰ CFU/mL. The results of this experiment show that the probes 1 to 10 according to the present invention have high sensitivity to the target species to be detected, and thus can be applied to a gene wafer for rapidly detecting micro pathogens present in foods.

All of the patents and documents cited in this specification are hereby incorporated by reference in their entirety. In the event of a conflict, the detailed description of the case (including definitions) will prevail.

While the invention has been described with respect to the specific embodiments of the invention, it will be understood that many modifications and changes can be made without departing from the scope and spirit of the invention. It is therefore intended that the invention be limited only by the scope of the appended claims.

<110> Hongguang University of Science and Technology

<120> Nucleic acid molecules, test kits, biochips, and methods for detecting food pathogens

<130> CP-26026

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<170> PatentIn version 3.5

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<223> McF primer for detecting M. catarrhalis

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Figure 1 shows the multiplex PCR when a genomic DNA mixture 1-7 derived from a milk sample is used as a template and a combination of primers consisting of SAU3/SAU4, Esa1/Esa2 and Sspp1S/Sspp1A is performed using a biotin-labeled primer. The result of agarose gel electrophoresis analysis of the obtained PCR product, wherein the diameter M represents a DNA ladder marker, and the diameter 1 to the diameter 7 represent a genomic DNA mixture 1 to 7, respectively;

Figure 2 shows the multiplex PCR obtained by using genomic DNA mixture 8-14 from milk samples as a template and using a combination of primers consisting of HAF1/HAR1, VP33/VP32 and PFF1PFR by biotin-labeled primers. Agarose gel electrophoresis analysis of the PCR product, wherein the diameter M represents a DNA ladder marker, and the diameter 1 to the diameter 7 represent a genomic DNA mixture 8 to 14, respectively;

3 shows the position of the DNA probe spot of the biochip 11, wherein B2 represents the probe 1; B4 represents the probe 3; D2 represents the probe 2; D4 represents the probe 4; and A1, A5, C3, E1 and E5 represent Positive control group; and D3 means negative control group;

4 shows the position of the DNA probe spot of the biochip 12, wherein A2 and A4 represent probes 7 and 5, respectively; B1 and B4 both represent the positive control group; B2 and B3 both represent the negative control group; and C1 to C3 respectively represent the probe. Needle 8 to 10; and C4 represents probe 6;

5A to 5D respectively show genomic DNA of M. catarrhalis, P. septicum, Aeromonas hydrophila, and Enterobacter aerogenes obtained from milk samples as templates, and respectively using biotin The hybridization profile observed when the PCR product obtained by performing PCR on McF/McR, PmF/PmR, AeGF/AeGR, and EnGF/EnGR is subjected to hybridization reaction with biochip 11;

Figures 6A to 6F show genomic DNA of Staphylococcus aureus, Enterobacter sakazakii, Escherichia coli O157:H7, Vibrio cholerae, Salmonella typhimurium, and Pseudomonas fluorescens from milk samples as templates And PCR products obtained by performing PCR on SAU3/SAU4, Esa1/Esa2, HAF1/HAR1, VP33/VP32, Sspp1S/Sspp1A, and PFF/PFR using biotin-labeled primers, respectively, are taken with biochip 12 a hybridization profile observed when performing a hybridization reaction;

7A to 7G respectively show bacterial genomic DNA mixtures 1 to 7 obtained from milk samples as templates, and primers composed of biotin-labeled primer pairs SAU3/SAU4, Esa1/Esa2, and Sspp1S/Sspp1A, respectively. a hybridization map observed when the PCR product obtained by combining the multiplex PCR is subjected to a hybridization reaction with the biochip 12;

Figures 8A to 8G show the bacterial genomic DNA mixture 8-14 from the milk sample as a template, respectively, and the primers composed of biotin-labeled primers for HAF1/HAR1, VP33/VP32 and PFF/PFR, respectively. The hybridization pattern observed when the PCR product obtained by the multiplex PCR was combined to carry out a hybridization reaction with the biochip 12 was carried out.

Claims (17)

A nucleic acid molecule reagent for detecting a food pathogen, comprising a nucleic acid molecule for detecting Enterobacter sakazakii, the nucleic acid molecule having a sequence selected from: sequence identification number: 9, sequence identification number: 10, and sequence identification number : nucleotide sequence shown in 21. The nucleic acid molecule reagent of claim 1, further comprising at least one nucleic acid molecule: (a) a nucleic acid molecule for detecting M. catarrhalis having a sequence selected from, for example, a sequence identification number : 1 and a nucleotide sequence of sequence identification number: 2; (b) a nucleic acid molecule for detecting Pasteurella septicum having a sequence selected from, for example, sequence identification number: 3 and sequence identification number: 4 a nucleotide sequence as shown; (c) a nucleic acid molecule for detecting a species of Aeromonas, having a nucleotide selected from the group consisting of: Sequence ID: 5 and Sequence ID: a sequence; (d) a nucleic acid molecule for detecting an Enterobacter species having a nucleotide sequence selected from the group consisting of: Sequence Identification Number: 7 and Sequence Identification Number: 8; (e) one for detection a nucleic acid molecule of Pseudomonas fluorescens having a nucleotide sequence selected from the group consisting of: Sequence ID: 11 and Sequence ID: 12; (f) a nucleic acid molecule for detecting Staphylococcus aureus , having a selected from, for example, a sequence identification number: 13 Series Identification Number: 14, and Sequence identification number: nucleotide sequence shown in 22; (g) a nucleic acid molecule for detecting E. coli O157:H7 having a sequence selected from, for example, sequence identification number: 15, sequence identification number: 16 and sequence identification Nucleotide sequence indicated by 23; (h) a nucleic acid molecule for detecting Vibrio cholerae having a sequence selected from, for example, sequence identification number: 17, sequence identification number: 18, and sequence identification number: 24 a nucleotide sequence; and (i) a nucleic acid molecule for detecting a Salmonella species having a nucleic acid selected from the group consisting of: Sequence ID: 19, Sequence ID: 20, and Sequence ID: Nucleotide sequence. A method for detecting the presence or absence of a pathogen in a food sample, comprising: subjecting the food sample to a DNA amplification reaction using a nucleic acid molecule reagent, wherein the nucleic acid molecule reagent comprises a method for detecting Enterobacter sakazakii a pair of primers comprising a forward sequence of a nucleotide sequence as shown in sequence identification number: 9 and a reverse primer having a nucleotide sequence as shown in SEQ ID NO: 10; A DNA fragment amplified by using the primer pair is detected, wherein the presence of the DNA fragment indicates the presence of a pathogen corresponding to the primer pair. The method of claim 3, wherein the nucleic acid molecule reagent further comprises at least one primer pair: (a) a primer pair for detecting M. catarrhalis, comprising a sequence identification Number: 1 before the nucleotide sequence a primer, and a reverse primer having a nucleotide sequence as shown in sequence identification number: 2; (b) a primer pair for detecting Pasteurella septicum, comprising a sequence identification number The nucleotide sequence shown in 3 is preceded by a primer, and a reverse primer having a nucleotide sequence as shown in SEQ ID NO: 4; (c) one for detecting a species of Aeromonas a primer pair comprising a forward sequence of a nucleotide sequence as shown in sequence identification number: 5, and a reverse primer having a nucleotide sequence as shown in SEQ ID NO: 6; (d) A primer pair for detecting an Enterobacter species comprising a nucleotide sequence as shown in sequence identification number: 7 and a nucleotide having a sequence number: 8 a reverse primer of the sequence; (e) a primer pair for detecting Pseudomonas fluorescens, comprising a nucleotide sequence as shown in sequence identification number: 11 and a primer Sequence identification number: reverse nucleotide of the nucleotide sequence shown in (f) a primer pair for detecting S. aureus comprising a nucleotide sequence as shown in sequence identification number: 13, and a sequence having a sequence identification number: 14 a reverse primer of a nucleotide sequence; (g) a primer pair for detecting E. coli O157:H7, comprising a nucleotide sequence having a sequence number: 15 a forward primer, and a reverse primer having a nucleotide sequence as shown in sequence identification number: 16; (h) a primer pair for detecting Vibrio cholerae, comprising a sequence identification number: The nucleotide sequence shown in Figure 17 is preceded by a primer, and a reverse primer having a nucleotide sequence as shown in SEQ ID NO: 18; and (i) a primer pair for detecting a Salmonella species, It comprises a forward sequence of a nucleotide sequence as shown in sequence identification number: 19, and a reverse primer having a nucleotide sequence as shown in SEQ ID NO: 20. The method of claim 3, wherein the food sample is selected from the group consisting of: fluid dairy, beverage, meat, seafood, animal feed, water, dairy, vegetable, fruit And canned food. The method of claim 3, wherein the total genomic DNA is extracted from the food sample as a template before the DNA amplification reaction is carried out. The method of claim 3, wherein the DNA amplification reaction is carried out by using at least one of the following methods: polymerase chain reaction, reverse transcriptase polymerase chain reaction (RT-PCR) , Real time quantitative PCR, nested PCR, hot-start PCR, in-situ PCR, degenerate oligonucleotide primers PCR (degenerate oligonucleotide primer PCR, DOP PCR), micro PCR (micro PCR), multiplex polymerase chain reaction, restriction fragment length polymorphism nucleic acid sequence-based amplification (NASBA), transcription-regulation expansion Transcription-mediated amplification (TMA), loop-mediated isothermal amplification (LAMP), and rolling circle amplification (RCA). The method of claim 4, wherein the detecting of the DNA fragment is carried out by a hybridization reaction using a probe. The method of claim 8, wherein the probe is selected from the group consisting of: (a) a probe for detecting M. catarrhalis having a sequence selected from, for example, Identification number: 1 and nucleotide sequence shown in sequence identification number: 2; (b) a probe for detecting Pasteurella septicum having a sequence selected from, for example, sequence identification number: 3 and sequence identification number a nucleotide sequence of: 4; (c) a probe for detecting a species of Aeromonas, having a core selected from the group consisting of: sequence identification number: 5 and sequence identification number: (d) a probe for detecting an Enterobacter species having a nucleotide sequence selected from the group consisting of: Sequence ID: 7 and Sequence ID: 8. (e) a probe for detecting Enterobacter sakazakii having a nucleotide sequence selected from the group consisting of: sequence identification number: 9, sequence identification number: 10, and sequence identification number: 21; (f) A probe for detecting Pseudomonas fluorescens having a nucleotide sequence selected from the group consisting of: Sequence ID: 11 and Sequence ID: 12; (g) one for detecting golden grapes a probe for cocci having a nucleotide sequence selected from, for example, sequence identification number: 13, sequence identification number: 14 and sequence identification number: 22; (h) one for detecting E. coli O157:H7 a probe having a nucleotide sequence selected from the group consisting of: sequence identification number: 15, sequence identification number: 16 and sequence identification number: 23; (i) a probe for detecting Vibrio cholerae, Having a nucleotide sequence selected from the group consisting of: sequence identification number: 17, sequence identification number: 18, and sequence identification number: 24; and (j) a probe for detecting Salmonella species having one Selected from sequence identification number: 19, sequence identification number: 20, and sequence identification Identification number: nucleotide sequence shown in 25. The method of claim 9, wherein the probe is selected from the group consisting of: (a) a probe for detecting M. catarrhalis having a sequence identification number: a nucleotide sequence shown in 1; (b) a probe for detecting Pasteurella septicum having a nucleotide sequence as shown in SEQ ID NO: 4; (c) a probe for detecting a species of Aeromonas, having a nucleotide sequence as shown in SEQ ID NO: 6; (d) a probe for detecting Enterobacter species, It has a nucleotide sequence as shown in SEQ ID NO: 7; (e) a probe for detecting Enterobacter sakazakii having a nucleotide sequence as shown in SEQ ID NO: 21; f) a probe for detecting Pseudomonas fluorescens having a nucleotide sequence as shown in SEQ ID NO: 11; (g) a probe for detecting S. aureus having A nucleotide sequence as shown in SEQ ID NO: 22; (h) a probe for detecting E. coli O157:H7 having a nucleotide sequence as shown in SEQ ID NO: 23; a probe for detecting Vibrio cholerae having a nucleotide sequence as shown in SEQ ID NO: 24; and (j) a probe for detecting a species of Salmonella having a sequence Identification number: nucleotide sequence shown in 25. The method of claim 8, wherein the hybridization reaction is carried out on a biochip selected from the group consisting of: a gene wafer, a microfluidic wafer, and a wafer laboratory. A method for detecting the presence or absence of a pathogen in a food sample, comprising: subjecting the food sample to a hybridization reaction using a nucleic acid molecule reagent, wherein the nucleic acid molecule reagent comprises a probe for detecting Enterobacter sakazakii The probe has a sequence selected from, for example, a sequence identification number: 9, Column identification number: 10 and a nucleotide sequence of sequence identification number: 21; and detecting whether there is a hybrid formed by performing hybridization reaction using the probe, wherein the presence of the hybrid indicates that there is a corresponding The presence of the pathogen of the probe. The method of claim 12, wherein the nucleic acid molecule reagent further comprises at least one probe of: (a) a probe for detecting M. catarrhalis having a sequence selected from the group consisting of Identification number: 1 and nucleotide sequence shown in sequence identification number: 2; (b) a probe for detecting Pasteurella septicum having a sequence selected from, for example, sequence identification number: 3 and sequence identification number a nucleotide sequence of: 4; (c) a probe for detecting a species of Aeromonas, having a core selected from the group consisting of: sequence identification number: 5 and sequence identification number: (d) a probe for detecting an Enterobacter species having a nucleotide sequence selected from the group consisting of: Sequence ID: 7 and Sequence ID: 8; (e) a probe for detecting Pseudomonas fluorescens having a nucleotide sequence selected from, for example, sequence identification number: 11 and sequence identification number: 12; (f) one for detecting Staphylococcus aureus a probe having a selected from, for example, a sequence identification number: 13, a sequence identification number: 14 Sequence Column identification number: nucleotide sequence shown in 22; (g) a probe for detecting E. coli O157:H7 having a sequence selected from, for example, sequence identification number: 15, sequence identification number: 16 and sequence identification Nucleotide sequence indicated by 23; (h) a probe for detecting Vibrio cholerae having a sequence selected from, for example, sequence identification number: 17, sequence identification number: 18, and sequence identification number: 24 a nucleotide sequence; and (i) a probe for detecting a Salmonella species having a probe selected from the group consisting of: Sequence ID: 19, Sequence ID: 20, and Sequence ID: Nucleotide sequence. The method of claim 12, wherein the hybridization reaction is carried out on a biochip selected from the group consisting of: a gene wafer, a microfluidic wafer, and a wafer laboratory. The method of claim 13, wherein the at least one probe is selected from the group consisting of: (a) a probe for detecting M. catarrhalis having sequence recognition a nucleotide sequence represented by 1; (b) a probe for detecting Pasteurella septicum having a nucleotide sequence as shown in SEQ ID NO: 4; (c) one for A probe for detecting Aeromonas species having a nucleotide sequence as shown in SEQ ID NO: 6; (d) a probe for detecting Enterobacter species having sequence recognition Nucleotide sequence shown in number 7; (e) a probe for detecting Enterobacter sakazakii having a sequence Identification number: nucleotide sequence shown in 21; (f) a probe for detecting Pseudomonas fluorescens having a nucleotide sequence as shown in SEQ ID NO: 11; (g) a probe for detecting S. aureus having a nucleotide sequence as shown in SEQ ID NO: 22; (h) a probe for detecting E. coli O157:H7 having sequence recognition Nucleotide sequence indicated by 23; (i) a probe for detecting Vibrio cholerae having a nucleotide sequence as shown in sequence identification number: 24; and (j) one for detecting A probe for a Salmonella species having a nucleotide sequence as shown in SEQ ID NO: 25. A test kit for detecting a food pathogen comprising a nucleic acid molecule reagent as claimed in claim 1 or 2. A biochip for detecting a food pathogen comprising a nucleic acid molecule reagent as claimed in claim 1 or 2.