KR20070032094A - Ethiene Gene Oligonucleotide Primer for Detection of Salmonella Species and Detection Method Using the Same - Google Patents

Abstract

The present invention is useful for rapid and specific screening of Salmonella (Salmonella), Salmonella enterotoxin gene (stn) gene-specific sequence number to 1 to 21 oligonucleotide primers; And also a method for rapidly and specifically detecting the Salmonella enterotoxin gene (stn) gene for the presence of the parasite Salmonella in a patient using an oligonucleotide primer of SEQ ID NOs: 1 to 21 in a polymerase chain reaction (PCR). The method comprises the steps of preparing a DNA template of the gene, amplifying the template by PCR using the primers, running the PCR product on a gel, and detecting the parasite.

Salmonella, enterotoxin gene, stn

Description

STN GENE OLIGONUCLEOTIDE PRIMERS FOR DETECTING SALMONELLA SPECIES AND DETECTION PROCESS USING THE SAME}

1 shows a 1% agarose gel showing all four PCR products used for the detection of Salmonella. Lane 1: 1 kb DNA ladder (Fermentas); Lane 2: PCR amplification with primers QVR133 and QVR134 (200 bp); Lane 3: PCR amplification with primers QVR135 and QVR136 (207 bp); Lane 4: PCR amplification with primers QVR137 and QVR138 (1318 pb); Lane 5: PCR amplification with primers QVR139 and QVR140 (450 bp); Lane 6: negative control with primers QVR139 and QVR140.

2 shows a 1% agarose gel showing detection of different numbers of Salmonella cells from blood using primers QYR137 and QVR138. Lane 1: 1 kb DNA ladder (Fermentas); Lane 2: positive control; Lane 3: blood sample embedded with approximately 1 cell and pre-strengthened for 5 hours; Lane 4: negative control (unseeded blood sample); Lane 5: blood sample embedded with less than 10 cells and preliminary unenriched PCR run; Lane 6: blood sample embedded with 10 cells and preliminary unenriched PCR run; Lane 7: Blood sample embedded with 10 ³ cells and preliminary unenriched PCR run.

3 shows a 1% agarose gel showing the PCR amplification product of nested PCR for the detection of Salmonella. The first PCR reaction was carried out using primers QVR137 and QVR138 and the reaction in nested PCR was carried out using primers QVR139 and QVR140. Lane 1: 1 kb DNA ladder (Fermentas); Lanes 2-7: different serotypes of Salmonella and lane 8: negative controls.

Salmonella species are familial intracellular parasites that invade epithelial cells and spread to humans mainly through water, meat, egg and poultry products. Selmonella infection is the most common food-borne gastrointestinal disease that spreads to humans in animals. Typhoid is still an endemic disease in many developing countries, and non-typhoid salmonella is also a major foodborne disease worldwide and is estimated to cause more than 500 deaths annually, with US $ 1 trillion to $ 1.5 trillion annually alone. Cost (Threlfall 1996; Mead et al., 1999). The numbers in India are not well documented and are expected to be much higher. Good monitoring and screening programs are needed to prevent Salmonella infection. Detection of Salmonella by conventional bacteriological methods is time consuming and usually requires 5 days. Therefore, many researchers have made efforts to reduce the time required for the detection of Salmonella and to increase the sensitivity of the detection method (Notermans et al., 1997; Ferretti et al 2001; Carli et al. 2001).

As public awareness of the health-related and economic impacts of food-borne contamination and disease increases, more efforts have been made to develop more sensitive methods of detection and identification of pathogens. Advances in molecular biotechnology, particularly polymerase chain reaction (PCR), have allowed more reliable microbial identification and supervision. PCR is also an invaluable tool for investigating and identifying food-borne outbreaks of the pathogens that cause microbial epidemics. PCR techniques provided increased sensitivity, allowed for faster treatment times, and improved detection of bacterial pathogens. In addition to food analysis, PCR has also been successfully applied for the detection and identification of pathogenic microorganisms in clinical and environmental samples (Simon 1999; White, 1992).

Enterotoxin production is one of the obvious pathological characteristics of diarrhea-causing bacteria. Salmonella serotypes are known to be associated with their gastroenteritis and diarrhea in humans and animals and have also been shown to produce enterotoxins. The stn gene is Salmonella lyphimurium ) approximately 89 minutes on the chromosome and the presence of the complete stn gene significantly contributes to the overall virulence of Salmonella.

The invention relates to a method for rapidly detecting the useful, Salmonella enterotoxin gene (stn) gene and enterotoxin gene of Salmonella in specific screening of Salmonella (Salmonella).

The present invention provides oligonucleotide primers of SEQ ID NOS: 1 to 21 specific for the Salmonella enterotoxin gene (stn) gene, useful for rapid and specific selection of Salmonella; And also using the oligonucleotide primers of SEQ ID NOs: 1 to 21 for polymerase chain reaction (PCR) to quickly and specifically detect the Salmonella enterotoxin gene (stn) gene for the presence of the parasite Salmonella in a patient. The method comprises the steps of preparing a DNA template of the gene, amplifying the template by PCR using the primers, running the PCR product on a gel, and detecting the parasite.

Embodiments of the present invention are oligonucleotide primers of SEQ ID NOS: 1 to 21 specific for the Salmonella enterotoxin gene (stn) gene, useful for rapid and specific selection of Salmonella.

Another embodiment of the present invention uses oligonucleotide primers of SEQ ID NOs: 1 to 21 for polymerase chain reaction (PCR) to rapidly and specific the Salmonella enterotoxin gene (stn) gene for the presence of the parasite Salmonella in patients. As a method of detecting automatically, the method

Preparing a DNA template of the gene,

Amplifying the template by PCR using the primers,

Running the PCR product on a gel, and

Detecting the parasite

Include them.

In yet another embodiment of the invention, said gene is sequenced from the serotype of Salmonella.

In yet another embodiment of the present invention, the primers can detect up to about one cell per few ml with or without any enrichment.

In yet another embodiment of the invention, the primers can detect up to 10 cells per ml of blood with or without any enrichment.

In yet another embodiment of the invention, the primers can detect up to one cell per gram of food and clinical sample with or without any enrichment.

In yet another embodiment of the invention, DNA from blood and background microflora does not interfere with PCR analysis.

In yet another embodiment of the invention, the method can be used as a water quality control of water and the diagnosis of bacterial Salmonella.

In yet another embodiment of the invention, the patient is a human and an animal.

In yet another embodiment of the invention, the primer sets of SEQ ID NOs: 1 and 2 produce 200 bp of amplification products.

In yet another embodiment of the invention, the primer sets of SEQ ID NOs: 3 and 4 produce 207 bp amplification products.

In yet another embodiment of the invention, the primer sets of SEQ ID NOs: 5 and 6 produce 1318 bp of amplification products.

In yet another embodiment of the invention, the primer sets of SEQ ID NOs: 7 and 8 produce 450 bp of amplification products.

In yet another embodiment of the invention, the concentration of primer is in the range of 1 pM to 100 pM.

In yet another embodiment of the invention, PCR products are visualized on gels in concentrations ranging from 0.3% to 2.7%.

In yet another embodiment of the present invention, PCR is performed for 30 seconds to 7 minutes of initial denaturation at 93 to 97 ° C. and for 3 seconds to 2 minutes at 93 to 97 ° C., for 10 seconds to 2 minutes at 50 to 67 ° C., at 70 to 70 ° C. 23 to 50 cycles of 10 seconds to 2 minutes at 75 ° C. and final extension of 2 to 10 minutes at 70 to 75 ° C.

In yet another embodiment of the invention, the DNA is isolated from pure culture, water, food and clinical samples.

In yet another embodiment of the invention, the Stn gene has been sequenced from multiple serotypes of Salmonella and the sequence has been found to be conserved. A PCR based protocol for the detection of Salmonella was developed using specific primers based on the Salmonella enterotoxin gene (stn). These primers and PCR protocols were envisioned by the inventors and are being reported for the first time. The method is very specific and can detect up to one cell per ml of water and less than 10 cells per ml of blood without any preliminary enrichment. The primers do not show any tendency to amplify non-salmonella DNA. DNA from blood and background microbiota does not interfere with the PCR assay by generating any non-specific PCR amplification product or inhibiting the PCR assay. The method can be used as a water quality control and diagnosis of bacteremia by Salmonella in humans and animals.

In yet another embodiment of the present invention, oligonucleotide primers envisioned based on the stn gene (salmonella enterotoxin gene) and used in the polymerase chain reaction may use Ifg to 10ug of DNA and any thermostable DNA polymerase enzyme. DNA was isolated from pure culture, water, food, and clinical samples by any known method suitable for PCR, and then the PCR products were visualized in the range of 0.5% to 2.5% on agarose gel, followed by 93-97 ° C. Initial denaturation for 30 seconds to 7 minutes and 23 to 50 cycles and 70 to 75 for 3 seconds to 2 minutes at 93 to 97 ° C., 10 seconds to 2 minutes at 50 to 67 ° C., 10 seconds to 2 minutes at 70 to 75 ° C. Useful for rapid and specific detection of Salmonella by a PCR protocol consisting of a final extension of 2 minutes to 10 minutes at < RTI ID = 0.0 >

In yet another embodiment of the invention, the primers selected in the rapid detection method of Salmonella are as follows:

Below are the primer sets 1 of SEQ ID NOs: 1 and 2, respectively. Also referred to as QVR133 and QVR134.

5'GAAGCAGCGCCTGTAAAATC3 '/ 5'TGGCTGTGGTGCAAAATATC3';

Below are primer sets 2 of SEQ ID NOs: 3 and 4, respectively. Also referred to as QVR135 and QVR136.

5'GCCACCAGCTTTTCTTTACG3 '/ 5'ACGAACCAGCGAAACAAACT;

Below are primer sets 3 of SEQ ID NOs: 5 and 6, respectively. Also referred to as QVR137 and QVR138.

5'GGTCAAAATCCAGCGGTTTA3 '/ 5'TTGCTGCTAACGGCGAGA3';

Below are primer sets 4 of SEQ ID NOs: 7 and 8, respectively. Also referred to as QVR139 and QVR140.

In yet another embodiment of the present invention, the concentration of the primers as claimed in claims 1 and 2 and the primers used in the rapid method for the detection of Salmonella can range from 1 pM to 100 pM, set 1, set 2, Set 3 and Set 4 yield 200 abp, 207 pb, 1318 pb and 450 pb of specific amplification products, respectively (FIG. 1).

In yet another embodiment of the present invention, primers for the detection of Salmonella and rapid detection methods in various sources, such as food, clinical (FIG. 3), with or without enhancement of all types of serotypes of Salmonella (FIG. 3). And 2) and less than one cell per ml or g per few samples. Detection from multiple sources containing one cell per 25 ml or 25 g of sample without enhancing or enhancing all types of serotypes of Salmonella in the primers and claims for claims 4 to 4 for the detection of Salmonella can do.

In yet another embodiment of the present invention, chromosomal DNA isolation by any known method, including detection of pure cultures of Salmonella and preparation of heat shock lysates in primers, is followed by PCR followed by agarose in the range of 0.5% to 2.5%. It can be used to visualize PCR products on gels.

methodology

The protocol consists of three main steps:

Preparation of template DNA

Polymerase chain reaction

Visualization of the product on agarose gel

PCR for someone DNA  Isolation:

Liquid culture or agar medium Cell pellets from 1 ml of single colonies on the suspension were suspended in 100 μl of sterile Millipore water and incubated in a dry bath at 100 ° C. for 5 minutes. The mixture was immediately transferred to an ice bath and left for 3 minutes. The resulting cell culture was centrifuged at 7000 rpm for 3 minutes and 2 μl of supernatant was used directly for a 20 μl PCR reaction.

If necessary, the assay was performed using isolated chromosomal DNA. To isolate pure chromosomal DNA, cultures were grown for 6 hours in nutrient liquid medium and DNA was isolated by GES method (Pitcher et al., 1989). 10 ng of DNA was commonly used for 20 μl reaction analysis.

Preparation of molds from blood:

DNA isolation kit (M / S Bio Basic, Canada) was used to isolate DNA from blood (FIG. 2).

Mold production from water:

To isolate DNA from aqueous bacteria, 1 ml of sample was centrifuged for 3 min at 7000 rpm in a 5 ml Eppendorf tube, the supernatant was discarded and 10 μl of autoclaved Millipores was added to each tube. The resulting suspension was vortexed and incubated for 5 min in anhydrous bath at 100 ° C. and the total volume was used for 20 μl PCR reaction.

reinforce:

For blood, 250 μl of sample aliquots were inoculated into 5 ml liquid medium and for water, 1 ml aliquots were inoculated into 1 ml dual strength brain heart infusion liquid medium (HiMedia, India) and incubated at 37 ° C. for 5 hours. . Cell lysates were prepared as above.

We provide the use of the stn gene as a detection marker of Salmonella.

The primary object of the present invention is to develop primers for the detection of the parasite Salmonella.

Another object of the present invention is to develop a method for rapid and efficient detection of the parasite Salmonella.

Still another object of the present invention is to develop a method for detecting parasite Salmonella from food, biological samples and the like.

<110> Council of Scientific and Industrial Research <120> STN GENE OLIGONUCLEOTIDE PRIMERS FOR DETECTION SALMONELLA SPECIES          AND DETECTION PROCESS USING THE SAME <160> 21 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> QVR133 <400> 1 gaagcagcgc ctgtaaaatc 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> QVR 134 <400> 2 tggctgtggt gcaaaatatc 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> QVR135 <400> 3 gccaccagct tttctttacg 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> QVR 136 <400> 4 acgaaccagc gaaacaaact 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> QVR137 <400> 5 ggtcaaaatc cagcggttta 20 <210> 6 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> QVR 138 <400> 6 ttgctgctaa cggcgaga 18 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> QVR139 <400> 7 gccggctttc aacgcctcta c 21 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> QVR 140 <400> 8 gaccaaagct gacgggacag 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide primer <400> 9 acgcctctac cgccgtttcc 20 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide primer <400> 10 cgaccaaagc tgacgggaca g 21 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide primer <400> 11 cgtttccacg ctggaaaatg c 21 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide primer <400> 12 gccggctttc aacgcctcta c 21 <210> 13 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide primer <400> 13 catggcggcg cgattaagg 19 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide primer <400> 14 aatcggaatg gcgggattga g 21 <210> 15 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide primer <400> 15 tgccgttcat aatcaaaatc g 21 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide primer <400> 16 gattttacag gcgctgcttc 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide primer <400> 17 ggtcaaaatc cagcggttta 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide primer <400> 18 gctcaggtgc gtgagaaagt 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide primer <400> 19 gttcgagcaa ttcgcttacc 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide primer <400> 20 gcttgatgca atgaagcgta 20 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide primer <400> 21 ttcccgctat cggtaacagt 20  

Claims (11)

Primer sets of SEQ ID NOs: 7 and 8 specific for the enterotoxin gene of Salmonella . In the method of detecting the Salmonella enterotoxin gene for the presence of the parasite Salmonella in animals other than humans using the primer set of claim 1, (Iii) preparing a DNA template of the gene, (Ii) amplifying the template by PCR using the primers, (Iii) running the PCR product on a gel, and (Iii) detecting a 450 bp amplification product indicating the presence of Salmonella Salmonella enterotoxin gene detection method comprising a. 3. The method of claim 2, wherein the gene is sequenced from the serotype of Salmonella. 3. The method of claim 2, wherein the primer is capable of detecting up to one cell per ml of water. 3. The method of claim 2, wherein the primer can detect up to 10 cells per ml of blood. 3. The method of claim 2, wherein the primer is capable of detecting up to one cell per gram of food and clinical sample. The method for detecting a Salmonella enterotoxin gene according to claim 2, which can be used for water quality control and diagnosis of bacterial Salmonella. The method of claim 2, wherein the concentration of the primer is in the range of 1 pM to 100 pM. 3. The method of claim 2, wherein the PCR product is visualized at a concentration ranging from 0.3% to 2.7% on the gel. The method according to claim 2, wherein the PCR is subjected to initial denaturation at 93 to 97 ° C. for 30 seconds to 7 minutes, 3 seconds to 2 minutes at 93 to 97 ° C., 10 seconds to 2 minutes at 50 to 67 ° C., and 10 at 70 to 75 ° C. 23 to 50 cycles of seconds to 2 minutes and a final extension of 2 to 10 minutes at 70 to 75 ° C. for detecting a Salmonella enterotoxin gene. 3. The method of claim 2, wherein the DNA is isolated from pure culture, water, food, and clinical samples.

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