WO2018199279A1 - Method for determining life or death of microorganism using ribosomal rna precursor - Google Patents

Abstract

The present invention addresses the problem of providing a means whereby life or death of microbial species over a broad range can be quickly and exactly determined. Provided are: an oligonucleotide set for determining life or death of a microorganism, said set comprising oligonucleotides designed in two ribosomal RNA gene regions between which the spacer region of a ribosomal RNA precursor (pre-rRNA) is sandwiched; a kit for determining life or death of a microorganism, said kit comprising the oligonucleotide set; and a method for determining life or death of a microorganism using the oligonucleotide set.

Description

Method for determining the life and death of microorganisms using ribosomal RNA precursors

The present invention relates to an oligonucleotide set for determining the survival of microorganisms and a method for determining the survival of microorganisms.

When microorganisms are mixed in the manufacturing process of aseptic preparations, the manufactured product cannot be used, and the production line is contaminated. Therefore, microbial contamination inspection is indispensable for the manufacture of sterile preparations. However, the “sterility test method”, which is a general test method of the Japanese Pharmacopoeia, stipulates that the culture should be carried out for 14 days or more, and has a drawback that it takes a long time for the test. Therefore, the need for an inspection method that can quickly grasp the contamination status of microorganisms is very high. On the other hand, the method of testing microorganisms using gene amplification reaction (PCR) is rapid, but there are many cases where materials and reagents themselves are contaminated with dead microorganisms or DNA. It cannot be said that it can be done. Therefore, establishment of a test system that can distinguish between live bacteria and dead bacteria is required.

A ribosomal RNA precursor (pre-rRNA) is a product transcribed from a ribosomal RNA gene, and becomes a mature ribosomal RNA (rRNA) molecule after cleavage and chemical modification. Methods for detecting microorganisms that target the sequence of pre-rRNA, which is the primary transcript before maturation of rRNA, have been reported so far. For example, Patent Document 1 discloses a method for detecting mycobacteria using a probe that hybridizes with rDNA, precursor rRNA, or rRNA target sequence of mycobacteria. However, in this method, the detection target is only mycobacteria, and there is no specific description of the target sequence of the precursor rRNA. Patent Document 2 discloses a method for detecting a living microorganism in a sample based on a difference in expression level of a target rRNA precursor between a nutritionally stimulated aliquot and a non-stimulated control aliquot. Patent Document 3 discloses a method in which primers specific to the ribosome region and spacer region of a pre-rRNA of a microorganism are designed and the presence of viable bacteria in a sample is detected by reverse transcription and quantitative PCR. However, in these conventional methods, the types of bacteria that can be detected are limited, and a special treatment is required before measurement, so that the methods are not quick and accurate.

Special Table 2001-501825 Japanese Patent No.5768282 WO 2013/049437 Publication

Therefore, an object of the present invention is to provide means capable of quickly and accurately determining the viability of a wide range of microbial species.

As a result of intensive studies to solve the above problems, the present inventors have found that a ribosomal RNA precursor (pre-rRNA) transcribed from a ribosomal RNA gene of a microorganism such as a bacterium or a fungus is rapidly cleaved and matured. Focusing on the fact that pre-rRNA can be detected more rapidly than when targeting microbial DNA, because pre-rRNA is synthesized in large quantities, and is rapidly degraded when the microorganism dies. We thought that if we target -rRNA, we can establish a test system that can perform comprehensive detection of the presence or absence of microorganisms and the determination of life and death simultaneously and quickly. Therefore, as a result of aligning and examining the base sequences of pre-rRNA of dozens of representative bacteria including E. coli and dozens of representative fungi including Aspergillus genus, the spacer region was determined for bacteria. A region that has a base sequence common to bacteria in the 23S rRNA gene and 5S rRNA gene, and a base sequence common to fungi in the 18S rRNA gene and 5.8S rRNA gene with the spacer region in between Each region was found, and an oligonucleotide set composed of a primer pair and a probe was designed based on the base sequence in the region. As for bacteria, pre-rRNA in E. coli-containing samples sterilized using the designed oligonucleotide set was measured by real-time PCR, and it was confirmed that no pre-rRNA was detected after 3 days at the latest. We obtained the knowledge that the viability of bacteria can be determined quickly from the presence or absence of pre-rRNA detection. The present invention has been completed based on this finding.

That is, the present invention includes the following inventions.
(1) A set of oligonucleotides for determining the survival of microorganisms, comprising oligonucleotides designed in two ribosomal RNA gene regions sandwiching the spacer region of a ribosomal RNA precursor (pre-rRNA).
(2) The oligonucleotide set for determining the viability of the microorganism according to (1), wherein the microorganism is a bacterium or a fungus.
(3) The oligonucleotide set for determining the viability of a microorganism according to (1) or (2), wherein the oligonucleotide is used as a primer and a probe.
(4) The microorganism is a bacterium, and the oligonucleotide set includes a primer pair comprising the following oligonucleotide (a) and the oligonucleotide (b), and a probe comprising the following oligonucleotide (c): The oligonucleotide set for determining the viability of a microorganism according to (1), which is configured.
(A) an oligonucleotide consisting of a base sequence containing at least 15 consecutive bases in the base sequence shown in SEQ ID NO: 1 (b) an oligonucleotide consisting of a base sequence containing at least 15 contiguous bases in the base sequence shown in SEQ ID NO: 2 and a sequence (C) Oligo consisting of a base sequence containing at least 15 bases in the base sequence shown in SEQ ID NO: 4 or its complementary sequence Nucleotide (5) The microorganism is a fungus, and the oligonucleotide set comprises a primer pair comprising the following oligonucleotide (d) and the oligonucleotide (e), and a probe comprising the following oligonucleotide (f): The microorganism according to (1), comprising Life and death decision for oligonucleotide set.
(D) an oligonucleotide consisting of a base sequence containing at least 15 consecutive bases in the base sequence shown in SEQ ID NO: 5 (e) an oligonucleotide consisting of a base sequence containing at least 15 bases continuous in the base sequence shown in SEQ ID NO: 6; An oligonucleotide consisting of a base sequence containing at least 15 consecutive bases in the base sequence shown in No. 7, an oligonucleotide consisting of a base sequence containing at least 15 consecutive bases in the base sequence shown in SEQ ID No. 8, and a base shown in SEQ ID No. 9 A mixture of oligonucleotides comprising a base sequence comprising at least 15 consecutive bases in the sequence (f) an oligonucleotide comprising a base sequence comprising at least 15 consecutive bases in the base sequence shown in SEQ ID NO: 10 or its complementary sequence (6) (1 ) To (5) The viability determination oligonucleotide set of microorganisms crab according comprises at least one set, microbial viability determination kit.
(7) Using the RNA extracted from the specimen as a template, RT-PCR amplification is performed using the oligonucleotide set for determining the viability of microorganisms as described in any one of (1) to (5), and the resulting amplification product is detected. And a method for determining the viability of a microorganism, characterized by quantifying.
(8) The microorganism viability determination method according to (7), wherein the RT-PCR is real-time RT-PCR.
(9) The microorganism life / death determination method according to (7) or (8), wherein the specimen is a food or drink, a medicine, a medical material or a medical instrument, a biological sample, or an environmental sample.

This application claims the priority of Japanese Patent Application No. 2017-087847 filed on Apr. 27, 2017, and includes the contents described in the specification of the patent application.

According to the present invention, there are provided an oligonucleotide set that can quickly and accurately determine whether a microorganism in a specimen is viable or dead, and a method for determining a living microorganism using the oligonucleotide set. Therefore, the present invention is useful for microbial contamination inspection at food factories and medical sites.

FIG. 1A shows a design region of a bacterial life / death determination oligonucleotide (forward primer, probe) in the 23S rRNA gene of a bacterial ribosomal RNA precursor (pre-rRNA). FIG. 1B shows the design region of a bacterial life / death judgment oligonucleotide (reverse primer) in the 5S rRNA gene of a bacterial ribosomal RNA precursor (pre-rRNA). Fig. 2-1 shows that E. coli is sterilized by heating at 65 ° C for 30 minutes, then at 37 ° C for a certain time [1 hour (1 hr), 3 hours (3 hr), 6 hours (6 hr), 1 day (d1), 2 days (d2), 3 days (d3), 4 days (d4), 5 days (d5), 6 days (d6)] amplification obtained by oligonucleotide set 1 for bacteria (product of the present invention) A curve is shown (DW: negative control). Fig. 2-2 shows that Escherichia coli is sterilized by heating at 65 ° C for 30 minutes, and then at 37 ° C for a certain period of time [1 hour (1 hr), 3 hours (3 hr), 6 hours (6 hr), 1 day (d1), 2 days (d2), 3 days (d3), 4 days (d4)] shows amplification curves obtained by performing real-time PCR using oligonucleotide set 2 (comparative product) for the incubated samples (NC: negative control) ). Fig. 3A shows Kanamycin of Escherichia coli, high temperature heat sterilization (95 ° C for 10 minutes), low temperature heat sterilization (65 ° C for 30 minutes), and then at 37 ° C for a certain time [1 hour (1h), 3 hours (3h), 6 hours (6h), 1 day (day 1), 2 days (day 2), 3 days (day 3), 4 days (day 4)] copies of ribosomal RNA precursor (pre-rRNA) measured by incubation Indicates a number. Fig. 3B shows the results of sterilization treatment of Escherichia coli kanamycin, high temperature heat sterilization (95 ° C for 10 minutes), and low temperature heat sterilization (65 ° C for 30 minutes) at 37 ° C for a certain time [1 hour (1h), 3 hours (3h), 6 hours (6h), 1 day (day 1), 2 days (day 2), 3 days (day 3), 4 days (day 4)] shows the number of viable bacteria measured by incubation. Fig. 4-1 shows the design region of fungal ribosomal RNA precursor (pre-rRNA) 18S rRNA gene oligonucleotides (forward primer, probe) and fungal ribosomal RNA precursor (pre-rRNA) 3) shows the design region of the oligonucleotide (reverse primer) for fungal viability determination in the 5.8S rRNA gene. Fig. 4-2 shows the design region of fungal ribosomal RNA precursor (pre-rRNA) 18S rRNA gene, and the fungal ribosomal RNA precursor (pre-rRNA). 3) shows the design region of the oligonucleotide (reverse primer) for fungal viability determination in the 5.8S rRNA gene. Fig.4-3 shows the design region of fungal ribosomal RNA precursor (pre-rRNA) 18S rRNA gene for oligonucleotides (forward primer, probe) for fungal life and death, and fungal ribosomal RNA precursor (pre-rRNA) 3) shows the design region of the oligonucleotide (reverse primer) for fungal viability determination in the 5.8S rRNA gene.

1. Oligonucleotide set for determining the viability of microorganisms The oligonucleotide set for determining the viability of microorganisms of the present invention is composed of oligonucleotides designed in two ribosomal RNA gene regions sandwiching the spacer region of a ribosomal RNA precursor (pre-rRNA). The The base length of the oligonucleotide is not limited, but is usually 15 to 30 bases, preferably 18 to 25 bases in the case of a primer, and 10 to 30 bases in the case of a probe.

The oligonucleotide set for life / death determination of microorganisms of the present invention, when the life / death determination target is a bacterium, the 23S rRNA gene and the 5S rRNA gene base sequence across the spacer region of the bacterial ribosomal RNA precursor (pre-rRNA) It is composed of two or more kinds of oligonucleotides designed based on the found common base sequences.

In addition, the oligonucleotide set for determining the viability of the microorganism of the present invention, when the life / death determination target is a fungus, includes an 18S の rRNA gene and a 5.8S rRNA gene with a spacer region of a fungal ribosomal RNA precursor (pre-rRNA) sandwiched between them. It is composed of two or more kinds of oligonucleotides designed based on highly common base sequences found in the base sequences.

In order to design the oligonucleotide of the present invention, first, for typical bacteria, the base region information of the 23S rRNA gene and the 5S ん で rRNA gene with the spacer region in between, and the typical fungus with the spacer region in between. Obtain the base sequence information of 18S rRNA gene and 5.8S rRNA gene. Base sequence information may be obtained from a database (DDBJ, NCBI, etc.), or may be obtained by directly analyzing the base sequence. The base sequence having high commonality can be specified by aligning and comparing a plurality of obtained base sequences. For alignment of base sequences, alignment software (for example, CLUSTALW, URL: http://www.ddbj.nig.ac.jp/) published on the Internet can be used.

Next, a primer is designed based on the identified highly common base sequence. Primers are designed in consideration of oligonucleotide length, GC content, Tm value, complementarity between oligonucleotides, secondary structure in oligonucleotides, and the like. For the primer design, commercially available software for primer design, such as Oligo ™ [manufactured by National Biosciences Inc. (USA)], GENETYX [manufactured by Software Development Co., Ltd. (Japan)], etc. can also be used.

The specific design criteria for the primers adopted are as follows.
(a) The primer length is in the range of 15 to 30 bases in order to allow specific annealing with the template cDNA.
(b) Avoid complementary sequences between both primers so as not to form dimers or conformations.
(c) Avoid self-complementary sequences to prevent the formation of hairpin structures within the primer.
(d) To ensure stable binding to the template cDNA, the GC content should be about 50% so that GC-rich or AT-rich is not unevenly distributed in the primer.
(e) The homology between the nucleotide sequence at the 3 ′ end of the primer and the template cDNA sequence is high.
(f) Since the annealing temperature depends on the Tm (melting temperature), in order to obtain highly specific PCR products, primers that are close to each other at a Tm value of 50 to 70 ° C, preferably 55 to 65 ° C, should be selected.

The probe design is based on software that comes with commercial real-time PCR instruments such as ABI Prism 7900HT Real-time PCR System (Life Technologies Japan Co., Ltd.) and TaqMan Universal PCR Master Master (Life Technologies Japan Co., Ltd.). What is necessary is just to carry out based on the protocol attached to a commercially available real-time PCR reagent. In general, the probe design criteria are that the GC content should be within the range of 20-80%, avoid the continuation of G or C of 4 bases or more in the sequence, and the Tm value of the corresponding primer pair. Also, it may be set higher by about 8 to 10 ° C.

Based on the above criteria, the following was established as an oligonucleotide set for determining the viability of microorganisms of the present invention.
(Bacteria life / death oligonucleotide set)
Forward primer: 5'-acgygagytg ggttya-3 '(SEQ ID NO: 1) (y = t / c)
Reverse primer: 5'-gcttracttc yskgttcg-3 '(SEQ ID NO: 2) (r = g / a, y = t / c, s = g / c, k = g / t) and 5'-gtttcacttc tgagttcg-3' Mixed primer of (SEQ ID NO: 3) Probe: 5'-cgtcgygaga cagktyggtc yctat-3 '(SEQ ID NO: 4) (y = t / c, k = g / t)
(Oligonucleotide set for fungal viability)
Forward primer: 5'-gattacgtcc ctgccctttg ta-3 '(SEQ ID NO: 5)
Reverse primer: 5'-cgagagccra gagatccrtt-3 '(SEQ ID NO: 6) (r = g / a), 5'-cgagaaccaa gagatccgtt-3' (SEQ ID NO: 7), 5'-cgaaagccga gagatccatt-3 '(SEQ ID NO: 8) ) And 5′-ccggaaccaa gagatccrtt-3 ′ (SEQ ID NO: 9) (r = g / a) mixed primer Probe: 5′-acaccgcccg tcgctactac cg-3 ′ (SEQ ID NO: 10)

The oligonucleotides that serve as the primers or probes described above are prepared by methods known in the art as methods for synthesizing oligonucleotides, such as the phosphoramidite method, the H-phosphonate method, etc., using a commonly used automatic DNA synthesizer. It is possible to synthesize. *

The oligonucleotide constituting the oligonucleotide set of the present invention is an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 1, 2, or 3 above, an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 4 or a complementary sequence thereof, or In addition to the oligonucleotide consisting of the base sequence shown in SEQ ID NO: 5, 6, 7, 8, or 9 above, the oligonucleotide consisting of the base sequence shown in SEQ ID NO: 10 or a complementary sequence thereof, as well as microorganisms based on pre-rRNA detection Those homologous oligonucleotides may be used as long as they can function as a life or death judgment primer or probe. Therefore, the oligonucleotide constituting the oligonucleotide set (for bacteria) of the present invention is an oligonucleotide consisting of a base sequence comprising at least 15 consecutive bases in the base sequence shown in SEQ ID NO: 1, and a continuous in the base sequence shown in SEQ ID NO: 2. An oligonucleotide consisting of a base sequence containing at least 15 bases, an oligonucleotide consisting of a base sequence containing at least 15 consecutive bases in the base sequence shown in SEQ ID NO: 3, a base sequence shown in SEQ ID NO: 4 or at least a contiguous sequence thereof It is an oligonucleotide consisting of a base sequence containing 15 bases. Further, the oligonucleotide constituting the oligonucleotide set (for fungus) of the present invention is an oligonucleotide comprising a base sequence comprising at least 15 consecutive bases in the base sequence shown in SEQ ID NO: 5, and a continuous in the base sequence shown in SEQ ID NO: 6. An oligonucleotide consisting of a base sequence containing at least 15 bases, an oligonucleotide consisting of a base sequence containing at least 15 consecutive bases in the base sequence shown in SEQ ID NO: 7, and at least 15 consecutive bases in the base sequence shown in SEQ ID NO: 8 An oligonucleotide consisting of a base sequence, an oligonucleotide consisting of a base sequence containing at least 15 bases in the base sequence shown in SEQ ID NO: 9, a base containing at least 15 bases in a base sequence shown in SEQ ID NO: 10 or a complementary sequence thereof Is an oligonucleotide that consists of a sequence. The base sequence of the homologous oligonucleotide may include at least 15 consecutive bases in the base sequences shown in SEQ ID NOs: 1 to 10 or its complementary sequence, but the total length is preferably 30 bases or less. There are no particular limitations on the site (3 'terminal side, 5' terminal side) in the oligonucleotide having at least 15 consecutive nucleotides.

Further, the probe of the present invention may be labeled with a labeling substance for detecting and quantifying a fluorescent signal derived from a target amplification product of bacteria to be detected. Examples of the labeled probe include a TaqMan probe, a molecular beacon probe, and a cycling probe, and a TaqMan probe is preferable. The TaqMan probe is a probe in which the 5 ′ end is modified with a fluorescent substance (reporter fluorescent dye) and the 3 ′ end is modified with a quenching substance (quencher fluorescent dye). The molecular beacon probe is a probe that can have a stem-loop structure in which the 5 ′ end is modified with a fluorescent substance (reporter fluorescent dye) and the 3 ′ end is modified with a quenching substance (quencher fluorescent dye). The cycling probe is a probe composed of RNA and DNA in which the 5 ′ end of an oligonucleotide is modified with a fluorescent substance (reporter fluorescent dye) and the 3 ′ end is modified with a quenching substance (quencher fluorescent dye). Examples of reporter fluorescent dyes for the TaqMan probe include 6-FAM (6-carboxyfluorescein), TET (6-carboxy-4, 7, 2 ', 7'-tetrachlorofluorescein), HEX (6-carboxy-2' , 4 ', 7', 4,7-hexachlorofluorescein) and the like, and examples of quencher fluorescent dyes include 6-carboxytetramethylrhodamine (TAMRA), 6-carboxy-X-rhodamine Rhodamine fluorescent dyes such as (ROX), [(4- (2-nitro-4-methyl-phenyl) -azo) -yl-((2-methoxy-5-methyl-phenyl) -azo)]-aniline ( BHQ) and the like. These fluorescent dyes are known and can be used because they are included in commercially available real-time PCR kits.

2. Microorganism Life / Death Determination Kit The oligonucleotide set can be made into a kit. The kit of the present invention only needs to contain at least one of the above-mentioned oligonucleotide sets, and if an appropriate set is appropriately selected from these oligonucleotide sets depending on the type and number of microorganisms for determining life and death, Good. In addition, the kit of the present invention includes molecular weight markers that can be used for gene amplification and confirmation of amplification products, RNA extraction reagents, PCR buffers such as PCR buffers and DNA polymerase, labeling substances, and specimens as necessary. (Standard strains, etc.), sterilized water, instructions, etc. may be included. The reagent in the kit may be a solution or a lyophilized product.

3. Method for Determining Microorganism Life and Death According to the present invention, there is also provided a method for determining microbe life and death in a specimen using the above-described oligonucleotide set for life and death determination of microorganisms based on pre-rRNA detection.

Specifically, a method for determining the life or death of bacteria is a method using an RNA extracted from a specimen as a template, an oligonucleotide (forward primer) comprising a base sequence containing at least 15 consecutive bases in the base sequence shown in SEQ ID NO: 1, Mixture (reverse primer) of an oligonucleotide consisting of a base sequence containing at least 15 bases in the base sequence shown in SEQ ID NO: 2 and an oligonucleotide consisting of a base sequence containing at least 15 bases in the base sequence shown in SEQ ID NO: 3 A probe comprising an oligonucleotide comprising a step of performing RT-PCR amplification using a primer pair comprising: and a base sequence comprising at least 15 consecutive bases in the base sequence shown in SEQ ID NO: 4 or a complementary sequence thereof. And detecting and quantifying using

In addition, a method for determining whether a fungus is alive or dead is based on an RNA (forward primer) comprising a base sequence comprising at least 15 consecutive bases in the base sequence shown in SEQ ID NO: 5 using RNA extracted from a specimen as a template, and SEQ ID NO: 6 An oligonucleotide consisting of a base sequence containing at least 15 bases in the base sequence shown in FIG. 8, an oligonucleotide consisting of a base sequence containing at least 15 bases in the base sequence shown in SEQ ID NO: 7, and a continuation in the base sequence shown in SEQ ID NO: 8. RT- using a primer pair consisting of a mixture of oligonucleotides comprising a base sequence comprising at least 15 bases and an oligonucleotide comprising a base sequence comprising at least 15 consecutive bases in the base sequence shown in SEQ ID NO: 9 (reverse primer) Perform PCR amplification Including process and the resulting detected using a probe consisting of an oligonucleotide consisting of the nucleotide sequence comprising at least 15 consecutive bases of the amplified product in the nucleotide sequence or its complementary sequence shown in SEQ ID NO: 10 and quantifying step.

The type of “specimen” is not particularly limited as long as there is a possibility that microorganisms are mixed or infected, and the presence can be detected by amplification of a specific region by PCR. Examples include pharmaceuticals, medical materials or medical instruments, biological samples, and environmental samples. Examples of the medical material or medical instrument include catheters, stents, artificial blood vessels, artificial organs, blood bags, bone prostheses, surgical tools, embolic devices, guide wires, and the like, and biological samples include blood (whole blood, plasma, Serum), saliva, spinal fluid, urine, milk and other body fluids, tissues, cell cultures, and the like, and environmental samples include air, soil, water, and the like. The sample may be the sample or product itself as described above, or may be a diluted or concentrated sample or a pretreated sample. Examples of the pretreatment include heat treatment, filtration, and centrifugation.

The bacterium capable of determining whether it is alive or dead by the method of the present invention includes both gram-positive bacteria and gram-negative bacteria. Specifically, Escherichia genus, Bacillus genus, Salmonella genus, Pseudomonas genus, Clostridium genus, Neisseria genus, Streptococcus genus, Staphylococcus genus (Staphylococcus) genus, Mycobacterium genus, Enterobacter genus, Enterococcus genus, Porphyromonas genus, Yersinia genus, Haemophilus genus, Helicobacter genus Genus, Citrobacter genus, Campylobacter genus, Borrelia genus, Serratia genus, Deinococcus genus, Shigella genus, Aeromonas genus, Eikenella Genus, Lactobacil (Lactobacillus) genus, Actinobacillus genus, Actinomyces genus, Bacteroides genus, Moraxella genus, Propionibacterium genus, Bartonella genus, Rickettsia ) Genus, Coxiella genus, Capnocytophaga genus, Klebsiella genus, Halobacillus genus, Fusobacterium genus, Erwinia genus, Elbenella genus, Lact Lactobacillus genus, Listeria genus, Mannheimia genus, Peptococcus genus, Prevotella genus, Proteus genus, Veillonella genus, Acholeplasma genus Bifidobacterium Bordetella genus, Bacteroides genus, Coxiella genus, Chlamiadia genus, Legionella genus, Leptospira genus, Mycoplasma genus, Nocardia genus, Examples include, but are not limited to, bacteria belonging to the genus Prevotella, the genus Providencia, the genus Rickettsia, the genus Ureaplasma and the genus Vibrio.

Examples of fungi that can be determined by the method of the present invention include Aspergillus spp., Alternaria spp., Absidia spp., Basidobolus spp., Candida spp., Chlamydoabsidia spp. , Conidobolus genus, Cokeromyces genus, Cryptococcus genus, Cunninghamella genus, Emericella genus, Echinos sporangium genus, Fusarium genus The genus Paula, Lichtheimia, Microsporum, Mucor, Malassezia, Moltierella, Pseudallescheria, Paecilomyces ,new Pneumocystis, Rhizopus, Rhizomucor, Rhodotorula, Saksenaea, Trichosporon, histoplasma, cocidiodes, C (Blastomyces) genus, Paracoccidioides genus, Penicillium genus, Sporothrix genus, Trichophyton genus, Epidermophyton genus, Malassezia genus, Hyalora And fungi belonging to the genus Cladosporium, but are not limited thereto.

As a method for extracting RNA from a specimen, a method known in the art, for example, an RNA extraction method using guanidine isothiocyanate, phenol and chloroform can be used. Alternatively, a commercially available RNA extraction reagent may be used.

A method for synthesizing cDNA from RNA is also well known in the art. A commercially available random primer is used as a primer, and cDNA is synthesized using reverse transcriptase in the presence of dNTPs. As the reverse transcriptase, for example, SuperScript II (manufactured by Invitrogen), Thermoscript RT, AMV reverse transcriptase, MMLV reverse transcriptase, etc. can be used. Commercially available reagents or kits for reverse transcription can also be used as appropriate.

The reaction conditions for the reverse transcription reaction can be appropriately set according to the reverse transcriptase used. For example, the reaction can be performed at 42 ° C. for 40 to 60 minutes. Further, after the reverse transcription reaction, the reverse transcriptase may be inactivated. Inactivation of reverse transcriptase can be performed by heat treatment or chemical treatment.

Next, PCR amplification is performed using the synthesized cDNA as a template and the primer pairs included in the oligonucleotide set of the present invention. PCR amplification is not particularly limited except that the above primer pairs are used, and may be performed according to a conventional method. Specifically, a cycle including denaturation of template DNA, annealing of the primer to the template, and primer extension reaction using a thermostable enzyme (DNA polymerase such as Taq polymerase or Tth DNA polymerase from Thermus thermophilus) is repeated. To amplify a fragment containing a specific gene sequence of bacterial or fungal pre-rRNA. The composition of the PCR reaction solution and the PCR reaction conditions (temperature cycle, number of cycles, etc.) should be determined by a person skilled in the art based on preliminary experiments, etc. under conditions such that a PCR amplification product can be obtained with high sensitivity in PCR using the above primer pairs. Can be selected and set appropriately. Methods for selecting appropriate PCR reaction conditions based on the Tm of the primer are well known in the art, for example, first a denaturation reaction at 94 ° C. for 5 minutes and then at 94 ° C. for 50 seconds (denaturation). The extension reaction can be carried out at 55 ° C. for 50 seconds (annealing), 72 ° C. for 1 minute (extension) for 30 cycles, and finally 72 ° C. for 1 minute. However, the conditions shown here are only examples, and the enzyme used, the reaction temperature, the reaction time, the number of cycles, etc. can be appropriately changed. Such a series of PCR operations can be performed using a commercially available PCR kit or PCR apparatus according to the operating instructions. For example, GeneAmp PCR System 9700 (Applied Biosystems), GeneAmp PCR System 9600 (Applied Biosystems) and the like can be used as the PCR apparatus. In the method of the present invention, RNA is converted to cDNA and PCR is performed ( RT-PCR) LightCycler DX480 (Roche) and Takara One Step RT-PCR Kit AMV (TAKARA), which can be carried out continuously in one tube, can be preferably used.

The above PCR is preferably performed by real-time PCR. Real-time PCR is a technique in which PCR is performed using a real-time PCR apparatus, and amplification by PCR is measured over time to quantify DNA as a template based on the amplification rate. In this method, a device dedicated to real-time PCR, in which a thermal cycler and a spectrofluorometer are integrated, is usually used. First, PCR is performed using a known amount of DNA that has been serially diluted as a standard, and based on this, the number of cycles (threshold cycle; Ct value) that results in a constant amount of amplification product in the region where amplification occurs exponentially Is plotted on the vertical axis and the initial DNA amount is plotted on the horizontal axis to create a calibration curve. A sample having an unknown concentration can be reacted under the same conditions to obtain a Ct value, and the target DNA amount in the sample can be measured from this value and a calibration curve. The real-time PCR method includes a method using a fluorescently labeled probe and a method using a fluorescent reagent depending on the detection method of the gene amplification product. Examples of methods using fluorescently labeled probes include TaqMan method, molecular beacon probe, TaqMan method using molecular probe, molecular beacon method, and cycling probe method. Methods using fluorescent reagents include double-stranded DNA together with primer pairs. Examples include an intercalator method in which an intercalator such as SYBR Green I, which is a compound that emits fluorescence by binding to, is added to the PCR reaction system. Among the above methods, the TaqMan method is preferable.

In the TaqMan method, PCR is performed by adding an oligonucleotide probe modified with a fluorescent substance (such as FAM) at the 5 ′ end and a quencher substance (such as TAMRA) at the 3 ′ end to the PCR reaction system. In the TaqMan method, the TaqMan probe specifically hybridizes to the template DNA under the conditions used for the polymerase extension reaction in the PCR amplification reaction, and is degraded as the DNA strand extends, that is, the template DNA is amplified. By releasing the substance, the amount of fluorescence in the PCR solution increases. This increase in the amount of fluorescence serves as an index for amplification of the template DNA, and the state of amplification in PCR can be easily detected and quantified in real time. The real-time PCR method can be carried out using a commercially available real-time PCR kit or real-time PCR apparatus based on the usual methods known to those skilled in the art, except that the above-mentioned oligonucleotide set is used, according to the operating instructions attached to them. That's fine. For example, QuantiTect SYBR Green Mix (Qiagen), ABI Prism 7500 FastAppReal-Time PCRTaSystem (Applied Biosystems), Takara-Bio 社 Thermal Cycler Dice Real Time System II etc. be able to.

Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

(Example 1) Preparation of Bacterial Life / Death Oligonucleotide Set Preparation The following bacterial ribosomal RNA that can be determined for the design of a bacterial life / death determination oligonucleotide set (primer and probe) based on rRNA precursor detection Precursor sequence information was downloaded from GenBank, and alignment was performed using Allele ID v7.80 (premier biosoft) to search for regions having a base sequence highly common among bacterial species.

(Bacterial species used for designing oligonucleotides: a total of 39 species)
Acholeplasma laidlawii
Bacillus subtilis
Bacillus cereus
Bifidobacterium bifidum
Bartonella henselae
Bordetella pertussis
Bacteroides fragilis
Coxiella burnetii
Clostridium perfringens
Clostridium tetani
Chlamydia trachomatis
Campylobacter jejuni
Escherichia coli
Enterobacter cloacae
Enterococcus faecalis
Fusobacterium nucleatum
Haemophilus influenzae
Klebsiella pneumoniae
Legionella pneumophila
Leptospira interrogans
Moraxella catarrhalis
Mycoplasma pneumoniae
Mycobacterium tuberculosis
Mycobacterium kansasii
Nocardia asteroides
Neisseria meningitidis
Pseudomonas aeruginosa
Propionibacterium acnes
Prevotella melaninogenica
Providencia stuartii
Rickettsia japonica
Streptococcus pneumoniae
Staphylococcus aureus
Staphylococcus epidermidis
Salmonella typhi
Serratia Bizio
Treponema pallidum
Ureaplasma urealyticum
Vibrio mimicus

As a result of the search, the region from 6804 to 6780th in the nucleotide sequence of the 23S rRNA gene identified as a region having a highly common nucleotide sequence (FIG. 1A), and 7644 to the nucleotide sequence of the 5S rRNA gene. Specific primers and probes were designed using Allele v 7.80 (premier biosoft) based on the base sequence of the region up to the 7685th region (Fig. 1B).

As a result, the oligonucleotide consisting of the base sequence shown in SEQ ID NO: 1 as a 23S rRNA common forward primer for general bacterial rRNA precursors, and the base sequences shown in SEQ ID NO: 2 and SEQ ID NO: 3 as 5S rRNA common reverse primers for general bacterial rRNA precursors The following oligonucleotide (oligonucleotide set 1) consisting of the base sequence shown in SEQ ID NO: 4 was designed as a 23S rRNA common probe for general bacterial rRNA precursors. The forward primer and the probe are the same as described above, and the reverse primer is an oligonucleotide having the nucleotide sequences shown in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14 Set 2) was designed.

(Bacterial oligonucleotide set 1)
Forward primer 1: 5'-acgygagytg ggttya-3 '(SEQ ID NO: 1) (y = t / c)
Reverse primer BR1: 5'-gcttracttc yskgttcg-3 '(SEQ ID NO: 2) (r = g / a, y = t / c, s = g / c, k = g / t)
Reverse primer BR2: 5'-gtttcacttc tgagttcg-3 '(SEQ ID NO: 3)
Probe: 5'-cgtcgyg aga cagktyggtc yctat-3 '(SEQ ID NO: 4) (y = t / c, k = g / t)
(Bacterial oligonucleotide set 2)
Forward primer 1: 5'-acgygagytg ggttya-3 '(SEQ ID NO: 1) (y = t / c)
Reverse primer BR3: 5'-cttctgwgtt cggsawgg-3 '(SEQ ID NO: 11) (w = a / t, s = g / c)
Reverse primer BR4: 5'-cttctctgtt cggaatgg-3 '(SEQ ID NO: 12)
Reverse primer BR5: 5'-cttccgggtt cggaatrg-3 '(SEQ ID NO: 13) (r = g / a)
Reverse primer BR6: 5'-cttcttggtt cgggatgg-3 '(SEQ ID NO: 14)
Probe: 5'-cgtcgyg aga cagktyggtc yctat-3 '(SEQ ID NO: 4) (y = t / c, k = g / t)

Example 2 Determination of E. coli life or death Method (1) Preparation of Test Sample A viable cell suspension (1 × 10 ⁶ CFU / ml) of E. coli JCM1649 was prepared using physiological saline. Place 500 μL of this Escherichia coli suspension in a 1.5 ml tube, (i) high-temperature sterilization at 95 ° C. for 10 minutes, (ii) low-temperature sterilization at 65 ° C. for 30 minutes, (iii) kanamycin antibiotic A test sample was prepared by sterilizing each agent under conditions of sterilization (300 μg / ml).

(2) Analysis of test sample by RT-PCR After each sterilization treatment, the test sample is kept at 37 ° C, 1 hour, 3 hours, 6 hours, 1 day, 2 days, 3 days, 4 days, After 5 days and 6 days, the rRNA precursor (pre-rRNA) in each test sample was measured as follows.

First, the test sample was centrifuged at 5 krpm for 5 minutes, and after removing the supernatant, 500 μL of RLT buffer containing 1% mercaptoethanol was added and suspended using RNeasy mini kit (Qiagen), and then zirconia beads ( It was transferred to a 2 ml tube containing 1 piece (diameter 0.2 mm 0.5 g). After treatment with a crusher μT-12 (manufactured by TAITEC) at 3500 rpm for 5 minutes, 350 μL was transferred to a new 1.5 ml tube, and RNA was extracted according to the manual attached to the kit (including optional DNase treatment).

The eluted RNA sample 50 μL was treated with DNase I 5 U (Takara Bio) and 1 × Buffer 30 ° C. for 30 minutes. After the treatment, RNA was purified again. The operation method was in accordance with RNA “clean up” in the attached manual (including optional DNase treatment). In order to quantify the rRNA precursor (pre-rRNA) in the obtained RNA sample, a master mix (when using oligonucleotide set 1) shown in Table 1 below was prepared, and real-time PCR amplification was performed.

For real-time PCR amplification, the primer pair of the oligonucleotide set 1 designed in Example 1 and the primer pair of the mixed primer of reverse primer BR1 and reverse primer BR2, or the forward primer of oligonucleotide set 2 and reverse primer BR3, reverse Using a primer pair of mixed primers of primer BR4, reverse primer BR5, and reverse primer BR6, each was performed once using a real-time PCR apparatus (LightCycler DX480; Roche) under the following PCR thermal cycle conditions. As a negative control, 5 μl of sterilized water was used as a template.

(RT-PCR conditions)
After a reverse transcription (RT) reaction at 42 ° C. for 10 minutes, 45 cycles were carried out, first at 95 ° C. for 10 minutes and then at 95 ° C. for 5 seconds and 60 ° C. for 1 minute.

The detection and quantification of the amplification product was performed using the probe designed in Example 1 (fluorescent dye 6-FAM added to the 5 'end of the oligonucleotide and quenching dye BHQ added to the 3' end). The length of the amplified product was about 400 bp.

Also, in order to confirm DNA remaining after DNase treatment, only TaKaRa primescript RT enzyme in Table 1 was compared. If a positive signal is detected with TaKaRa primescript RT enzyme- (without reverse transcription), DNA remains, and the rRNA precursor may not be accurately quantified. However, when it was detected with a negative control, it was cut off around the same number of cycles due to the enzyme host-derived DNA.

(3) Viable count (CFU / ml)
For test samples sterilized under the conditions of high-temperature heat sterilization at 95 ° C for 10 minutes and low-temperature heat sterilization at 65 ° C for 30 minutes, the specified time after sterilization (1 hour, 3 hours, 6 hours, 1 day, 2 days , 3 days, 4 days), the test samples were used for the viable count. For test samples sterilized with kanamycin antibiotic (300 μg / ml), remove the supernatant by centrifuging at 5 krpm for 5 minutes, then add 500 μL of LB medium without the antibiotic and add E. coli cells. Was suspended in LB medium, E. coli cells at each stage were seeded on LB agar medium and incubated overnight at 37 ° C., and cfu / ml was measured by colony count.

2. Results Amplification curves obtained by performing real-time PCR using oligonucleotide set 1 and oligonucleotide set 2 are shown in FIGS. 2-1 and 2-2, respectively. Originally, although a large amount of host-derived rRNA and genomic DNA was mixed in reverse transcriptase, it was not detected by RT-PCR (with reverse transcription) in oligonucleotide set 2 (FIG. 2-2). , Dotted arrow), oligonucleotide set 2 was judged to have low sensitivity. On the other hand, in the oligonucleotide set 1, since rRNA and genomic DNA derived from the host were detected in both RT-PCR (with reverse transcription) and PCR (without reverse transcription) (FIG. 2-1, dotted arrows), the sensitivity was high. It was judged to be expensive. Therefore, the oligonucleotide set 1 was established as an oligonucleotide set for determining the viability of the microorganism (bacteria) of the present invention.

Quantitative results of rRNA precursors by real-time PCR (using oligonucleotide set 1) are shown in FIG. 3A, and the viable count results are shown in FIG. 3B. As shown in FIG. 3A, the rRNA precursor was found to be 72 hours after high-temperature sterilization at 95 ° C. for 10 minutes, 48 hours after low-temperature heat sterilization at 65 ° C. for 30 minutes, and 72 hours after kanamycin treatment. It was below the detection limit. In addition, as shown in FIG. 3B, all the bacteria that passed 1 hour or more after any sterilization treatment were killed. From this result, when more than 72 hours have passed since the death of live bacteria, the rRNA precursor is below the detection limit, and even if bacterial DNA is detected, it is possible to distinguish between live and dead bacteria if no rRNA precursor is detected It was proved.

(Example 3) Preparation of oligonucleotide set for determination of fungal life and death The following fungal ribosomal RNAs that can be determined for the design of oligonucleotide set (primer and probe) for determination of fungal life and death based on rRNA precursor detection Precursor sequence information was downloaded from GenBank, and alignment was performed using Allele ID v7.80 (premier biosoft) to search for regions having a base sequence highly common between fungal species.

(Fungus species used in oligonucleotide design: 95 species in 27 genera)
Aspergillus genus
Alternaria genus
Absidia genus
Genus Basidobolus
Candida genus
Chlamydoabsidia genus
Genus Conidobolus
Cokeromyces genus
Genus Cryptococcus
Cunninghamella genus
Emericella genus
Echinosporangium genus
Genus Fusarium
Genus Lomentospora
Lichtheimia genus
Genus Microsporum
Genus Mucor
Genus Malassezia
Genus Moltierella
Pseudallescheria genus
Paecilomyces genus
Pneumocystis genus
Genus Rhizopus
Rhizomucor genus
Rhodotorula
Saksenaea genus
Genus Trichosporon

As a result of the search, it was specified that the region has a highly common base sequence, the region from 1017 to 1071 of the base sequence of the 18S rRNA gene (Figs. 4-1 to 3), and the region of the 5.8S rRNA gene Specific primers and probes were designed using Allele v 7.80 (premier biosoft) based on the nucleotide sequence (Figs. 4-1 to 3) in the region up to the 2434-2463th of the nucleotide sequence.

As a result, an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 5 as an 18S rRNA common forward primer for fungal general rRNA precursors, and SEQ ID NOs: 6, 7, 8 and 9 as a 5.8S rRNA common reverse primer for fungal general rRNA precursors. The following oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 10 was designed as an 18S か ら rRNA 配 列 common probe for fungal general rRNA precursors, and the oligonucleotide set of the present invention was prepared.

(Fungo oligonucleotide set)
Forward primer: 5'-gattacgtcc ctgccctttg ta-3 '(SEQ ID NO: 5)
Reverse primer FR1: 5'-cgagagccra gagatccrtt-3 '(SEQ ID NO: 6) (r = g / a)
Reverse primer FR2: 5'-cgagaaccaa gagatccgtt-3 '(SEQ ID NO: 7)
Reverse primer FR3: 5'-cgaaagccga gagatccatt-3 '(SEQ ID NO: 8)
Reverse primer FR4: 5'-ccggaaccaa gagatccrtt-3 '(SEQ ID NO: 9)) (r = g / a)
Probe: 5'-acaccgcccg tcgctactac cg-3 '(SEQ ID NO: 10)

Table 2 below shows the master mix composition when real-time PCR amplification is performed using the above-mentioned oligonucleotide set for fungi.

The present invention can be used for microbial contamination inspection in the field of food and pharmaceutical manufacturing and medical practice.

All publications, patents and patent applications cited in this specification shall be incorporated into this specification for reference.

Claims (9)

A set of oligonucleotides for determining the viability of microorganisms composed of oligonucleotides designed in two ribosomal RNA gene regions sandwiching the spacer region of ribosomal RNA precursor (pre-rRNA). The oligonucleotide set for life / death determination of microorganisms according to claim 1, wherein the microorganisms are bacteria or fungi. The oligonucleotide set for determining whether a microorganism is alive or dead according to claim 1 or 2, wherein the oligonucleotide is used as a primer and a probe. The microorganism is a bacterium, and the oligonucleotide set is composed of a primer pair consisting of the following (a) oligonucleotide and (b) oligonucleotide, and a probe consisting of the following (c) oligonucleotide: The oligonucleotide set for life / death determination of microorganisms according to claim 1.
(A) an oligonucleotide consisting of a base sequence containing at least 15 consecutive bases in the base sequence shown in SEQ ID NO: 1 (b) an oligonucleotide consisting of a base sequence containing at least 15 contiguous bases in the base sequence shown in SEQ ID NO: 2 and a sequence (C) Oligo consisting of a base sequence containing at least 15 bases in the base sequence shown in SEQ ID NO: 4 or its complementary sequence nucleotide The microorganism is a fungus, and the oligonucleotide set is composed of a primer pair consisting of the following (d) oligonucleotide and (e) oligonucleotide, and a probe consisting of the following (f) oligonucleotide: The oligonucleotide set for life / death determination of microorganisms according to claim 1.
(D) an oligonucleotide consisting of a base sequence containing at least 15 consecutive bases in the base sequence shown in SEQ ID NO: 5 (e) an oligonucleotide consisting of a base sequence containing at least 15 bases continuous in the base sequence shown in SEQ ID NO: 6; An oligonucleotide consisting of a base sequence containing at least 15 consecutive bases in the base sequence shown in No. 7, an oligonucleotide consisting of a base sequence containing at least 15 consecutive bases in the base sequence shown in SEQ ID No. 8, and a base shown in SEQ ID No. 9 Mixture of oligonucleotides comprising a base sequence comprising at least 15 consecutive bases in the sequence (f) Oligonucleotide comprising a base sequence comprising at least 15 consecutive bases in the base sequence shown in SEQ ID NO: 10 or its complementary sequence A kit for determining the viability of microorganisms, comprising at least one oligonucleotide set for determining viability of microorganisms according to any one of claims 1 to 5. Using RNA extracted from a specimen as a template, RT-PCR amplification is performed using the oligonucleotide set for determining whether a microorganism is alive according to any one of claims 1 to 5, and the obtained amplification product is detected and quantified. A method for judging whether a microorganism is alive or dead. The microorganism viability determination method according to claim 7, wherein the RT-PCR is real-time RT-PCR. The method for determining whether a microorganism is alive or dead according to claim 7 or 8, wherein the specimen is a food or drink, a medicine, a medical material or a medical instrument, a biological sample, or an environmental sample.

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