JP2016042802A - Method for examining presence or absence of food poisoning pathogenic bacteria - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for efficiently examining the presence or absence of Salmonella, enterohemorrhagic Escherichia coli (EHEC), and Shigella.SOLUTION: The efficiency of a screening method for a carrier with a multiplex PCR method can be increased by using the multiplex PCR method, in which the positive rate (positive number/all number of specimens) is 0.5% or less, as a method for examining the presence or absence of Salmonella, enterohemorrhagic Escherichia coli, and Shigella in a healthy person. Further, upon trying to increasing the efficiency of the screening, we found out that a detection method using a multiplex PCR from pool feces and a melting profile analysis is effective.SELECTED DRAWING: None

Description

The present invention relates to the field of nucleic acid amplification. More specifically, the present invention relates to a method for examining the presence or absence of Salmonella, enterohemorrhagic Escherichia coli (EHEC), and Shigella that are food poisoning causative bacteria.

  In preventing food poisoning, it is important to find a carrier of a food poisoning germ at an early stage. For this reason, food cooks are inspected for Salmonella, enterohemorrhagic Escherichia coli (EHEC), and Shigella that are food poisoning causative bacteria.

Conventionally, Salmonella, enterohemorrhagic Escherichia coli (EHEC), and Shigella have been tested by a smear culture method. Specifically, after culturing the specimen under a predetermined condition and growing to a certain extent, culturing in a selective medium that allows only the bacteria to be detected to grow, and observing the colonies formed by the growth It is a method of detecting.

  However, in such a conventional method, based on (1) the culturing step, it takes time until results are obtained, (2) low detection sensitivity, and (3) colony observation results. Since it is difficult to make an objective determination, there are problems such as difficulty in ensuring the objectivity of the detection result.

  Accordingly, detection methods using the PCR method have been developed in order to solve the problems of these detection methods (Non-Patent Document 1). In the PCR method, (1) it can be detected quickly because it does not go through the culturing step, (2) it has high detection sensitivity, and (3) it is determined whether the target DNA is amplified by whether the target DNA has been amplified. Therefore, there is an advantage that bacteria can be detected objectively.

  As for the inspection of Salmonella, enterohemorrhagic Escherichia coli (EHEC), and Shigella by PCR method, a definitive inspection is generally performed by a smear culture method (Non-patent Document 2). This is because there is a high possibility of false positives in the PCR method. In fact, even in the case of PCR positive, a phenomenon that becomes negative in the smear culture method is recognized. The cause is various factors such as occurrence of non-specific amplification.

  Although the PCR method has a higher positive rate than the smear culture method, it is used as a primary screening. This is due to the fact that it is easier and more efficient to smear and culture PCR positive specimens after screening by the PCR method. However, there is no clear indicator for the positive rate of the PCR method, leaving room for improvement in more efficient screening methods for detection of Salmonella, enterohemorrhagic Escherichia coli (EHEC), and Shigella using the PCR method. ing.

TaKaRa BIO VIEW 55, 48 pages (2008) Journal of Infectious Diseases 86: 741-748, 2012

  Screening for Salmonella, enterohemorrhagic Escherichia coli (EHEC), Shigella using the PCR method needs to have a higher positive rate than the smear culture method because no omission occurs, but from the viewpoint of efficiency, A high correlation with the smear culture method is required. Therefore, the problem to be solved by the present invention is to provide a screening method for Salmonella, enterohemorrhagic Escherichia coli (EHEC), and Shigella using PCR method more efficiently.

  In view of the above circumstances, the present inventor has found that, as a result of earnest research, efficient screening can be carried out by reducing the positive rate of healthy human Salmonella, enterohemorrhagic Escherichia coli (EHEC), and Shigella. It was completed. Furthermore, the present inventors have found that a screening method from pooled stool and a detection method using melting curve analysis are effective in improving the efficiency of screening.

That is, the present invention has the following configuration.
[1] A method for examining the presence or absence of one or more of Salmonella, enterohemorrhagic Escherichia coli, and Shigella by using a multiplex PCR method. A test method comprising the steps of 1) and (2) and having a positive rate of 0.5% or less.
(1) A step of performing multiplex PCR targeting the three bacterial species (2) A step of analyzing the obtained amplification product and detecting the presence or absence of any one or more of the three bacterial species [2 ] The test | inspection method as described in [1] whose positive rate is 0.2% or less.
[3] The examination method according to [1] or [2], wherein the examination object is a stool specimen.
[4] The test method according to any one of [1] to [3], wherein the test target is a pooled sample of stool samples collected from stool of X types (X represents an integer of 2 or more).
[5] The inspection method according to [4], wherein X is 20 or more.
[6] The inspection method according to any one of [1] to [5], wherein the detection method in step (2) is a melting curve analysis using a fluorescent intercalator.
[7] In the step (2), Salmonella, enterohemorrhagic Escherichia coli (EHEC) and Shigella are separated and detected based on differences in Tm values of amplification products. Inspection method.
[8] The detection method according to [1], including an internal control (internal standard) that gives a Tm value different from any of the amplification products of the three bacterial species in step (2).
[9] In the step (1), multiplex PCR is performed using as a primer set three primer pairs designed so that Tm values of the amplification products of the three bacterial species are separated from each other by 1 ° C. or more. Detection method.
[10] The detection method according to [9], wherein the primer set is a combination of three primer pairs selected one by one from the following three groups (I) to (III).
(I) Salmonella amplification primer pair (a) represented by the following (a) or (b) (a) “an oligonucleotide consisting of 15 bases or more in SEQ ID NO: 1” and “oligo consisting of 15 bases or more in SEQ ID NO: 2” Complementary bases to each base sequence of the primer pair of oligonucleotide pair (b) (a) consisting of at least one of the group consisting of “nucleotide” and “oligonucleotide consisting of 15 bases or more of SEQ ID NO: 3” Oligonucleotide pair having sequence (II) Primer pair for intestinal hemorrhagic Escherichia coli (EHEC) amplification represented by (c) or (d) below
(C) at least one of the group consisting of “an oligonucleotide consisting of 15 bases or more in SEQ ID NO: 4” and “an oligonucleotide consisting of 15 bases or more in SEQ ID NO: 5”; Each nucleotide sequence of the primer pair of oligonucleotide pair (d) (c) consisting of at least one of the group consisting of “an oligonucleotide consisting of 15 bases or more” and “an oligonucleotide consisting of 15 bases or more of SEQ ID NO: 7” Oligonucleotide pair (III) having a complementary base sequence to the primer pair for amplification of Shigella shown by (e) or (f) below
(E) each of the primer pair of the oligonucleotide pair (f) (e) consisting of “oligonucleotide consisting of 15 bases or more in SEQ ID NO: 8” and “oligonucleotide consisting of 15 bases or more in SEQ ID NO: 9” Oligonucleotide pair having a complementary base sequence to the base sequence [11] One or more of Salmonella, Enterohemorrhagic Escherichia coli (EHEC) and Shigella, including the following (A) to (D) Reagent for inspecting the presence or absence of bacteria.
(A) thermostable DNA polymerase (B) primer set according to any of [9] or [10] (C) DNA synthesis substrate (deoxynucleotide triphosphate (dNTP)), and
(D) The reagent according to [11], further comprising a buffer solution [12] containing at least one member selected from the group consisting of magnesium ions, ammonium ions, and potassium ions, and [E].
(E) Fluorescent intercalator

  The present invention makes it possible to increase the efficiency of screening for Salmonella, enterohemorrhagic Escherichia coli (EHEC), and Shigella by PCR. Thereby, since it is possible to increase the number of negative samples that can be confirmed in several hours, a rapid and efficient test report can be expected. Furthermore, in the present invention, since detection can be performed quickly and efficiently, it can be expected to be used in industrial applications that require processing of a large number of samples.

It is a figure of the result of having detected three bacterial species, Salmonella, enterohemorrhagic Escherichia coli (EHEC), and Shigella from genomic DNA and a positive specimen. It is a figure of the result of having detected three bacterial species, Salmonella, enterohemorrhagic Escherichia coli (EHEC), and Shigella from pool feces.

Hereinafter, the present invention will be described in detail.
The present invention is characterized in that screening efficiency is remarkably improved by using a multiplex PCR method with a low positive rate. Focusing on this positive rate, there are no examples of Salmonella, enterohemorrhagic Escherichia coli (EHEC) and Shigella testing, and this low positive rate was a major factor in increasing detection efficiency.

One embodiment of the present invention is a method for testing the presence or absence of any one or more of Salmonella, enterohemorrhagic Escherichia coli, and Shigella by using a multiplex PCR method. The test method includes the following steps (1) and (2) and has a positive rate of 0.5% or less.
(1) A step of performing multiplex PCR targeting the three bacterial species (2) A step of analyzing the obtained amplification product and detecting the presence or absence of any one or more of the three bacterial species

The multiplex PCR method is a method capable of amplifying a plurality of targets at the same time, and is performed by mixing all the primer sets for the plurality of targets and performing PCR. That is, the multiplex PCR in the present invention is to perform PCR with one solution by mixing all the primers for amplifying Salmonella, enterohemorrhagic Escherichia coli (EHEC), and Shigella.

  The positive rate in the test method of the present invention means the ratio of positive samples to the total number of samples when healthy subjects are tested. That is, even when pool stool described later is used, the positive rate is calculated by dividing the individual samples. For example, suppose that 5000 samples are pooled 50 by sample and 100 pools are examined. At this time, when there are five positive pools, the positive rate is calculated as 5 ÷ 5000, which is 0.1%.

  The low multiplex PCR positive rate in the test method of the present invention is preferably 0.5% or less from the viewpoint of efficiency. More preferably, it is 0.2% or less. With regard to efficiency, the allowable range of the number of cells requiring smear culture due to PCR positive is calculated for the total number of specimens, taking into account two points of time efficiency and profitability.

The inspection method of the present invention is intended to inspect for the presence or absence of any one or more of the three bacterial strains of Salmonella, enterohemorrhagic Escherichia coli (EHEC), and Shigella that are food poisoning causative bacteria. Is not particularly limited, but it is preferable to use a stool specimen. For example, a stool specimen can be used. A stool specimen refers to a stool specimen collected from a stool. The method for collecting a stool specimen is not particularly limited, and can be obtained, for example, by scraping a part of the stool. Samples collected for stool can be used as stool samples.

  In the test method of the present invention, the stool specimen is not particularly limited. For example, a stool specimen collected from a mammalian stool can be mentioned. Although it does not specifically limit as a mammal, For example, a human, a cow, a pig, a dog, a cat etc. can be mentioned. Further, the stool specimen to which the present invention is applied may have been subjected to dilution, filtration, and other physicochemical treatment on the premise that DNA destruction is suppressed for the purpose of storage and the like. Good.

  In the test method of the present invention, genomic DNA extracted from the stool specimen can be amplified as a template. The separation and purification of this genomic DNA is not limited, but commercially available MagExtractor-Genome- (TOYOBO), NucleoSpin (registered trademark) Tissue (Takara Bio), Wizard (registered trademark) Genomic DNA Purification Kit (Promega), FlexiGene DNA Kit (Qiagen) and the like can be used.

  As described above, in the test method of the present invention, the extracted genomic DNA can be used as a template. However, from the viewpoint of simplicity and speed of the test, it is possible to use a stool specimen that has not been subjected to separation and purification of genomic DNA. preferable. For example, a method in which a solution in which a stool specimen is suspended as it is is heat-treated and centrifuged, and then the supernatant is used as a template. The ratio of the fecal sample used for PCR to the PCR reaction solution is not particularly limited as long as PCR is appropriately performed. For example, it can be appropriately adjusted within a range of 1 to 20 v / v%.

  Furthermore, when multiple specimen tests are required, it is more preferable to use a pool specimen as a stool specimen. The addition ratio of the stool specimen used for PCR is not particularly limited as long as PCR is appropriately performed. For example, it can be appropriately adjusted within a range of 1 to 20 v / v%. In the present invention, the pooled specimen is composed of a stool specimen collected from stool of X types (X represents an integer of 2 or more).

  The X-type stool referred to here may be each stool excreted by the X-type individual, or may be X-type stool excreted by the same individual. X is not particularly limited, but the detection rate of Salmonella to be detected, enterohemorrhagic Escherichia coli (EHEC), Shigella species (of one stool sample out of 100 stool samples) When the bacteria are detected in the sample, the detection rate is assumed to be 1%). For example, a stool sample collected from human stool is used as a stool sample, and the detection rate of Salmonella, enterohemorrhagic Escherichia coli (EHEC), and Shigella is about 0.05%. In this case, if detection is performed using a pooled specimen made of stool specimens collected from 1000 types of stool, the probability that these three bacterial species will be detected is 1000 (sample) × 0.05% = 50%. . Therefore, when different pool samples are sequentially replaced and detected, three bacterial species are detected with a probability of once every two times. In the same case, if detection is performed using a pooled sample made of stool samples collected from 100 types of stool, three bacterial species are detected with a probability of once in 20 times. In the same case, if detection is performed using a pooled sample consisting of stool test samples collected from 10 types of stool, three bacterial species are detected with a probability of once in 200 times. Considering the probability of Salmonella, enterohemorrhagic Escherichia coli (EHEC), Shigella can be efficiently proceeded when detecting by sequentially replacing different pool specimens, The type of stool specimen contained in the pool specimen can be determined. Usually, it is preferable to detect three bacterial species with a probability of once every several tens of times. Therefore, X is preferably 20 or more, more preferably 30 or more, more preferably 40 or more, and more preferably 50 or more.

  The positive rate in the multiplex method is most important in calculating efficiency when using pool specimens. For example, when a test is performed in which 50 samples of samples having a positive rate of 0.2% in the multiplex PCR method are pooled, the number of smears can be 10% of the total number of samples. Since 10% is a numerical value that is significant in terms of time and profitability, in the present invention, the positive rate is more preferably 0.2% or less.

In the test method of the present invention, the method of detecting the presence or absence of any one or more of the three bacterial species by analyzing the obtained amplification product after multiplex PCR may be electrophoresis, Although melting curve analysis using a fluorescent intercalator may be used, it is preferable to use melting curve analysis from the viewpoint of simplicity of inspection. Furthermore, in the melting curve analysis, the three bacterial species can be separated and detected by making the Tm values of the amplification products different from each other among the three bacterial species. Therefore, the three bacterial species can be separated and detected by the Tm value. It is more preferable to use a primer.

  In the test method of the present invention, the primer design in the multiplex PCR method may be performed so as to lower the positive rate of Salmonella, enterohemorrhagic Escherichia coli (EHEC), Shigella, and satisfy the above-mentioned positive rate. There is no particular limitation.

Although the primer used for multiplex PCR is not specifically limited, The length of the DNA fragment to be amplified is, for example, 20 to 1000 bases, more preferably 50 to 700 bases, and still more preferably 100 to 500 bases. Furthermore, the length of the primer in the present invention is preferably 13 to 35 bases, more preferably 16 bases or more, and 30 bases or less.

  The primer set used in the present invention uses a primer set capable of amplifying genes common to many groups so that any group of Salmonella, enterohemorrhagic Escherichia coli (EHEC), and Shigella can be detected. It is preferable.

  For example, as Salmonella, 2 bacterial species (S. enterica and S. bongori), 6 subspecies, and 2501 serotypes are known. When detecting Salmonella, it is preferable to use a primer set capable of amplifying the gene of the cell-invasive protein invA.

  For example, enterohemorrhagic Escherichia coli (EHEC) including O-157 is known as VT1, VT2, VT2vha, VT2vhb, and VT2vp as verotoxins. When detecting enterohemorrhagic Escherichia coli (EHEC), it is preferable to use a primer set capable of amplifying VT1 and VT2 genes.

  For example, A to D groups (13 serotypes as group A; 6 serotypes as group B; 18 serotypes as group C; 1 serotype as group D1) are known as Shigella. When trying to detect Shigella, it is preferable to use a primer set that can amplify the gene of pathogenic factor ipaH.

  As a method for enabling detection of any group of Salmonella, enterohemorrhagic Escherichia coli (EHEC), and Shigella, a plurality of primers can be used. For example, in order to amplify enterohemorrhagic Escherichia coli (EHEC), a total of four primer sets for VT1 amplification and VT2 amplification are used as a primer set for amplifying enterohemorrhagic Escherichia coli (EHEC). be able to.

  A degenerate primer can also be used to enable detection of any group of Salmonella, enterohemorrhagic E. coli (EHEC), and Shigella. By using degenerate primers, it is possible to design a primer that includes all possible base sequences in any group.

In addition to the conditions described above, the primer set used in the test method of the present invention is preferably capable of being separated and detected by the difference in Tm values of the amplification products of three species of Salmonella, enterohemorrhagic Escherichia coli (EHEC), and Shigella. The difference in Tm value is 1 ° C. or higher, more preferably 2 ° C. or higher, and more preferably 3 ° C. or higher. The Tm value of each amplification product is determined by each real-time PCR apparatus and analysis software.

  Although the primer set in this invention is not specifically limited, The primer set shown to sequence number 1-9 can be mentioned as an example. The primers described in SEQ ID NOs: 1 and 2 are salmonella amplification forward primers, and the primer described in SEQ ID NO: 3 is a salmonella amplification reverse primer. The primers described in SEQ ID NOs: 4 and 5 are forward primers for amplification of enterohemorrhagic E. coli (EHEC), and the primers described in SEQ ID NOs: 6 and 7 are reverse primers for amplification of enterohemorrhagic Escherichia coli (EHEC). The primer set forth in SEQ ID NO: 8 is a forward primer for amplifying Shigella, and the primer set forth in SEQ ID NO: 9 is a reverse primer for amplifying Shigella.

As a primer set in this invention, you may use the primer shown to these sequence number 1-9, and a complementary sequence. Moreover, in order to respond | correspond to a variation | mutation, substitution of several bases of the sequence shown to sequence number 1-9 may be included. The term “several bases” as used herein refers to 10 bases or less, more preferably 5 bases or less, more preferably 4 bases or less, more preferably 3 bases or less, more preferably 2 bases or less, more preferably 1 base or less.

In addition, at least one of the primers for amplifying each of the above three bacterial species is a base sequence (or a complementary sequence thereof) described in any of SEQ ID NOs: 1 to 9 above. It may consist of a part of. For example, in at least one primer pair of Salmonella, Enterohemorrhagic Escherichia coli (EHEC) and Shigella, the forward primer and / or the reverse primer consists of 15 bases or more, more preferably 18 bases or more of each SEQ ID NO: What is necessary is just to be comprised from the oligonucleotide.
In other words, the longest of the base sequences described in SEQ ID NOs: 1 to 23 is 23 bases and the shortest is 19 bases. Most preferably, it has the full length of the sequence represented by each base sequence. As long as the “part of the base sequence” is within a range in which the total length is not less than 15 bases, at least one of the ends may be missing on the premise of maintaining the continuity of the sequence. In that case, the length of the base sequence is preferably longer. For example, in the case of SEQ ID NO: 1, it may be 15 bases or more, more preferably 16 bases or more, more preferably 17 bases or more, more preferably 18 bases or more, more preferably 19 bases or more, more preferably 20 bases or more. More preferably 21 bases or more, more preferably 22 bases or more, and most preferably 23 bases. For example, in the case of SEQ ID NO: 4, it may be 15 bases or more, more preferably 16 bases or more, more preferably 17 bases or more, more preferably 18 bases or more, and most preferably 19 bases.
Furthermore, the primer pair for amplifying each of the above three bacterial species may have several bases added to either end, as long as it includes the base sequence described in any of the above. By adding a base to any terminal, the total length of the primer is preferably 18 bases or more, more preferably 20 bases or more, and more preferably 23 bases or more. In this case, the upper limit of the total primer length is preferably 35 bases, more preferably 32 bases, and even more preferably 30 bases.

  The primer concentration used in the present invention is not limited and may be adjusted as appropriate. However, it is preferable to lower the primer ratio that gives an amplification product that increases the peak and to balance the peak. If necessary, the concentration of the primer is set higher than that in the case of performing normal PCR.

  In the present invention, it is more preferable to include an internal control (internal standard) in order to prevent the occurrence of false negatives. In the negative case, only the amplification of the internal control (internal standard) is observed, and it can be confirmed that the PCR was successful. On the other hand, when PCR inhibition or forgetting to add a reagent occurs, amplification of the internal control (internal standard) is not recognized, so it can be confirmed that PCR has failed.

  The internal control (internal standard) is not particularly limited as long as Tm values or amplification lengths of Salmonella, enterohemorrhagic Escherichia coli (EHEC), and Shigella are different. If the Tm value is different, the Tm value of the internal control (internal standard) may be any one that gives a Tm value different from any of the amplification products of Salmonella, enterohemorrhagic Escherichia coli (EHEC), and Shigella. It may be lower or higher than the Tm value of all the bacterial species. Moreover, the intermediate | middle of either of three bacterial species may be sufficient. If the amplification length is different, the amplification length of the internal control (internal standard) is shorter or shorter than that of Salmonella, enterohemorrhagic Escherichia coli (EHEC), and Shigella. Also good. Moreover, the intermediate | middle of either of three bacterial species may be sufficient.

  As an internal control (internal standard), a target not included in the test target and a primer set for amplifying the target may be used. In addition, a template that gives a Tm value or amplification length different from the three bacterial species when amplified using Salmonella, enterohemorrhagic Escherichia coli (EHEC), and Shigella amplification primers without adding a primer for internal control. It is good also as an internal control (internal standard).

In the test method of the present invention, the composition during the multiplex PCR reaction is not particularly limited as long as the positive rate is satisfied, and conventionally known components are included, and the ratio thereof is not limited. As the composition component, at least one selected from the group consisting of a heat-resistant DNA polymerase, a DNA synthesis substrate (for example, one or more types of deoxynucleotide triphosphates or derivatives of deoxynucleotide triphosphates), a buffer, and a salt. It is preferable to contain.

The thermostable DNA polymerase is not particularly limited, and a conventionally known thermostable bacteria-derived polymerase can be used. Specifically, Taq DNA polymerase and Tth DNA polymerase belonging to family A (PolI type), KOD DNA polymerase, Pfu DNA polymerase, Pwo DNA polymerase, Ultimate DNA polymerase, PrimeSTAR (registered trademark) belonging to family B (α type) Examples include DNA polymerase. The thermostable DNA polymerase may be a mutant having an amino acid sequence in which one or several amino acids are substituted, deleted, added or inserted in the amino acid sequence, and is not particularly limited.

Examples of the buffer include, for example, Tris (TRIS), Tricine (TRICINE), Bis-Tricine (BIS-TRICINE), Hepes (HEPES), Mops (MOPS), Tes (TES), Tapes (TAPS), Pipes (PIPES). ), And caps (CAPS), but are not particularly limited.
The reaction buffer preferably contains Mg ²⁺ at a concentration of about 1.0 to 5 mM, preferably about 1.5 to 2.5 mM. Furthermore, KCl may be included.
Moreover, you may contain surfactant as needed.

  Furthermore, the reaction buffer may contain albumin, glycerol, heparin, trehalose, betaine and the like. What is necessary is just to add these addition ratios in the range which does not inhibit PCR reaction.

When the detection method is a melting curve analysis using a fluorescent intercalator, a fluorescent intercalator is added. The fluorescent intercalator may be added in advance to the PCR reaction solution or may be added after PCR, but it is preferably added in advance to the PCR reaction solution from the viewpoint of simplicity.

A fluorescent intercalator is any molecule that binds to a nucleic acid in a reversible, non-covalent manner by insertion (intercalation) into double-stranded DNA, thereby indicating the presence and quantity of the nucleic acid. Point to. In general, an intercalator is a dye that emits fluorescence when inserted into double-stranded DNA.

  A number of intercalators are known in the art. For example, Ethidium bromide, cyanine dyes (eg, TOTO (registered trademark), YOYO (registered trademark), BOBO and POPO), SYBR (registered trademark) Green I, SYBR (registered trademark) Green ER, SYBR (registered trademark) Green Gold , SYBR (R) DX, PicoGreen (R), LCGreen (R), EvaGreen (R), SYTOX (R) Green, ResoLight, propidium iodide, Acidine orange, 7-amino-actinomycin D, CyQUANT (registered trademark) GR, SYTO (registered trademark) 9, SYTO (registered trademark) 10, SYTO (registered trademark) 13, SYTO (registered trademark) 14, SYTO (registered trademark) 2, etc. FUN-1, and the like, but not particularly limited.

  In order to separate and detect Salmonella, enterohemorrhagic Escherichia coli (EHEC), and Shigella in the present invention, an instrument capable of melting curve analysis is used. A device capable of performing a melting curve analysis includes, but is not limited to, a real-time PCR device. Specific examples include Light Cycler (registered trademark) of Roche Diagnostics, ABI PRISM (registered trademark) 7000/7700, 7500/7500 FAST real-time PCR system, 7900HT Fast real-time PCR system, StepOne / StepOnePlus real-time PCR system, Qiagen's Rotor Gene, Takara Bio's Thermal Cycler Dice (registered trademark) Real Time System, Bio-Rad's MiniOpticon, CFX384 Touch, CFX96 000M Is mentioned. As for the PCR reaction, the above-mentioned real-time PCR apparatus may be used, but a thermal cycler may be used independently of the melting curve analysis.

  The temperature cycle in PCR can be appropriately set according to the primers used. In addition, for melting curve analysis, the temperature range of the acquired data is Salmonella, enterohemorrhagic Escherichia coli (EHEC), Shigella, internal control (internal standard) Tm value of internal control (internal standard) The temperature rise rate (number of data points) and the like may be set to conditions suitable for each real-time PCR apparatus.

Hereinafter, based on an Example, this invention is demonstrated more concretely. However, the present invention is not limited to the following examples.

Example 1: Detection of three bacterial species from genomic DNA and positive specimens Primer set capable of separation and detection by Tm value as a primer for detecting three bacterial species of Salmonella, enterohemorrhagic Escherichia coli (EHEC), Shigella 1-9) was designed. The primers described in SEQ ID NOs: 1 and 2 are salmonella amplification forward primers, and the primer described in SEQ ID NO: 3 is a salmonella amplification reverse primer. The primers described in SEQ ID NOs: 4 and 5 are forward primers for amplifying enterohemorrhagic Escherichia coli (EHEC), and the primers described in SEQ ID NOs: 6 and 7 are reverse primers for amplifying enterohemorrhagic Escherichia coli (EHEC). The primer described in SEQ ID NO: 8 is a forward primer for amplifying Shigella, and the primer described in SEQ ID NO: 9 is a reverse primer for amplifying Shigella. In order to prevent false positives, primers for internal control amplification (SEQ ID NO: 10 and SEQ ID NO: 11) were designed to have a Tm value lower than that of Salmonella, enterohemorrhagic Escherichia coli (EHEC), and Shigella. . This internal control amplification primer uses pBR322 as a template.

In this example, Blend Taq-Plus- (TOYOBO) was used as a PCR reagent, and EvaGreen (Biotium) was used as a fluorescent intercalator. In addition, the primer concentration was equally distributed and added so that the primer concentration for detecting each bacterium was 1.2 μM. That is, as a primer set for detecting Salmonella, enterohemorrhagic Escherichia coli (EHEC), and Shigella, 3.6 μM of primer is included in the PCR reaction solution. The primer concentration for internal control amplification was 0.5 μM each for the forward primer and the reverse primer, and 2.5 μM / μL pBR322 was added as a template.

In this example, the PCR reaction solution was prepared in a 20 μL system. To this 20 μL reaction solution, a positive real specimen as a test object was suspended in water so that the concentration was about 10%, and 2 μL of the supernatant liquid after heat treatment at 95 ° C. for 5 minutes and centrifugation was added. As a control, amplification was performed using purified Salmonella, enterohemorrhagic Escherichia coli O157, and Shigella genomic DNA 100 copies as a template instead of feces.

  The prepared sample was subjected to an initial denaturation at 94 ° C. for 2 minutes in Bio-Rad MiniOpticon, followed by 40 cycles of denaturation: 94 ° C., 10 seconds, annealing: 55 ° C., 15 seconds, extension: 68 ° C., 30 seconds. After performing PCR, melting curve analysis was performed at 0.5 ° C./Step.

  These results are shown in FIG. Salmonella, enterohemorrhagic Escherichia coli (EHEC), and Shigella were able to be detected, and each could be detected separately.

Example 2: Detection of three bacterial species from pooled stool In this example, pooled stool containing one positive real sample was examined under the PCR reaction solution and cycle conditions described in Example 1. Pool stool is prepared by suspending a small amount of stool in a stool collection tube in a test tube containing sterilized water (about 1 mL) for each stool collection tube. The stool specimen pooled in one tube (about several μL) was heat-treated at 95 ° C. for 5 minutes, and the supernatant after centrifugation was used.

  These results are shown in FIG. As a result, the same result was obtained for pooled stool, and it was shown that it was possible to test multiple samples more easily and quickly.

Example 3: Confirmation of positive rate and verification of its effectiveness 4700 real specimens were pooled 50 by the method described in Example 2. 94 pooled cases were inspected under the conditions described in Example 1. In order to verify the effectiveness, all 4,700 cases were individually examined by the smear culture method.

  As a result, 5 cases of Salmonella and 3 cases of enterohemorrhagic Escherichia coli were determined to be positive, and the positive rate by multiplex PCR was 8/4700 samples, which was 0.17%. From this result, it was confirmed that the primer design and reaction solution of this example satisfy the positive rate condition.

  Therefore, this effectiveness was verified. As a result of examining 4700 specimens by the smear culture method, 4300 specimens for 86 pools judged negative by the PCR method were judged negative. Moreover, positive samples were recognized in 2 pools out of 8 pools determined to be PCR positive. From this result, it was confirmed that false negatives did not occur in the multiplex PCR method, and the effectiveness of the multiplex PCR screening using pooled stool was confirmed.

  In the case of the present example, since there are 8 multiplex PCR positives in 94 pools, the ratio that requires smear culture is 8 × 50/4700 = 8.5% with respect to the total number of samples. . This value was achieved by using a multiplex PCR method in which the positive rate was controlled, and the result was sufficiently significant in terms of efficiency of the screening method by the multiplex PCR method.

As a method for examining the presence or absence of Salmonella, enterohemorrhagic Escherichia coli and Shigella in healthy individuals, a multiplex PCR method with a positive rate (positive number / total number of samples) of 0.5% or less can be used. The efficiency of the primary screening method for carriers using the plex PCR method could be improved. Thereby, since it is possible to increase the number of negative samples that can be confirmed in several hours, a rapid and efficient test report can be expected. Furthermore, in the present invention, since detection can be performed quickly and efficiently, it can be expected to be used in industrial applications that require processing of a large number of samples.

Claims (12)

A method for examining the presence or absence of any one or more of Salmonella, enterohemorrhagic Escherichia coli and Shigella using a multiplex PCR method, comprising the following (1) and A test method comprising the step (2) and having a positive rate of 0.5% or less.
(1) A step of performing multiplex PCR targeting the three bacterial species (2) A step of analyzing the obtained amplification product and detecting the presence or absence of any one or more of the three bacterial species The test | inspection method of Claim 1 whose positive rate is 0.2% or less. The test method according to claim 1 or 2, wherein the test object is a stool specimen. The test method according to any one of claims 1 to 3, wherein the test object is a pooled sample of stool samples collected from stool of X types (X represents an integer of 2 or more). The inspection method according to claim 4, wherein X is 20 or more. The inspection method according to any one of claims 1 to 5, wherein the detection method in the step (2) is a melting curve analysis using a fluorescent intercalator. The method according to any one of claims 1 to 6, wherein in the step (2), three species of Salmonella, enterohemorrhagic Escherichia coli (EHEC), and Shigella are separated and detected based on differences in Tm values of amplification products. The detection method according to claim 1, further comprising an internal control (internal standard) that gives a Tm value different from any of the amplification products of the three bacterial species in step (2). The detection method according to claim 7, wherein in step (1), multiplex PCR is performed using three primer pairs designed so that Tm values of the amplification products of the three bacterial species are separated from each other by 1 ° C. or more. . The detection method according to claim 9, wherein the primer set is a combination of three primer pairs each selected one by one from the following three groups (I) to (III).
(I) Salmonella amplification primer pair (a) represented by the following (a) or (b) (a) “an oligonucleotide consisting of 15 bases or more in SEQ ID NO: 1” and “oligo consisting of 15 bases or more in SEQ ID NO: 2” Complementary bases to each base sequence of the primer pair of oligonucleotide pair (b) (a) consisting of at least one of the group consisting of “nucleotide” and “oligonucleotide consisting of 15 bases or more of SEQ ID NO: 3” Oligonucleotide pair having sequence (II) Primer pair for intestinal hemorrhagic Escherichia coli (EHEC) amplification represented by (c) or (d) below
(C) at least one of the group consisting of “an oligonucleotide consisting of 15 bases or more in SEQ ID NO: 4” and “an oligonucleotide consisting of 15 bases or more in SEQ ID NO: 5”; Each nucleotide sequence of the primer pair of oligonucleotide pair (d) (c) consisting of at least one of the group consisting of “an oligonucleotide consisting of 15 bases or more” and “an oligonucleotide consisting of 15 bases or more of SEQ ID NO: 7” Oligonucleotide pair (III) having a complementary base sequence to the primer pair for amplification of Shigella shown by (e) or (f) below
(E) each of the primer pair of the oligonucleotide pair (f) (e) consisting of “oligonucleotide consisting of 15 bases or more in SEQ ID NO: 8” and “oligonucleotide consisting of 15 bases or more in SEQ ID NO: 9” Oligonucleotide pair having a base sequence complementary to the base sequence A reagent for examining the presence or absence of at least one of Salmonella, enterohemorrhagic Escherichia coli (EHEC), and Shigella, comprising the following (A) to (D).
(A) thermostable DNA polymerase (B) primer set according to any of [9] or [10] (C) DNA synthesis substrate (deoxynucleotide triphosphate (dNTP)), and
(D) Buffer solution containing one or more of the group consisting of magnesium ion, ammonium ion and potassium ion Furthermore, the reagent of Claim 11 containing the following (E).
(E) Fluorescent intercalator