JP2018093756A - Improved method for screening enterobacteria - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a screening method for enterobacteria which can improve the screening efficiency of fecal specimen examination, early determine negative specimens, and further narrow down positive specimens.SOLUTION: The method includes the following steps (1) to (5) and is for examining for the existence of any of three bacterial strains from Salmonella, intestinal hemorrhagic E. coli and Shigella in a fecal sample: (1) a step of pooling X stool sample specimen suspensions (X is an integer of 10 or more) to prepare pool feces; (2) a step of performing primary screening by multiplex PCR using pooled feces prepared in (1); (3) a step of selecting pool faeces in which any one of the three bacterial species has become positive in (2); (4) a step of preparing pool feces consisting of Y feces (Y is an integer smaller than X) stool specimen suspension using individual feces used for pool feces selected in (3); (5) a step of conducting secondary screening by PCR using pools feces produced in (4).SELECTED DRAWING: None

Description

The present invention relates to a screening method for food poisoning causative bacteria in a stool sample by nucleic acid amplification.

The nucleic acid amplification method is a technique for amplifying several copies of a target nucleic acid to a visualization level, that is, several hundred million copies or more. A typical nucleic acid amplification method is PCR (Polymerase Chain Reaction). PCR includes (1) DNA denaturation by heat treatment (dissociation from double-stranded DNA to single-stranded DNA), (2) annealing of primer to template single-stranded DNA, (3) the primer using DNA polymerase This is a method of amplifying a target nucleic acid in a sample by repeating three cycles of three steps of extension as one cycle.

Tests using PCR technology are widely used in the field of forensic medicine such as genetic diagnosis and clinical diagnosis, or microbiological tests in foods and the environment. For the detection of PCR amplification products, a real-time PCR method that can easily detect a small amount of nucleic acid without performing electrophoresis is widely used.

  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.

  In order to solve the problems of the smear culture method, a detection method using the PCR method has been developed (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 has been amplified by whether or not the target DNA has been amplified. Therefore, there is an advantage that bacteria can be detected objectively.

The stool test for food poisoning causal bacteria is generally performed by first screening by the PCR method, removing the negative sample, and then performing a definite test for the positive sample by the smear culture method (Non-patent Document 2). . In addition, in the stool test of healthy people, the retention rate of food poisoning causative bacteria is very low, and the majority of specimens are negative. In order to make a rapid determination, pooled stool is generally used for screening by the PCR method, with about 10 to about 50 samples as one pool (Patent Document 1). After screening from the pool stool by PCR, positive specimens are determined by smearing each specimen contained in the positive pool individually.

Screening by the PCR method is a method of detecting the presence or absence of food poisoning causative bacteria by omitting the DNA extraction operation from the stool sample and subjecting the sample suspension subjected to heat treatment to PCR. At this time, by omitting DNA extraction, a PCR reaction inhibiting substance such as a polysaccharide contained in the stool sample is brought in. Therefore, various ideas have been made to reduce these effects.

Reagents for stool examination by PCR for Salmonella, enterohemorrhagic Escherichia coli, Shigella are “Intestinal pathogen gene detection reagent kit” manufactured by Shimadzu Corporation, “TaKaRa enteropathogenic bacteria gene detection kit” manufactured by Takara Bio. Toyobo's “Intestinal Bacterial Gene Detection Kit—High-Speed Fluorescence Detection” is commercially available.

These commercial kits perform melting curve analysis after the PCR reaction, and determine the positive bacterial species based on the difference in Tm value of the melting curve analysis result. In melting curve analysis, when a peak corresponding to a certain bacterial species is detected and a positive determination is made, it is impossible to determine that a bacterial species that has not been detected is a negative determination (Non-Patent Document 3). This is because, in a pooled stool (co-infected pool) containing a plurality of bacterial species, it may not be detected due to superiority or inferiority of amplification. Therefore, it was necessary to smear each specimen contained in the positive pool on each bacterial species detection medium.

  In addition, these commercially available kits are screened using 50 specimens as one pool. After screening by PCR, positive specimens are determined by smearing each specimen contained in the positive pool individually. The positive rate by screening using a commercially available PCR kit is about 10%, and about 90% of negative samples can be determined. However, since 50 samples are pooled, even if only positive pools are selected, 98% of them are negative samples. Therefore, screening efficiency is improved, early determination of negative samples and further narrowing down of positive samples are required.

Japanese Patent Laying-Open No. 2006-042802

TaKaRa BIO VIEW No. 55, page 48 (issued in 2008) Journal of Infectious Diseases Volume 86: 741-748 (issued in 2012) Enteric Bacterial Gene Detection Kit-High Speed Fluorescence Detection-Instruction Manual (December 2015)

  An object of the present invention is to improve screening efficiency of a stool test, to early determine negative samples, and to further narrow down positive samples.

  In light of the above circumstances, the present inventors conducted extensive research, and after conducting primary screening using multiplex PCR method from pool feces, by carrying out secondary screening only for positive pools, It was found that it is possible to narrow down positive specimens easily. Further, by adopting a method capable of separating and detecting each bacterial species using a fluorescent probe for primary screening, it has been found that screening can be performed more efficiently, and the present invention has been completed.

That is, the present invention has the following configuration.
Item 1. A method for examining the presence or absence of any one of three species of Salmonella, Enterohemorrhagic Escherichia coli (EHEC) and Shigella in a stool sample, comprising the following steps (1) to (5) An inspection method characterized by comprising.
(1) Pooling stool specimen suspensions of X pieces (X represents an integer of 10 or more) and producing pooled stool (2) Using the pooled stool prepared in step (1), Step (3) of performing primary screening by multiplex PCR targeting three bacterial species (3) Step of selecting pool feces in which any one of the three bacterial species is positive (4) ) Using the individual stool used for the pool stool selected in step (3), further creating a pool stool comprising Y stool specimen suspensions (Y is an integer smaller than X) (5) A step of performing secondary screening by PCR using one of the three bacterial species as a target, using the pooled stool prepared by the step (4). Item 2. The inspection method according to Item 1, wherein in the multiplex PCR in step (2), a positive bacterial species is identified by utilizing a difference in fluorescence wavelength of the fluorescent probe.
Item 3. The test method according to Item 1 or 2, wherein the primary screening method separates and detects each bacterial species from pooled feces in which two or more of the three bacterial species are present.
Item 4. Item 4. The inspection method according to any one of Items 1 to 3, wherein X is an integer of 20 or more.
Item 5. Item 5. The inspection method according to any one of Items 1 to 4, wherein Y is an integer of 30 or less.
Item 6. Item 6. The test method according to any one of Items 1 to 5, wherein the reagent used for the multiplex PCR in step (2) contains an internal standard gene.
Item 7. Item 7. The inspection method according to any one of Items 1 to 6, wherein the reagent used for PCR in the step (5) contains an internal standard gene.

  With the screening method of the present invention, more negative specimens can be determined early. In addition, since the number of smear cultures can be reduced, the efficiency of the inspection can be improved.

FIG. 3 is a diagram of an amplification curve in Example 1. FIG. 6 is a diagram of an amplification curve in Example 2. FIG. 10 is a diagram of an amplification curve in Example 3. It is a figure of the amplification curve in Example 4.

Hereinafter, the present invention will be described in more detail while showing embodiments of the present invention.

The inspection method of the present invention includes the following steps (1) to (5). As a result, the presence or absence of any one of the three bacterial species of Salmonella, Enterohemorrhagic Escherichia coli (EHEC), and Shigella in the stool sample can be efficiently examined.
(1) Pooling stool specimen suspensions of X pieces (X represents an integer of 10 or more) and producing pooled stool (2) Using the pooled stool prepared in step (1), Step (3) of performing primary screening by multiplex PCR targeting three bacterial species (3) Step of selecting pool feces in which any one of the three bacterial species is positive (4) ) Using the individual stool used for the pool stool selected in step (3), further creating a pool stool comprising Y stool specimen suspensions (Y is an integer smaller than X) (5) A step of performing secondary screening by PCR targeting any one of the three bacterial species using the pool feces prepared in step (4)

  As a sample to be used in the PCR reaction solution, a food poisoning cause is obtained by performing a heat treatment from a stool sample such as a stool test at 75 ° C. to 100 ° C., more preferably 85 ° C. to 95 ° C. for 30 seconds to 20 minutes, more preferably 3 minutes to 10 minutes. Although the sample which inactivated the microbe is mentioned, it does not specifically limit.

Furthermore, when multiple specimens need to be examined, a pooled stool in which a plurality of specimens are pooled is used. The addition ratio of the stool test sample used for PCR is not particularly limited as long as PCR can be appropriately performed. For example, it can be appropriately adjusted within a range of 1 to 20 v / v%. In the present invention, the pooled stool comprises a stool suspension of type A (A represents an integer of 2 or more) suspended in water at 1 to 20 v / v%.

Here, the A-type stool may be each stool excreted by the A-type individual or A-type stool excreted by the same individual. Although it does not specifically limit about A, A is 10 or more, Preferably A is 20 or more, More preferably, it is 30 or more.

  In the present invention, the PCRs in steps (2) and (5) are preferably performed under the same temperature cycle conditions. The preferred PCR reaction time is within 1 hour, preferably within 50 minutes, more preferably within 40 minutes.

The PCR reaction solution used in the present invention includes a buffer, an appropriate salt, a magnesium salt or a manganese salt, deoxynucleotide triphosphate, a detection target region of a food poisoning bacterium to be detected, and a primer set and fluorescence corresponding to the internal standard gene. A probe and, if necessary, an additive may be contained.

In the PCR reagent used in the test method of the present invention, it is more preferable to include an internal control (internal standard) in the PCRs of steps (2) and (5) 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.

  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, only a template having an amplification region different from the three bacterial species when amplified using primers for amplification of Salmonella, Enterohemorrhagic Escherichia coli (EHEC), Shigella, is not added for internal control. However, internal control (internal standard) may be used.

The primer used in the present invention is not particularly limited, but a primer having 15 to 30 bases, preferably 18 to 25 bases, is preferably used. If necessary, a complementary strand of the base sequence described in the sequence listing may be used. Moreover, when the target nucleic acid consists of subtypes, a degenerate primer may be included.

The fluorescent probe used in the present invention is not particularly limited. In the present invention, it is preferable to use a fluorescent probe of 15 to 30 bases, preferably 18 to 25 bases. Moreover, when the target nucleic acid consists of subtypes, a degenerate probe may be included.

The fluorescent dye used as the fluorescent probe is not particularly limited. For example, TaqMan (registered trademark) hydrolysis probe (US Pat. No. 5,210,015, US Pat. No. 5,538,848, US Pat. No. 5,487,972 and US Pat. No. 5,804,375).

The primary screening in the present invention refers to a step of screening using PCR from a pooled stool of X stool specimens. X is not particularly limited as long as it is 10 or more, but is preferably 30 or more, more preferably 40 or more. The upper limit value of X is not particularly limited, but is preferably about 100.

The PCR reagent used for the primary screening is not particularly limited, but is preferably a reagent that can simultaneously detect three species of Salmonella, Enterohemorrhagic Escherichia coli (EHEC), and Shigella in a multiplex. As a preferred mode, the fluorescence wavelength of the fluorescent probe for detecting the internal standard gene and the fluorescence wavelength of the fluorescent probe for detecting food poisoning cause bacteria do not overlap, and the detection can be performed by another channel. More preferably, the fluorescent dye is selected so that the fluorescence wavelength of the fluorescent probe for detecting the internal standard gene and the fluorescence wavelength of each of the fluorescent probes for detecting Salmonella, enterohemorrhagic Escherichia coli, and Shigella are not overlapped.

The multiplex PCR in the primary screening is preferably a method for identifying positive bacterial species by utilizing the difference in fluorescence wavelength of the fluorescent probe. Moreover, the primary screening method isolate | separates and detects each bacterial species from the pool feces in which 2 or more types of bacterial species among the said 3 bacterial species exist. It may be.

The secondary screening in the present invention refers to a step of screening using PCR from a pooled stool of Y stool specimens. The Y stool specimens selected here are selected from the stool specimens included in the pool that became positive in the primary screening. Y is an integer smaller than X and is not particularly limited, but Y is preferably 30 or less. More preferably, it is 20 or less, More preferably, it is 10 or less. Furthermore, Y may be 1, and can be applied to individual screening. It is performed for the purpose of further narrowing down positive samples from stool samples contained in the pool that became positive in the primary screening.

The PCR reagent used for the secondary screening is not particularly limited. Preferably, there is no particular limitation as long as it has a reagent configuration capable of detecting any one species selected from Salmonella, Enterohemorrhagic Escherichia coli (EHEC), and Shigella.

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

Example 1. Separation and detection example of co-infection pool model (1) Using enterobacterial gene detection kit-high-speed fluorescence detection- (TOYOBO), which is a commercially available melting curve analysis type food poisoning causative detection kit, Detection of a co-infection pool model was performed. According to the recommended protocol, each food poisoning causal bacteria DNA was detected. As a model of the co-infection pool, 10000 copies and 10 copies of DNA of two species selected from Salmonella, Enterohemorrhagic Escherichia coli, and Shigella were added, respectively, and both food poisoning causative bacteria DNA were detected.

(2) Results An amplification curve is shown in FIG. In any co-infection pool model of any food poisoning causative DNA combination, it was impossible to detect 10 copies of the gene.

Example 2. Separation and detection example of co-infection pool model using fluorescent probe method (1) Enterobacteriaceae gene detection kit-probe detection- (TOYOBO), which is a detection kit of food poisoning causal bacteria using a commercially available fluorescent probe The detection of the co-infection pool model was performed. According to the recommended protocol, each food poisoning causal bacteria DNA was detected. As a co-infection pool model, two types of bacterial species selected from Salmonella, enterohemorrhagic Escherichia coli, and Shigella were added in an amount of 10,000 copies and 10 copies, respectively.

(2) Results An amplification curve is shown in FIG. In any co-infection pool model of any combination of food poisoning causal bacteria, both 10000 and 10 copies of the gene could be isolated and detected simultaneously. Compared with the melting curve analysis type detection kit, it was shown that the detection kit using the fluorescent probe method has a higher ability to separate and detect co-infected specimens.

Example 3 FIG. Primary screening using pooled stool samples (1) Preparation of pooled stool samples
A pooled stool heat treatment specimen was prepared using 9,000 human stool specimens. Samples suspended at 10% (weight%) were mixed in 50 equal amounts, heat-treated at 95 ° C. for 10 minutes, centrifuged at 12,000 rpm for 5 minutes, and 180 pool fecal heat-treated samples were prepared. .

(2) Reaction The primary screening was performed by the multiplex PCR method targeting Salmonella, enterohemorrhagic Escherichia coli, and Shigella using the enterobacterial gene detection kit-probe detection- according to the recommended protocol.

(3) Results The amplification results are shown in FIG. Of the 180 pools, 15 pools were determined to be positive, and the positive rate was 8.3%. Of the 15 pools, 6 specimens were positive for Salmonella and 9 pools were positive for enterohemorrhagic E. coli. Of 9,000 samples, 91.7% negative samples were confirmed by primary screening.

Example 4 Secondary screening using pooled fecal samples (1) Preparation of pooled fecal samples
Ten samples were re-pooled from 300 samples contained in 6 pools determined to be positive for Salmonella in Example 1 to produce 30 Salmonella positive suspected pools. Similarly, 10 specimens were re-pooled from 450 specimens contained in 9 pools determined to be positive for enterohemorrhagic Escherichia coli to prepare 45 enterohemorrhagic Escherichia coli positive pools.

(2-1) Preparation of reaction solution A PCR reaction solution having the following composition was prepared, and Salmonella and enterohemorrhagic Escherichia coli were detected. A primer / probe solution for detecting Salmonella was used for the Salmonella positive suspected pool, and a primer / probe solution for detecting enterohemorrhagic Escherichia coli was used for the enterohemorrhagic Escherichia coli positive suspected pool.
1xBuffer (Blend Taq (registered trademark) attachment buffer (Toyobo))
3mg / ml BSA
Each 0.2 mM dATP, dGTP, dCTP, dTTP
1% gelatin 0.1 unit / μl rTth DNA polymerase (Toyobo)
0.02 μg / μl Anti-Tth antibody Salmonella detection primer / probe solution (Table 1)
Probe / probe solution for detecting enterohemorrhagic Escherichia coli (Table 2)

(3) Result Amplification results are shown in FIG. Of the 75 suspected positive pools, 18 were positive and the positive rate was 36%. Eight pools were positive for Salmonella and 10 were positive for enterohemorrhagic E. coli.
From the results of Example 3 and Example 4, out of 9,000 samples, the suspected positive samples could be narrowed down to 180 samples by combining the primary screening and the secondary screening, and 98% of the samples were negative.

  INDUSTRIAL APPLICABILITY The present invention is very useful in the field of forensic medicine such as genetic diagnosis and clinical diagnosis, or in a field in which a large number of samples are required to be examined quickly, such as in microbial tests in foods and the environment.

Claims (7)

A method for examining the presence or absence of any one of three species of Salmonella, Enterohemorrhagic Escherichia coli (EHEC) and Shigella in a stool sample, comprising the following steps (1) to (5) An inspection method characterized by comprising.
(1) Pooling stool specimen suspensions of X pieces (X represents an integer of 10 or more) and producing pooled stool (2) Using the pooled stool prepared in step (1), Step (3) of performing primary screening by multiplex PCR targeting three bacterial species (3) Step of selecting pool feces in which any one of the three bacterial species is positive (4) ) Using the individual stool used for the pool stool selected in step (3), further creating a pool stool comprising Y stool specimen suspensions (Y is an integer smaller than X) (5) A step of performing secondary screening by PCR targeting any one of the three bacterial species using the pool feces prepared in step (4) The test method according to claim 1, wherein in the multiplex PCR of step (2), a positive bacterial species is identified by utilizing a difference in fluorescence wavelength of the fluorescent probe. The test method according to claim 1 or 2, wherein the primary screening method separates and detects each bacterial species from pooled feces in which two or more of the three bacterial species are present. The inspection method according to claim 1, wherein X is an integer of 20 or more. The inspection method according to claim 1, wherein Y is an integer of 30 or less. The test method according to any one of claims 1 to 5, wherein the reagent used for the multiplex PCR in step (2) contains an internal standard gene. The test method according to any one of claims 1 to 6, wherein the reagent used for PCR in the step (5) contains an internal standard gene.