JP5224541B2 - Oligonucleotides for detecting Salmonella Hadar and rapid serotype identification method using them - Google Patents

Description

  The present invention relates to a rapid identification method of Salmonella serotype by multiplex detection of serotype-specific genes.

  Salmonella infection is not only a hindrance to livestock production but also causes human food poisoning through livestock products and is a public health threat. Since Salmonella is transmitted to livestock through feed, it is defined as a harmful microorganism by the feed safety law. For this reason, the Agricultural, Forestry and Fisheries Consumption Safety Technology Center and Fertilizer and Feed Safety Inspection Department are continuously conducting monitoring surveys of salmonella contamination in feed. Whether the isolated Salmonella is one of the six major serotypes (Gallinarum, Typhimurium, Dublin, Enteritidis, Choleraesuis, Abortusequi) defined as “livestock infectious disease” or “reported infectious disease” in the Livestock Infectious Disease Prevention Law Since subsequent responses differ, serotype identification is essential, but the current Salmonella serotyping method requires about two weeks to determine. For this reason, even if the said 6 serotype Salmonella is detected from a feed, the possibility that the said feed will be consumed before the determination result comes out is high. In addition, the slaughterhouse law was revised in 2004, and it was stipulated that the carcasses should be completely discarded if pathogens contained in surveillance infectious diseases were isolated. For this reason, in the meat hygiene inspection laboratory, it became necessary to test whether or not the above six serotypes were separated from materials in which gross lesions were observed. It takes about two weeks for the test results to come out, and during this time it is necessary to hold the carcass, which is a major public health problem. For these reasons, there is a strong demand for the development of a rapid identification method for major Salmonella serotypes in the field of feed inspection and meat hygiene inspection. If a rapid identification method for major Salmonella serotypes isolated from humans and livestock is put into practical use, it can be applied to daily inspections at livestock health centers, identification of serotype-impossible bacteria, screening tests, etc. It is an extremely effective tool for ensuring the safety and security of things.

JP2007-274934

Kim H.J. et al. 2006. Identification of Salmonella enterica serovar Typhimurium using specific PCR primers obtained by comparative genomics in Salmonella serovars. J. Food Prot. 69: 1653-1661.

Salmonella is classified into 1 genus, 2 bacterial species, and 6 subspecies based on biochemical properties, DNA homology, and the like. Apart from this classification under the international naming convention, Salmonella has established serotype classification based on the combination of fungus (O) antigen and flagellar (H) antigen from an early stage, and more than 2,500 serotypes have been reported to date. . Usually, one week is required for the isolation and identification of Salmonella, and if serotyping is performed, one week is required, that is, a total of two weeks are required to identify the serotype.
At the Agriculture, Forestry and Fisheries Consumption Safety Technology Center and meat hygiene inspection laboratories in each prefecture, Salmonella was isolated and identified in one week, and the group O serotype was determined. At this time, the present inventors can use a PCR method or the like together to detect a serotype-specific gene, thereby reducing the test time, which conventionally required two weeks, to one week. I thought. That is, an object of the present invention is to provide oligonucleotides for detecting Salmonella and a rapid serotype identification method using them.

Of the over 2,500 Salmonella serotypes, more than 1,500 serotypes are classified as subspecies I, including most of the serotypes that often cause infections in humans and livestock. Therefore, Salmonella we encounter daily have extremely high genetic similarity, and this has become a barrier to developing serotyping methods using differences in nucleotide sequences. On the other hand, more than 10 years have passed since the first report of the whole genome sequence of bacteria, and hundreds of bacteria have been reported. Even in Salmonella, the entire genome sequence of more than 20 strains can be referenced.
Therefore, in the present invention, genes specific to Salmonella major serotypes are comprehensively extracted in silico (on a computer), and over 3,700 Salmonella field isolates owned by the present inventors are used. By verifying its specificity, we tried to develop a rapid identification method for Salmonella serotypes using PCR.

Specifically, three genes with high specificity for each of the three serotypes of serotypes Salmonella Typhimurium (ST), Salmonella Hadar (SH), and Salmonella Infantis (SI) were selected by comparative genome analysis in silico . We established a multiplex PCR system that simultaneously detects these genes and four genes of invA known to be specific for Salmonella. When the specificity of the multiplex PCR method as the above three serotype identification method was verified using a field isolate, all three serotype-specific genes were amplified in the case of the serotype. There were no cases of positive 3 genes in the type, with some exceptions. That is, it was shown that the method of the present invention has sufficient specificity. By using this multiplex PCR method when Salmonella was isolated, it became possible to identify these three serotypes within one week from the start of the separation culture.

The present invention is based on such knowledge, and more specifically, relates to the invention described in [1] to [24] below.
[1] Any one set described in the following (1) to (10);
(1) an oligonucleotide comprising the base sequence described in SEQ ID NO: 1 and a set of oligonucleotides comprising the base sequence described in SEQ ID NO: 2,
(2) a set of oligonucleotides containing the base sequence set forth in SEQ ID NO: 3 and oligonucleotides containing the base sequence set forth in SEQ ID NO: 4;
(3) an oligonucleotide containing the base sequence set forth in SEQ ID NO: 5 and a set of oligonucleotides containing the base sequence set forth in SEQ ID NO: 6;
(4) a set of oligonucleotides containing the base sequence set forth in SEQ ID NO: 7 and oligonucleotides containing the base sequence set forth in SEQ ID NO: 8;
(5) an oligonucleotide comprising the base sequence set forth in SEQ ID NO: 9 and an oligonucleotide set comprising the base sequence set forth in SEQ ID NO: 10;
(6) an oligonucleotide comprising the base sequence set forth in SEQ ID NO: 11 and an oligonucleotide set comprising the base sequence set forth in SEQ ID NO: 12;
(7) an oligonucleotide comprising the base sequence set forth in SEQ ID NO: 13 and an oligonucleotide set comprising the base sequence set forth in SEQ ID NO: 14;
(8) a set of oligonucleotides containing the base sequence set forth in SEQ ID NO: 15 and oligonucleotides containing the base sequence set forth in SEQ ID NO: 16;
(9) an oligonucleotide comprising the base sequence set forth in SEQ ID NO: 17 and an oligonucleotide set comprising the base sequence set forth in SEQ ID NO: 18;
(10) A set of an oligonucleotide containing the base sequence set forth in SEQ ID NO: 19 and an oligonucleotide containing the base sequence set forth in SEQ ID NO: 20.
[2] Three sets of [1] (2) to (4).
[3] Three sets according to (5) to (7) of [1].
[4] Three sets according to (8) to (10) of [1].
[5] Six sets according to (2) to (7) of [1].
[6] A set of 6 sets according to (2) to (4) and (8) to (10) of [1].
[7] Six sets as set forth in (5) to (10) of [1].
[8] Nine sets according to (1) (2) to (10).
[9] Four sets according to (1) to (4) of [1].
[10] Four sets according to (1) and (5) to (7) of [1].
[11] Four sets according to (1) and (8) to (10) of [1].
[12] Seven sets according to (1) to (7) of [1].
[13] Seven sets of [1] (1) to (4) and (8) to (10).
[14] A set of 7 sets according to (1) and (5) to (10) of [1].
[15] Ten sets according to (1) to (10) of [1].
[16] A salmonella serotyping kit comprising the set according to any one of [1] to [15].
[17] A method for identifying a serotype of a Salmonella to be tested, comprising the step of comparing the types of PCR products described in (a) below and the types of PCR products described in (b) below: A method for determining that the serotype of the Salmonella to be tested is ST when the types of the PCR products compared in the above match;
(A) A type of PCR product amplified by PCR using the genomic DNA of a control Salmonella whose serotype is ST as a template and at least the set of [2] or [9] as a primer set, the control Salmonella And (b) the type of PCR product amplified by PCR using the genomic DNA of the test Salmonella as a template and the same set as (a) as a primer set.
[18] A method for identifying a serotype of a Salmonella to be tested, comprising the step of comparing the types of PCR products described in (a) below and the types of PCR products described in (b) below: A method for determining that the serotype of the Salmonella to be tested is SH when the types of the PCR products compared in the above match;
(A) A type of PCR product amplified by PCR using the genomic DNA of a control Salmonella whose serotype is SH as a template and at least the set of [3] or [10] as a primer set, the control Salmonella And (b) the type of PCR product amplified by PCR using the genomic DNA of the test Salmonella as a template and the same set as (a) as a primer set.
[19] A method for identifying a serotype of a Salmonella to be tested, comprising the step of comparing the types of PCR products described in (a) below and the types of PCR products described in (b) below: A method in which the serotype of the Salmonella to be tested is determined to be SI when the types of the PCR products compared in the above match;
(A) A type of PCR product amplified by PCR using the genomic DNA of a control Salmonella whose serotype is SI as a template and at least the set of [4] or [11] as a primer set, the control Salmonella And (b) the type of PCR product amplified by PCR using the genomic DNA of the test Salmonella as a template and the same set as (a) as a primer set.
[20] The method according to any one of [17] to [19], wherein the PCR is multiplex PCR.
[21] DNA fragment amplified by PCR using serotype ST Salmonella genomic DNA as a template and at least one set selected from the group consisting of (1) to (4) of [1] as a primer set .
[22] Amplified by PCR using serotype SH Salmonella genomic DNA as a template and at least one set selected from the group consisting of (1) and (5) to (7) of [1] as a primer set DNA fragment.
[23] Amplified by PCR using serotype SI Salmonella genomic DNA as a template and at least one set selected from the group consisting of (1) and (8) to (10) of [1] as a primer set DNA fragment.
[24] The DNA fragment according to any one of [21] to [23], wherein the PCR is multiplex PCR.

  According to the present invention, oligonucleotides for detecting Salmonella and rapid serotype identification methods using them have been developed. Conventionally, since it took about two weeks to identify Salmonella serotypes, there was a risk that the feed and meat to be examined were consumed before the serotypes were identified. By utilizing the present invention, it is possible to reduce the time required for Salmonella serotype identification by one week or more, and thus it is possible to avoid such a risk.

It is a photograph which shows identification of serotype ST by multiplex PCR method. Lane 1, 100 bp DNA Ladder; Lane 2, T1; Lane 3, T2; Lane 4, T3; Lane 5, T4; Lane 6, S1; Lane 7, S2; Lane 8, S3; Lane 9, S4

The present inventors analyzed genes specific to Salmonella serotypes ST, SH, and SI in silico and succeeded in finding genes specific to these serotypes. In addition, the present inventors have succeeded in finding primers for multiplex PCR that can specifically detect these serotypes. Based on such findings, the present invention provides any one set of oligonucleotides described in (1) to (10) below.
(1) an oligonucleotide comprising the base sequence described in SEQ ID NO: 1 and a set of oligonucleotides comprising the base sequence described in SEQ ID NO: 2,
(2) a set of oligonucleotides containing the base sequence set forth in SEQ ID NO: 3 and oligonucleotides containing the base sequence set forth in SEQ ID NO: 4;
(3) an oligonucleotide containing the base sequence set forth in SEQ ID NO: 5 and a set of oligonucleotides containing the base sequence set forth in SEQ ID NO: 6;
(4) a set of oligonucleotides containing the base sequence set forth in SEQ ID NO: 7 and oligonucleotides containing the base sequence set forth in SEQ ID NO: 8;
(5) an oligonucleotide comprising the base sequence set forth in SEQ ID NO: 9 and an oligonucleotide set comprising the base sequence set forth in SEQ ID NO: 10;
(6) an oligonucleotide comprising the base sequence set forth in SEQ ID NO: 11 and an oligonucleotide set comprising the base sequence set forth in SEQ ID NO: 12;
(7) an oligonucleotide comprising the base sequence set forth in SEQ ID NO: 13 and an oligonucleotide set comprising the base sequence set forth in SEQ ID NO: 14;
(8) a set of oligonucleotides containing the base sequence set forth in SEQ ID NO: 15 and oligonucleotides containing the base sequence set forth in SEQ ID NO: 16;
(9) an oligonucleotide comprising the base sequence set forth in SEQ ID NO: 17 and an oligonucleotide set comprising the base sequence set forth in SEQ ID NO: 18;
(10) A set of an oligonucleotide containing the base sequence set forth in SEQ ID NO: 19 and an oligonucleotide containing the base sequence set forth in SEQ ID NO: 20.

  In the present invention, the word “consisting of” can be used instead of the word “including”.

  The oligonucleotide set of the present invention is useful as a PCR primer used for identification of Salmonella serotypes, particularly as a primer for multiplex PCR. Multiplex PCR can detect multiple PCR products at once.

  For example, the oligonucleotide set (primer set) used to determine whether the Salmonella serotype is ST is at least (2) selected from the group consisting of (1) to (10) above. To (4), or at least (1) to (4).

  For example, the oligonucleotide set (primer set) used to determine whether the serotype of Salmonella is SH or not is selected from the group consisting of (1) to (10) above (5) To (7), or at least (1) and (5) to (7).

  For example, the oligonucleotide set (primer set) used to determine whether the Salmonella serotype is SI is at least (8) selected from the group consisting of (1) to (10) above. To (10), or at least (1) and (8) to (10).

  For example, as a set of oligonucleotides (primer set) used to determine whether the Salmonella serotype is ST, SH, or neither, the above (1) to (10) Examples include at least the set described in (2) to (7) or the set described in (1) to (7).

  For example, as a set of oligonucleotides (primer set) used to determine whether the serotype of Salmonella is ST, SI, or neither, (1) to (10) above Exemplified is a set according to at least (2) to (4) and (8) to (10) selected from the group consisting of, or at least a set according to (1) to (4) and (8) to (10) it can.

  For example, the oligonucleotide sets (primer sets) used to determine whether the Salmonella serotype is SH, SI, or neither are the above (1) to (10) The set as described in at least (5) to (10) selected from the group which consists of, or at least the set as described in (1) and (5) to (10) can be illustrated.

  For example, the oligonucleotide set (primer set) used to determine whether the serotype of Salmonella is ST, SH, SI, or neither is (1) Can be exemplified by at least the set described in (2) to (10) or the set described in (1) to (10).

That is, the present invention provides the sets described in (A) to (N) below in addition to any one set described in (1) to (10) above.
(A) Three sets according to (2) to (4) above.
(B) Three sets according to (5) to (7) above.
(C) Three sets according to (8) to (10) above.
(D) Six sets described in (2) to (7) above.
(E) Six sets according to (2) to (4) and (8) to (10) above.
(F) Six sets according to (5) to (10) above.
(G) Four sets according to (1) to (4) above.
(H) Four sets according to (1) and (5) to (7) above.
(I) The four sets described in (1) and (8) to (10) above.
(J) Seven sets as described in (1) to (7) above.
(K) A set of seven sets according to (1) to (4) and (8) to (10) above.
(L) A set of 7 sets according to (1) and (5) to (10) above.
(M) Nine sets according to (2) to (10) above.
(N) Ten sets as described in (1) to (10) above.

The invA gene, which is a Salmonella-specific gene, can be detected by PCR using the set (1) as a primer. That is, the above set (1) is useful as a primer set for detecting whether or not the test bacterium is Salmonella or for confirming that the test bacterium is Salmonella. The set (1) is also useful as a positive control for PCR reaction.

The present invention also provides a reagent for identifying a Salmonella serotype and a kit for identifying a Salmonella serotype. Such reagents and kits include at least the following component (i).
(I) At least one set described in (1) to (10) above

In addition to the component (i), the reagent or kit may further contain the following additional element (ii) or (iii).
(Ii) DNA polymerase: The reagent or kit of the present invention may contain a DNA polymerase that catalyzes a template-dependent complementary strand synthesis reaction. Various DNA polymerases used in known nucleic acid synthesis reactions such as PCR can be used in the present invention.
(Iii) Nucleotide substrate: The reagent or kit of the present invention may contain nucleotides used as a substrate for a nucleic acid complementary strand synthesis reaction. Specifically, at least one of dCTP, dGTP, dTTP, and dATP, usually all of these (dNTP) can be included in the kit. These nucleotides can utilize not only natural structures but also derivatives. Nucleotides modified with fluorescent substances or binding ligands are known. The above-mentioned reagents and kits can be accompanied by a document that describes the presence or absence of an amplification product by PCR and the size of the PCR product amplified by PCR. Moreover, the size information of the PCR product amplified by PCR can be converted into machine-readable data and added to a reagent or kit as a recording medium storing the data.

  For example, the reagent or kit used to identify whether or not the serotype of the test Salmonella is ST is described in at least (2) to (4) selected from (1) to (10) above. Or a reagent or kit containing at least the set described in (1) to (4).

  For example, the reagent or kit used for identifying whether or not the serotype of the test Salmonella is SH is described in at least (5) to (7) selected from (1) to (10) above. Or a reagent or kit containing at least the sets described in (1) and (5) to (7).

  For example, the reagent or kit used to identify whether or not the serotype of the test Salmonella is SI is described in at least (8) to (10) selected from (1) to (10) above. Or a reagent or kit containing at least the sets described in (1) and (8) to (10).

  For example, the reagent or kit used to determine whether the serotype of the test Salmonella is ST, SH, or neither is selected from (1) to (10) above. Examples of the reagent or kit include at least the set described in (2) to (7), or at least the set described in (1) to (7).

  For example, the reagent or kit used to determine whether the serotype of the Salmonella to be tested is ST, SI, or neither is selected from (1) to (10) above Examples of the reagent or kit include at least the set according to (2) to (4) and (8) to (10), or at least the set according to (1) to (4) and (8) to (10). .

  For example, the reagent or kit used to determine whether the serotype of the test Salmonella is SH, SI, or neither is selected from the above (1) to (10) Examples of the reagent or kit include at least the set described in (5) to (10), or at least the set described in (1) and (5) to (10).

  For example, the reagent or kit used to determine whether the serotype of Salmonella to be tested is ST, SH or SI, or any of them is selected from (1) to (10) above Examples of the reagent or kit include at least the set described in (2) to (10) or the set described in (1) to (10).

  The present invention also relates to a method for identifying Salmonella serotypes using at least one set of the oligonucleotide sets of the present invention. In the present invention, “at least one set” includes 3, 4, 6, 7, 8, 9, or 10 combinations. That is, the present invention relates to a method for identifying a Salmonella serotype described in (I) to (VII) below. In the present invention, the PCR is preferably a multiplex PCR method.

(I)
A method for identifying the serotype of a Salmonella to be tested, comprising the steps of comparing the types of PCR products described in (a) below and the types of PCR products described in (b) below. A method in which when the types of PCR products match, the serotype of the test Salmonella is determined to be the serotype (ST) of the control Salmonella.
(A) At least the set according to (2) to (4) selected from the group consisting of (1) to (10) above, or at least (1) to (1) using the genomic DNA of a control Salmonella as ST as a template 4) The types of PCR products amplified by PCR using the set described in (4) as a primer set, the types of PCR products specific to the control Salmonella, and (b) genomic DNA of the test Salmonella as a template, Types of PCR products amplified by PCR using the same set as in a) as a primer set.

(II)
A method for identifying the serotype of a Salmonella to be tested, comprising the steps of comparing the types of PCR products described in (a) below and the types of PCR products described in (b) below. A method in which when the PCR product types match, the serotype of the test Salmonella is determined to be the serotype (SH) of the control Salmonella.
(A) At least the set according to (5) to (7) selected from the group consisting of (1) to (10) above, using at least the genomic DNA of a control Salmonella that is SH, or at least (1) and ( 5) to the kind of PCR product amplified by PCR using the set described in (7) as a primer set, the kind of PCR product specific to the control Salmonella, and (b) genomic DNA of the test Salmonella Types of PCR products amplified by PCR using the same set as (a) as a primer set as a template.

(III)
A method for identifying the serotype of a Salmonella to be tested, comprising the steps of comparing the types of PCR products described in (a) below and the types of PCR products described in (b) below. A method in which, when the types of PCR products match, the serotype of the test Salmonella is determined to be the serotype (SI) of the control Salmonella.
(A) a set of at least (8) to (10) selected from the group consisting of the above (1) to (10), or at least (1) and ( The types of PCR products amplified by PCR using the sets described in 8) to (10) as primer sets, the types of PCR products specific to the control Salmonella, and (b) genomic DNA of the test Salmonella Types of PCR products amplified by PCR using the same set as (a) as a primer set as a template.

(IV)
A method for identifying the serotype of a Salmonella to be tested, comprising the steps of comparing the types of PCR products described in (a) below and the types of PCR products described in (b) below. A method in which, when the PCR product types match, the test Salmonella serotype is determined to be the control Salmonella serotype (ST or SH) that matches the PCR product type.
(A) a set of at least (2) to (7) selected from the group consisting of (1) to (10) above, using at least the genomic DNA of a control Salmonella that is ST or SH as a template, or at least (1) To the type of PCR product amplified by PCR using the set described in (7) as a primer set, and (b) the type of PCR product specific to the control Salmonella, and the genomic DNA of the test Salmonella as a template. The kind of PCR product amplified by PCR using the same set as (a) as a primer set.

(V)
A method for identifying the serotype of a Salmonella to be tested, comprising the steps of comparing the types of PCR products described in (a) below and the types of PCR products described in (b) below. A method in which, when the PCR product types match, the test Salmonella serotype is determined to be the control Salmonella serotype (SH or SI) with a matching PCR product type;
(A) a set of at least (5) to (10) selected from the group consisting of (1) to (10) above, wherein at least genomic DNA of a control Salmonella that is SH or SI is used as a template, or at least (1) And the kind of PCR product amplified by PCR using the set described in (5) to (10) as a primer set, and the kind of PCR product specific to the control Salmonella, and (b) the genome of the test Salmonella Types of PCR products amplified by PCR using DNA as a template and the same set as (a) as a primer set.

(VI)
A method for identifying the serotype of a Salmonella to be tested, comprising the steps of comparing the types of PCR products described in (a) below and the types of PCR products described in (b) below. A method in which when the PCR product type matches, the test Salmonella serotype is determined to be the control Salmonella serotype (ST or SI) with the PCR product type match;
(A) At least (2) to (4) and (8) to (10) selected from the group consisting of (1) to (10) above, using the genomic DNA of a control Salmonella that is ST or SI as a template Or a PCR product that is amplified by PCR using at least the sets described in (1) to (4) and (8) to (10) as a primer set, and that is specific for the control Salmonella And (b) the type of PCR product amplified by PCR using the genomic DNA of the test Salmonella as a template and the same set as (a) as a primer set.

(VII)
A method for identifying the serotype of a Salmonella to be tested, comprising the steps of comparing the types of PCR products described in (a) below and the types of PCR products described in (b) below. A method in which when the PCR product type matches, the test Salmonella serotype is determined to be the control Salmonella serotype (ST, SH, or SI) that matches the PCR product type.
(A) The control Salmonella genomic DNA selected from the group consisting of ST, SH, and SI is used as a template, and at least (2) to (10) are selected from the group consisting of (1) to (10) above Or the types of PCR products amplified by PCR using the 10 sets described in (1) to (10) as primer sets, the types of PCR products specific to the control Salmonella, and (b ) A kind of PCR product amplified by PCR using the test Salmonella genomic DNA as a template and the same set as (a) as a primer set.

In the method of the present invention, when the types of PCR products match, it is determined that the serotype of the test Salmonella is the serotype of the control Salmonella with the same PCR product type.
If the types of PCR products do not match, it is determined that the serotype of the test Salmonella is not the serotype of the control Salmonella.

In the present invention, “type of PCR product” means a pattern of a PCR product classified according to the presence or absence of the PCR product or the molecular size, and “type of PCR product” can be expressed as a “PCR pattern”.
A “primer set” can also be expressed as a “primer pair”.
Further, for example, “a set of an oligonucleotide including the base sequence described in SEQ ID NO: 1 and an oligonucleotide including the base sequence described in SEQ ID NO: 2” (the set described in (1) above) is “SEQ ID NO: 1 and a primer set (primer pair) comprising a primer including the base sequence described in SEQ ID NO: 2. Other sets of the present invention (the sets described in (2) to (10) above) can be expressed in the same manner.

  The “type of PCR product specific to control Salmonella” in the present invention can also be expressed as “type of PCR product specific to serotype of control Salmonella”.

  The control Salmonella in the present invention is amplified by PCR using the genomic DNA of the Salmonella as a template and selected from the group consisting of (1) to (10) above and using a specific set described herein as a primer set. A PCR product type that is specific for the control Salmonella. Examples of the control salmonella include, but are not limited to, at least one salmonella selected from the group consisting of ST, SH, and SI.

The entire genome sequence of ST is known as a sequence specified by accession number NC_003197. In addition, ST features are `` McClelland, M., Sanderson, KE, Spieth, J., Clifton, SW, Latreille, P., Courtney, L., Porwollik, S., Ali, J., Dante, M., Du, F., Hou, S., Layman, D., Leonard, S., Nguyen, C., Scott, K., Holmes, A., Grewal, N., Mulvaney, E., Ryan, E., Sun, H., Florea, L., Miller, W., Stoneking, T., Nhan, M., Waterston, R. And Wilson, RK Complete genome sequence of Salmonella enterica serovar Typhimurium LT2, Nature 413 (6858), 852 -856 (2001) ". In addition, the entire genome sequence information of SH and SI is ftp://ftp.sanger.ac.uk/pub/pathogens/Salmonella/HADAR.dbs, ftp://ftp.sanger.ac.uk/pub/pathogens, respectively. It can be referenced at /Salmonella/SIN.dbs.

In the present invention, examples of Salmonella to be tested include bacteria having the characteristics of Salmonella, bacteria having the potential of Salmonella, and bacteria closely related to Salmonella, but are not limited thereto. Salmonella is a Gram-negative facultative anaerobic gonococcus and is usually moved by pericytes, but there are non-motile serotypes or mutant strains. That is, the subject Salmonella of the present invention includes motility serotypes and non-motility serotypes or mutant strains. Most strains have the following properties. The subject Salmonella of the present invention includes Salmonella exhibiting the following characteristics and properties.
・ Gas is produced from glucose.
・ Negative oxidase, indole, phenylalanine deaminase.
-Shows lysine decarboxylase, ornithine decarboxylase, and hydrogen sulfide positive properties.

  In addition, when Salmonella infects humans and livestock, the infected humans and livestock exhibit symptoms such as typhoid, sepsis, pneumonia, miscarriage, acute gastroenteritis, and diarrhea. The subject Salmonella of the present invention includes Salmonella that causes such symptoms in humans and livestock.

Salmonella serotypes are classified according to the classification method of 6 subspecies under S. enterica , based on biochemical properties, DNA homology, etc., and 1 genus and 2 species of Salmonella enterica and Salmonella bongori . The “serotype” in the present invention means a serotype determined by a combination of the cell (O) antigen and flagellar (H) antigen according to the Kauffman-White format. References include 9th edition Antigenic Formulas of the Salmonella Serovars, 2007, Patrick AD Grimont & Francois-Xavier Weill (http://www.pasteur.fr/sante/clre/cadrecnr/salmoms/WKLM_2007.pdf).

  “Identification” in the present invention can also be expressed as “by type”, “detection”, “identification”, “discrimination”, “specification”, “appraisal”, or “determination”.

The inventions (I) to (VII) can also be expressed as follows. The invention of (I) is shown below as an example, but the inventions of (II) to (VII) can also be expressed in the same manner.
A method for identifying a serotype of a Salmonella to be tested comprising the steps of determining the identity of the PCR product described in (a) below and the PCR product described in (b) below, wherein the PCR product is A method in which, when identical, the test Salmonella serotype is determined to be the control Salmonella serotype (ST).
(A) At least the set according to (2) to (4) selected from the group consisting of (1) to (10) above, or at least (1) to (1) using the genomic DNA of a control Salmonella as ST as a template A PCR product amplified by PCR using the set described in 4) as a primer set, the PCR product specific for the control Salmonella, and (b) the genomic DNA of the test Salmonella as a template, the same as (a) PCR product amplified by PCR using the set as a primer set.
If the PCR products are identical, it is determined that the serotype of the control Salmonella and the serotype of the test Salmonella are the same.

The inventions (I) to (VII) can also be expressed as follows. The invention of (I) is shown below as an example, but the inventions of (II) to (VII) can also be expressed in the same manner.
The type of the PCR product described in the following (b) including the step of comparing the type of the PCR product described in the following (a) and the type of the PCR product described in the following (b) is described in the following (a) To determine whether or not the PCR product matches the type.
(A) At least the set according to (2) to (4) selected from the group consisting of (1) to (10) above, or at least (1) to (1) using the genomic DNA of a control Salmonella as ST as a template 4) The types of PCR products amplified by the PCR according to the set described in the above, wherein the types of PCR products specific to the control Salmonella, and (b) the genomic DNA of the test Salmonella as a template, Types of PCR products amplified by PCR using the same set as a primer set.
If it is determined that the types of PCR products match, it is determined that the serotype of the control Salmonella is the serotype (eg, ST) of the test Salmonella.

Although the method of this invention is illustrated below, it is not limited to this.
In the present invention, first, a specific salmonella selected from ST, SH and SI is used as a template, and the specific DNA described in the present specification is selected from the group consisting of (1) to (10) above. The type of PCR product amplified by PCR using the set as a primer set is detected (hereinafter referred to as PCR product X). Next, the type of PCR product (hereinafter referred to as PCR product Y) detected by PCR using the genomic DNA of the desired Salmonella whose serotype is unknown as a template and the same set as the control Salmonella as a primer set is detected. To do. A desired salmonella whose serotype is unknown is defined as a test salmonella. Next, PCR product X and PCR product Y are compared. When the compared PCR products match, the serotype of the control Salmonella is determined to be the serotype of the test Salmonella.

  Specifically, first, a specific set (primer set) described in the present specification selected from the group consisting of (1) to (10) is synthesized. Primers can be chemically synthesized according to the nucleotide sequence information. Methods for synthesizing oligonucleotides having an arbitrary base sequence are known. For example, the oligonucleotide can be synthesized by a DNA synthesizer. Specifically, DNA synthesizers based on principles such as the phosphoramidite method and the phosphonate method are now in practical use. These DNA synthesizers chemically link nucleotide monomers one by one on a solid phase according to a given base sequence. All reaction steps are automated, and synthesis is started by inputting target base sequence information. Therefore, the target DNA can be synthesized by giving the designed primer base sequence to such a DNA synthesizer.

Next, PCR is performed for each control Salmonella selected from ST, SH, and SI using genomic DNA as a template and the synthesized primer set. In the present invention, preferably, genomic DNA of control Salmonella is first prepared (extraction of genomic DNA). Extraction of genomic DNA from Salmonella is generally performed by those skilled in the art, for example, by the cetyltrimethylammonium bromide (CTAB) method (Murray and Thompson, Nucleic Acids Research 8, p.4321-4325, 1980). It can be carried out. In addition, genomic DNA can be extracted using DNeasy (registered trademark) Plant Mini Kit (QIAGEN) or the like. Moreover, it is also possible to carry out PCR without using a genomic DNA extraction process, for example, directly using a cell disruption solution or the like.
An example of the PCR method is a multiplex PCR method.

  Next, the presence or absence of the PCR product or the molecular size of the PCR product amplified by PCR is measured. Methods for measuring the presence or absence of a PCR product or the molecular size of a PCR product amplified by PCR include methods well known to those skilled in the art. Examples include, but are not limited to, a method using polyacrylamide gel electrophoresis, a method using agarose gel electrophoresis, a method using a fragment analyzer, and a microchip electrophoresis apparatus (Multina, Shimadzu).

  When measuring the molecular size of the PCR product, specifically, the PCR product is fluorescently labeled and then separated by capillary electrophoresis, and the molecular size of each PCR product separated is measured. Alternatively, PCR products are separated by electrophoresis (on polyacrylamide gel / agarose gel) and the molecular size of each separated PCR product is measured. Usually, the molecular size (for example, molecular weight or length) of each separated PCR product is measured using a DNA fragment (for example, a size marker) of a known size as a control. Next, the PCR products are classified by molecular size, and the kind of the PCR product is specified. The type of PCR product identified is specific for the serotype of the control Salmonella.

  Next, PCR is performed using the synthesized primer set using the genomic DNA of the test Salmonella as a template. Identification of a PCR product using the genomic DNA of the test Salmonella as a template is preferably performed by the same experimental method and experimental conditions as those of the control Salmonella.

  In the present invention, the type of PCR product specific to any control Salmonella selected from the group consisting of ST, SH and SI can be exemplified by the types of PCR products obtained by the method described in this example. However, the present invention is not limited to this. For example, the types of PCR products specific to these control Salmonella include PCR products detected when PCR products amplified under PCR conditions different from those in this example are separated by the same separation method and separation conditions as in this example. PCR products that correspond to these types of PCR products, or PCR that is detected when PCR products amplified under the same PCR conditions as in this example or different PCR conditions are separated using different separation methods and separation conditions from this example It may be a PCR product type corresponding to the product type.

The present invention further provides a PCR product amplified by PCR using ST genomic DNA as a template and at least one set selected from the group consisting of (1) to (4) as a primer set. Such PCR products are ST-specific PCR products. The PCR product is useful for determining whether or not the Salmonella serotype is ST.
As at least one set selected from the group consisting of (1) to (4) above, three sets described in (2) to (4) and four sets described in (1) to (4) Although a set can be illustrated, it is not limited to these.

Furthermore, the present invention provides a PCR product amplified by PCR using SH genomic DNA as a template and at least one set selected from the group consisting of (1) and (5) to (7) as a primer set. provide. Such PCR products are SH-specific PCR products. The PCR product is useful for determining whether or not the Salmonella serotype is SH.
As at least one set selected from the group consisting of (1) and (5) to (7) above, three sets as described in (5) to (7) and from (1) and (5) Although the four sets described in (7) can be exemplified, the present invention is not limited to these.

The present invention also provides a PCR product amplified by PCR using SI genomic DNA as a template and at least one set selected from the group consisting of (1) and (8) to (10) as a primer set. provide. Such PCR products are SI-specific PCR products. The PCR product is useful for determining whether or not the Salmonella serotype is SI.
As at least one set selected from the group consisting of (1) and (8) to (10) above, three sets as described in (8) to (10) and from (1) and (8) Although the four sets described in (10) can be exemplified, the present invention is not limited to these.
In the present invention, PCR is preferably multiplex PCR.

EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not restrict | limited to these Examples.
Using genome-wide sequences of serotypes ST, SH, and SI published on the method database, comparative genome analysis was performed between ST and SH, ST and SI, and SH and SI, and non-common regions Genes contained in the serotype-specific gene candidates. By conducting homology search using a gene database, serotype-specific genes were narrowed down and three genes were selected for each serotype. An attempt was made to establish a multiplex PCR method for simultaneously detecting these genes and four genes of the invA gene involved in intracellular entry of Salmonella. The invA gene is known to be specific to the genus Salmonella, and by simultaneously detecting this gene, it can be shown that the test bacterium is included in the genus Salmonella. In addition, by setting the amplification size of this gene to the maximum, it is possible to increase the consumption of dNTPs and to suppress the nonspecific amplification.
Next, ST125 strains (Table 1-1, 1-2), SH19 strains (Table 2), SI55 strains (Table 3), derived from different cases among approximately 3,700 field-isolated Salmonella collected over the past 30 years, Select one strain from each of the other 117 serotypes (Tables 4-1 and 4-2), and perform multiplex PCR using these genomic DNAs as templates to simultaneously detect the above genes, and serotypes in field-isolated Salmonella The specificity of this method was verified by examining specific gene candidate possession rates.

Results and Discussion As a result of comparative genome analysis based on ortholog analysis, 533, 297, and 268 non-matching ORFs were extracted by ST, SH, and SI, respectively. Among these genes, one gene was selected from the region considered to be an inserted sequence, homology search was performed, and three genes with less registration in other Salmonella were selected by ST, SH, and SI, respectively. Table 5 shows primers for detecting these genes and the invA gene by the multiplex PCR method, the base sequences of amplification products, and the like. In multiplex PCR for identifying ST, four gene regions of invA , TMP1, TMP2, and TMP3 were targeted. Similarly the SH invA, HMP1, HMP2, and 4 gene region HMP3, and the SI invA, IMP1, IMP2, and 4 gene region IMP3 target. As a result of studying the conditions, multiplex PCR was performed in a 50 μl reaction system containing DNA polymerase (KOD FX; Toyobo) 1 U, dNTPs 0.4 mM each, primers 0.3 μM each (4 pairs), and template DNA 100 ng. Thermal transformation 98 ° C., 10 seconds, annealing 52 ° C., 30 seconds, extension reaction 68 ° C., 30 seconds were set to 35 cycles. The size of the amplified product was confirmed by electrophoresis in a 2% agarose gel.

In the multiplex PCR for ST identification, all three serotype-specific genes were positive in the tested ST125 strain, whereas in the 117 serotypes other than ST, only three genes were present in serotype O4: i:-. A positive result was obtained (FIG. 1, Table 6). This serotype was considered to be a single-phase mutant of ST and could not be distinguished from ST by this method. The positive rate of each gene in the non-ST 117 strain was 13.7% for TMP1, 21.4% for TMP2, and 9.4% for TMP3 (Table 6). In the multiplex PCR for SH identification, all of the 19 strains tested were positive for 3 genes, whereas in the 117 serotypes other than SH, 3 gene positive strains were not found. The positive rate of each gene in the non-SH 117 strain was 0.9% for HMP1, 3.4% for HMP2, and 0.9% for HMP3 (Table 7). In the multiplex PCR for SI identification, all of the tested SI55 strains were 3 gene-positive, whereas in 117 serotypes other than SI, 3 gene-positive strains were not found. The positive rate of each gene in the non-SI 117 strain was 6.0% for IMP1, 0% for IMP2, and 4.3% for IMP3 (Table 8). From the above results, these multiplex PCR methods were considered to have sufficient specificity for use as identification methods for the above three serotypes.

Claims (8)

A method for identifying the serotype of a Salmonella to be tested, comprising the steps of comparing the types of PCR products described in (a) below and the types of PCR products described in (b) below. A method in which when the types of PCR products match, it is determined that the serotype of the Salmonella to be tested is SH;
(A) a kind of PCR product amplified by PCR using a genomic DNA of a control Salmonella having a serotype of SH as a template and at least a set described in (i) or (ii) below as a primer set, Types of PCR products specific to Salmonella, and (b) types of PCR products amplified by PCR using the genomic DNA of the test Salmonella as a template and the same set as (a) as a primer set;
(I) a set according to (1) to (3) below (1) an oligonucleotide comprising the base sequence set forth in SEQ ID NO: 9 and an oligonucleotide set comprising the base sequence set forth in SEQ ID NO: 10;
(2) a set of oligonucleotides containing the base sequence set forth in SEQ ID NO: 11 and oligonucleotides containing the base sequence set forth in SEQ ID NO: 12;
(3) an oligonucleotide comprising the base sequence set forth in SEQ ID NO: 13 and an oligonucleotide set comprising the base sequence set forth in SEQ ID NO: 14;
(Ii) a set described in (1) to (4) below (1) an oligonucleotide containing the base sequence described in SEQ ID NO: 1, and a set of oligonucleotides containing the base sequence described in SEQ ID NO: 2.
(2) a set of oligonucleotides containing the base sequence set forth in SEQ ID NO: 9 and an oligonucleotide containing the base sequence set forth in SEQ ID NO: 10;
(3) an oligonucleotide comprising the base sequence set forth in SEQ ID NO: 11 and an oligonucleotide set comprising the base sequence set forth in SEQ ID NO: 12;
(4) A set of oligonucleotides containing the base sequence set forth in SEQ ID NO: 13 and oligonucleotides containing the base sequence set forth in SEQ ID NO: 14. The method of claim 1, wherein the PCR is multiplex PCR. The following three sets described in (5) to (7) :
(5) an oligonucleotide comprising the base sequence set forth in SEQ ID NO: 9 and an oligonucleotide set comprising the base sequence set forth in SEQ ID NO: 10;
(6) an oligonucleotide comprising the base sequence set forth in SEQ ID NO: 11 and an oligonucleotide set comprising the base sequence set forth in SEQ ID NO: 12;
(7) A set of oligonucleotides containing the base sequence set forth in SEQ ID NO: 13 and oligonucleotides containing the base sequence set forth in SEQ ID NO: 14 . The following four sets as described in (1) and (5) to (7) :
(1) an oligonucleotide comprising the base sequence described in SEQ ID NO: 1 and a set of oligonucleotides comprising the base sequence described in SEQ ID NO: 2,
(5) an oligonucleotide comprising the base sequence set forth in SEQ ID NO: 9 and an oligonucleotide set comprising the base sequence set forth in SEQ ID NO: 10;
(6) an oligonucleotide comprising the base sequence set forth in SEQ ID NO: 11 and an oligonucleotide set comprising the base sequence set forth in SEQ ID NO: 12;
(7) A set of oligonucleotides containing the base sequence set forth in SEQ ID NO: 13 and oligonucleotides containing the base sequence set forth in SEQ ID NO: 14 . A salmonella serotype determination kit comprising the set according to claim 3 or 4 . A DNA fragment amplified by PCR using a serotype SH Salmonella genomic DNA as a template and the set according to claim 3 or 4 as a primer set. The DNA fragment according to claim 6 , wherein the PCR is multiplex PCR. A set including (1) to (10) below:
(1) an oligonucleotide comprising the base sequence described in SEQ ID NO: 1 and a set of oligonucleotides comprising the base sequence described in SEQ ID NO: 2,
(2) a set of oligonucleotides containing the base sequence set forth in SEQ ID NO: 3 and oligonucleotides containing the base sequence set forth in SEQ ID NO: 4;
(3) an oligonucleotide containing the base sequence set forth in SEQ ID NO: 5 and a set of oligonucleotides containing the base sequence set forth in SEQ ID NO: 6;
(4) a set of oligonucleotides containing the base sequence set forth in SEQ ID NO: 7 and oligonucleotides containing the base sequence set forth in SEQ ID NO: 8;
(5) an oligonucleotide comprising the base sequence set forth in SEQ ID NO: 9 and an oligonucleotide set comprising the base sequence set forth in SEQ ID NO: 10;
(6) an oligonucleotide comprising the base sequence set forth in SEQ ID NO: 11 and an oligonucleotide set comprising the base sequence set forth in SEQ ID NO: 12;
(7) A set of oligonucleotides containing the base sequence set forth in SEQ ID NO: 13 and oligonucleotides containing the base sequence set forth in SEQ ID NO: 14
(8) a set of oligonucleotides containing the base sequence set forth in SEQ ID NO: 15 and oligonucleotides containing the base sequence set forth in SEQ ID NO: 16;
(9) an oligonucleotide comprising the base sequence set forth in SEQ ID NO: 17 and an oligonucleotide set comprising the base sequence set forth in SEQ ID NO: 18;
(10) A set of oligonucleotides comprising the base sequence set forth in SEQ ID NO: 19 and oligonucleotides comprising the base sequence set forth in SEQ ID NO: 20.