JP5189747B2 - Oligonucleotides for Coagulase Type of Staphylococcus aureus - Google Patents

Description

  The present invention relates to a coagulase type oligonucleotide for S. aureus. Further, the present invention relates to a simple and rapid typing method for coagulase type of S. aureus and a reagent for coagulase type using an oligonucleotide.

Coagulase is an extracellular secreted protein that has been used for a long time to distinguish Staphylococcus aureus from other genera of Staphylococcus, and has the effect of coagulating human, monkey and rabbit plasma. This coagulase is known to have a difference in antigenicity, and the presence of 10 types (types I to X) of the coagulase type of Staphylococcus aureus has been reported. In staphylococcal food poisoning, it has been reported that coagulase type VII accounts for 60 to 70% of the total, followed by about type 10 and type II, respectively. As described above, it has been reported that even Staphylococcus aureus is a food poisoning-causing fungus that tends to be limited to a specific type, and typing the strains isolated from various materials determines the food-causing causative fungus. It is also very helpful. In addition, the source of contamination can be pursued by examining the interrelationship with other epidemiological markers such as phage type and toxin type. Actually, type-specific antisera are commercially available for epidemiological investigation of food poisoning and for diagnosis of Staphylococcus aureus-related diseases, and typing methods from type I to type VIII have been established.

The coagulase typing method using commercially available antiserum is described below. One colony of S. aureus of the test strain is inoculated into a Brain Heart Infusion (BHI) liquid medium and left to stand at 37 ° C. overnight under aerobic conditions. The culture solution is centrifuged at 3000 rpm for 30 minutes, and the supernatant is collected and used as a test antigen solution. After dispensing 0.1 mL of the test antigen solution into 9 small test tubes, add 0.1 mL of antiserum against each coagulase type (types I to VIII) and diluted normal rabbit serum as a control to each test tube. Incubate at 37 ° C for 1 hour. Add 0.2 mL of diluted rabbit plasma to all tubes and allow to react at 37 ° C. Determine plasma clotting by tilting the small test tube and continue observation for 1, 2, 4, 24, and 48 hours until only one of the 9 test tubes is not coagulated. The serotype of the test tube in which clotting is blocked represents the coagulase type of the test strain. Thus, it takes a lot of time to isolate the S. aureus strain and determine the coagulase type. In addition, depending on the strain, production of coagulase is weak and determination may be difficult. In that case, it is necessary to devise methods such as shaking culture for enhancing coagulase production, culture in a rabbit plasma-added liquid medium, and selection of colonies with high coagulase production using a rabbit plasma-added agar medium. In addition, depending on the strain, coagulase production is very strong, and coagulation is observed in all test tubes within a reaction time of 1 hour, which makes it difficult to determine. In that case, the coagulase test must be performed by serially diluting the test antigen solution by a factor of 2, diluting the test antigen to the highest dilution factor at which coagulation is observed, and performing the coagulase type test again.
Thus, since the coagulase type test using antiserum requires a lot of time and skill, it is very useful in epidemiology, but its use outside Japan is low.

  In relation to the reagent kit relating to Staphylococcus aureus, an oligonucleotide characterized by hybridizing with a nucleic acid sequence common to each coagulase type present in the coagulase gene, and a labeled oligonucleotide labeled with the oligonucleotide, And a detection method and detection reagent kit for Staphylococcus aureus using the oligonucleotide and the labeled oligonucleotide as a probe or primer are disclosed (for example, see Patent Document 1). This relates to a method for detecting Staphylococcus aureus in a sample and a reagent kit for detection, and does not enable coagulase I to VIII type classification.

The gene structure of coagulase type I to X has recently been clarified (for example, see Non-Patent Document 1). According to it, each type of coagulase gene base sequence had high homology of 67.2-90.2%.
However, no technique has been reported that makes it possible to perform typing by finding a base sequence almost specific to each of the coagulase type I to VIII genes and performing gene amplification using the oligonucleotide chain as a primer.
Japanese Patent Laid-Open No. 6-25845 Watanabe S. et al., Journal of Bacteriology, Vol. 187, p. 3698-3707, 2005

  It is an object of the present invention to provide a rapid and simple typing method for Saccharomyces aureus coagulase types I to VIII by typing the S. aureus coagulase type in a specimen.

As a result of intensive studies on the coagulase type of Staphylococcus aureus, the present inventors have found a base sequence almost specific for each of the coagulase I to VIII genes and perform gene amplification using the oligonucleotide chain as a primer. As a result, the technology that makes it possible to sort by mold has been established, and the present invention has been completed.
That is, the present invention relates to an oligonucleotide for specifically amplifying coagulase type I to VIII genes, and by using the oligonucleotide to determine the coagulase type of Staphylococcus aureus in a sample. The present invention relates to a quick and simple typing method for each type.

An oligonucleotide that specifically detects a coagulase type I gene of Staphylococcus aureus comprises at least 10 consecutive base sequences represented by SEQ ID NOs: 1 to 4 in the sequence listing or a complement thereof An oligonucleotide consisting of a sequence.
The oligonucleotide for specifically detecting the coagulase type II gene of Staphylococcus aureus comprises an oligonucleotide comprising at least 10 consecutive base sequences represented by SEQ ID NOs: 5 to 9, or An oligonucleotide consisting of its complementary sequence.
In addition, the oligonucleotide that specifically detects the coagulase type III gene of Staphylococcus aureus contains at least 10 consecutive bases of the base sequences represented by SEQ ID NOs: 10 to 11, or An oligonucleotide consisting of its complementary sequence.
In addition, the oligonucleotide that specifically detects the coagulase type IV gene of Staphylococcus aureus contains at least 10 consecutive bases of the base sequences represented by SEQ ID NOs: 12 to 20 in the sequence listing or An oligonucleotide consisting of its complementary sequence.
The oligonucleotide for specifically detecting the coagulase V type gene of Staphylococcus aureus includes at least 10 consecutive bases of the base sequences represented by SEQ ID NOs: 21 to 24, or An oligonucleotide consisting of its complementary sequence.
The oligonucleotide for specifically detecting the coagulase type VI gene of Staphylococcus aureus contains at least 10 consecutive bases of the base sequence represented by SEQ ID NO: 25-30, or An oligonucleotide consisting of its complementary sequence.
In addition, the oligonucleotide that specifically detects the coagulase type VII gene of Staphylococcus aureus contains at least 10 consecutive bases of the base sequence represented by SEQ ID NO: 31 to 33 in the sequence listing or An oligonucleotide consisting of its complementary sequence.
In addition, the oligonucleotide that specifically detects the coagulase type VIII gene of Staphylococcus aureus contains at least 10 consecutive bases of the base sequences represented by SEQ ID NOs: 34 to 35 in the sequence listing or An oligonucleotide consisting of its complementary sequence.

Furthermore, the present invention relates to a target coagulase gene in DNA in a sample using at least two kinds of oligonucleotides for each coagulase type among the oligonucleotides for each coagulase type shown in SEQ ID NOs: 1 to 35 in the sequence listing. It is a coagulase type method characterized by specifically amplifying a part.
The present invention also relates to a primer set comprising at least two types of oligonucleotides for each coagulase type among the oligonucleotides for each coagulase type shown in SEQ ID NOs: 1 to 35, SEQ ID NO: 1 to 35, thermostable DNA polymerase, dNTP and buffer solution Coagulase type reagent kit characterized by comprising:

  In the present invention, by using gene amplification for each of Staphylococcus aureus coagulases I to VIII, a simple, rapid and accurate typing test can be performed. In addition to isolated strains and viable bacteria as specimens, samples containing dead Staphylococcus aureus cells, that is, food specimens and clinical specimens containing S. aureus DNA, have high detection sensitivity due to gene amplification. Coagulase type classification is possible. Therefore, the present invention can be used as a useful technique in the pursuit of contamination sources during staphylococcal food poisoning, epidemiological investigations, and coagulase type tests conducted in the inspection and diagnosis of S. aureus related diseases. In addition, the provision of a coagulase type reagent kit characterized by containing a primer set, a thermostable DNA polymerase, dNTP and a buffer is extremely useful for molecular epidemiological analysis in the field.

For the coagulase type using the oligonucleotide of the present invention, Polymerase Chain Reaction method (PCR method) generally used as a nucleic acid amplification method is used. The length of the oligonucleotide chain used as a primer for PCR should be at least 10 bases in consideration of selectivity for template DNA, PCR detection sensitivity, and reproducibility, but generally 20 bases. Longitudinal length is desirable. When the oligonucleotide of the present invention is used as a primer for PCR, it is not necessary to use the entire sequence shown in SEQ ID NOs: 1 to 35 or their complementary sequences. By synthesizing a primer consisting of about 20 bases containing at least 10 consecutive bases of the oligonucleotide of the present invention on the 3 ′ end side of the primer, and performing PCR reaction according to the dissociation temperature (Tm value), An amplification product specific to the type can be obtained.
Oligonucleotides used as primers are synthesized using a commercially available DNA synthesizer using the phosphoramidide method, for example, Applied Biosystems 3400 DNA synthesizer. The synthesized oligonucleotide is purified by gel filtration or high performance liquid chromatography using a reverse phase column.
A thermostable DNA polymerase is used for PCR, but the origin of this enzyme may be derived from any species as long as it retains activity at a temperature of 90 to 95 ° C. The heat denaturation temperature is 90 to 95 ° C, the annealing temperature for hybridizing the primers is 37 to 65 ° C, the polymerization reaction is 50 to 75 ° C, and PCR is 20 to 45 with one cycle from this heat denaturation to the polymerization reaction. A cycle is performed to amplify the coagulase type I to VIII gene fragment of S. aureus DNA contained in the sample.

  The target amplification product is detected by subjecting the reaction solution after PCR to agarose gel electrophoresis as it is. From the length of the amplified nucleotide fragment, the presence or absence of a sequence to be recognized by the primer is confirmed. This result is the type-specific result of Staphylococcus aureus coagulase types I-VIII in the specimen. It is also effective to use other electrophoresis or chromatography to detect the amplified nucleotide fragment.

As a specimen in the present invention, a nucleic acid specimen such as DNA roughly extracted or purified from isolated and cultured cells is used. In addition, since the PCR method specifically amplifies the target gene, it can be directly used from a sample containing various microorganisms such as foods, clinical samples, or environmental samples without isolation and culture of bacteria. In addition, the bacteria in the samples do not have to be viable for all of the above samples, and can be inspected as long as the dead cells have DNA.
Alternatively, one of the oligonucleotides represented by SEQ ID NOs: 1 to 35 in the sequence listing may be used as a probe to selectively detect the target nucleotide sequence on the membrane or other support. In this case, the oligonucleotide is preferably modified with a label.
EXAMPLES The present invention will be described in more detail below with reference to examples and test examples, but these are merely illustrative and the present invention is not limited by these.

(By coagulase type by PCR method)
(1) Preparation of sample Staphylococcus aureus used a coagulase type I-VIII standard strain (heading next to Table 1). Each strain was cultured in a BHI (Brain Heart Infusion) liquid medium (BBL) at 35 ° C. for 24 hours. The cells were collected from 1 mL of the culture solution and suspended in 162 μL of Tris-EDTA Buffer. 18 μL of 15 mg / ml lysostaphin (Wako Pure Chemical Industries, Ltd.) was added to the bacterial suspension and incubated at 37 ° C. for 30 minutes. DNA was prepared from this lysostaphin treatment solution using Blood & Cell Culture DNA kit (Qiagen).

(2) Primer design From the base sequences of the coagulase type I to VIII genes, a specific sequence for each gene was found, and an oligonucleotide chain containing the region was designed as a primer. The oligonucleotide chain used as a primer was chemically synthesized. The combination of primers for detecting each coagulase type and the estimated number of bases of the amplification product are shown in the vertical heading of Table 2.

(3) PCR
The PCR reaction solution was prepared using a reagent from Takara Bio. That is, a total of 15 μL of PCR reaction solution containing 0.2 U Ex Taq Polymerase, 0.2 mM dNTP mixture, 1.5 μL of 10 × Ex Taq Buffer, 0.5 μM coagulase type primer, and 1 to 1000 ng of sample DNA. Prepared. The PCR reaction was carried out using Gene Amp 9700 (Applied Biotechnology), after initial denaturation at 94 ° C for 4 minutes, followed by one cycle of 94 ° C for 30 seconds, 52 ° C for 30 seconds, 72 ° C for 60 seconds. 30 cycles were performed, and further maintained at 72 ° C for 7 minutes, and then maintained at 4 ° C.

(4) Detection In order to detect the amplified nucleotide fragment, the PCR reaction solution was subjected to 3% agarose electrophoresis. After electrophoresis of 4.5 μL of the PCR reaction solution with 3% agarose and staining with ethidium bromide, fluorescence under ultraviolet irradiation was detected. At the same time, a molecular weight marker was also run, and the length of the detected nucleotide fragment was calculated by comparing the relative mobility.

(5) Results Since the oligonucleotide of the present invention used as a primer was designed based on the information of known coagulase type I to VIII gene base sequences, the length of the nucleotide fragment to be amplified by PCR can be estimated. . When the estimated base number of the amplification product shown in the vertical heading in Table 1 matches the length of the nucleotide fragment actually detected from the PCR reaction, each primer correctly amplifies the target region. It was judged as “+” in Table 1. On the other hand, "-" was shown for those in which no nucleotide amplification was observed. As a result, for the 39 primer combinations shown in Table 1, the coagulase type determined from the amplification product and the coagulase type indicated by the S. aureus coagulase standard strain all coincided. Moreover, it did not react at all with a coagulase-type DNA template different from the primer target. That is, oligonucleotides designed to detect each coagulase type can specifically amplify the target gene and accurately determine the coagulase type.

(By coagulase type by multiplex PCR)
According to the coagulase type by PCR shown in Example 1, 8 PCR reactions are required per sample, which is not suitable for testing of multiple specimens. Therefore, multiplex PCR was studied in which primers for detecting four coagulase types were mixed in a single PCR reaction solution. According to this method, coagulase types I to VIII can be classified by two PCR reactions.

(1) Preparation of sample The DNA prepared in Example 1 was used as a template.

(2) PCR
Primers used for multiplex PCR were divided into two sets. That is, primer set 1 comprises coagulase III, IV, VII, and VIII type detection primers, primer set 2 comprises I, II, V, and VI type detection primers, and each primer is 5 μM in the PCR reaction solution. It was prepared as follows. The combinations of each coagulase type detection primer and the estimated number of amplified product bases are shown in Table 2 below.

  Here, a positive control marker was introduced to confirm that the PCR reaction was performed correctly. That is, primers that detect the femA gene of Staphylococcus aureus (Forward primer: aaaaaagcac ataacaagcg, Reverse Primer: gataaagaag aaaccagcag, amplification product 132 bp, Mehrotra M. et al, J. Clin. Microbiol. 38, 1032-1035, 2000) Was added to each reaction solution to a concentration of 0.05 μM. Other reagents and PCR reaction conditions used for PCR were the same as in Example 1.

(3) Detection The detection was performed in the same manner as in Example 1.

(4) Results The results of subjecting the multiplex PCR product to 3% agarose gel electrophoresis are shown in FIG. In PCR using primer set 1, nucleotide fragments having the same size as the estimated number of bases were detected only from the reaction solution containing DNA of coagulase III, IV, VII, and VIII standard strains. On the other hand, in PCR using primer set 2, nucleotide fragments having the same size as the estimated number of bases were detected only from reaction solutions containing DNA of coagulase I, II, V, and VI standard strains. It did not react at all with the coagulase type DNA different from the target of each primer. On the other hand, the femA gene, which is a positive control marker, was detected in all samples except the negative control (no sample), confirming that PCR was performed correctly. That is, this multiplex PCR can specifically amplify the target gene and accurately determine the coagulase type by two PCR reactions per sample.

(Comparison of antiserum method and multiplex PCR method)
(1) Specimens A total of 126 strains were used: 40 strains of Staphylococcus aureus derived from food poisoning, 36 strains of clinically derived Staphylococcus aureus, and 50 strains of Staphylococcus aureus derived from raw milk. Each strain prepared DNA by the same method as in Example 1.

(2) PCR
Multiplex PCR was performed in the same manner as in Example 2.

(3) Detection The detection was performed in the same manner as in Example 1.

(4) Type of coagulase using antiserum The culture solution of the test bacteria was centrifuged at 3000 rpm for 30 minutes, and the supernatant was collected to obtain a test antigen solution. The test was carried out using a commercially available antiserum-based coagulase type kit (staphylococcal coagulase type immune serum “Seken”, Denka Seken Co., Ltd.) according to the attached instruction manual.

(5) Results Table 3 shows the relationship between the coagulase type determined using antiserum and the coagulase type determined by multiplex PCR for the S. aureus strain 126 used as a specimen. The coagulase type determined by both methods matched 125 strains, and the match rate was 99.2%.

A coagulase type reagent kit comprising the following parts was prepared.
(1) Coagulase type primer set The primer set shown in Table 2 consisting of at least two kinds of oligonucleotides for each coagulase type (each 10 μM).
(2) Thermostable DNA polymerase
Ex Taq Polymerase (5U / μL, Takara Bio Inc.)
(3) dNTP
dNTP Mixture solution (2.5 mM each, Takara Bio Inc.)
(4) Buffer solution
10 × Ex Taq Buffer (Takara Bio Inc.)

The result of having used the multiplex PCR product for 3% agarose gel electrophoresis is shown. (Example 2)

Claims (6)

Of the PCR primers wherein the specific detection of either Kano co Aguraze type gene of coagulase I~VIII type gene of Staphylococcus aureus, for each coagulase type using at least two PCR primers PCR reactions A coagulase typing method characterized by specifically amplifying a part of a target coagulase gene in DNA in a sample , wherein SEQ ID NOs: 1, 2, 5, 6, 10, 12, PCR primers including all range of the nucleotide sequence represented by 13,14,15,16,21,22,25,26,29,31,34 is Forward primer, SEQ ID NO: 3, 4, PCR primers including all range of the nucleotide sequence represented by 9,11,17,18,19,20,23,24,27,28,30,32,33,35 the R A method according to coagulase type, characterized in that a PCR reaction is performed in the presence of at least one forward primer and at least one reverse primer for each coagulase type, which are everse primers. Of the PCR primers wherein the specific detection of either Kano co Aguraze type gene of coagulase I~VIII type gene of Staphylococcus aureus, a primer set comprising at least two PCR primers for each of the coagulase type, A reagent kit for coagulase type, comprising a thermostable DNA polymerase, dNTP and a buffer , wherein SEQ ID NOs: 1, 2, 5, 6, 10, 12, 13, 14, 15, 16 , PCR primers including all range of the nucleotide sequence represented by 21,22,25,26,29,31,34 is Forward primer, SEQ ID NO: 3,4,7,8,9,11,17,18 , PCR primers including all range of the nucleotide sequence represented by 19,20,23,24,27,28,30,32,33,35 the Reverse Pla A coagulase type reagent kit , wherein the kit comprises at least one forward primer and at least one reverse primer for each coagulase type . A set of PCR primers comprising at least two of the following (1) to (4).
(1) A set of two PCR primers characterized by specifically detecting a coagulase type III gene and comprising the entire base sequence represented by SEQ ID NOs: 10 and 11, respectively, at the 3 ′ end (2) Coagulase type IV The gene is specifically detected, and a set of two PCR primers each containing the entire base sequence represented by SEQ ID NOs: 13 and 20 at the 3 ′ end. (3) A coagulase type VII gene is specifically detected. A set of two PCR primers containing the entire base sequence represented by SEQ ID NOs: 31 and 32 at the 3 ′ end, respectively, (4) specifically detecting a coagulase type VIII gene, A set of two PCR primers each containing the entire base sequence represented by SEQ ID NOs: 34 and 35 at the 3 ′ end A set of PCR primers comprising at least two of the following (5) to (8).
(5) A set of two PCR primers characterized by specifically detecting a coagulase type I gene, each containing the entire base sequence represented by SEQ ID NOs: 2 and 4 at the 3 ′ end (6) Coagulase type II It is characterized by specifically detecting a gene, and a set of two PCR primers each containing the entire base sequence represented by SEQ ID NO: 6 and 9 at the 3 ′ end (7) Specifically detecting a coagulase V-type gene A set of two PCR primers each containing the entire base sequence represented by SEQ ID NOS: 22 and 24 at the 3 ′ end, respectively, (8) characterized by specifically detecting a coagulase VI type gene, A set of two PCR primers each including the entire 3 ′ end of the base sequence represented by SEQ ID NOs: 29 and 30 at the 3 ′ end Set of PCR primers as claimed in claim 3, and / or, PCR was carried out in the presence of a set of PCR primers according to claim 4, for specifically detecting a gene of different types of coagulase of Staphylococcus aureus A coagulase type method characterized by that. Set of PCR primers as claimed in claim 3, and / or, coagulase typing kit characterized in that it comprises a set of PCR primers as claimed in claim 4.