Multiplex PCR primer combination for identifying serotypes of Streptococcus pasteurianus and its application

Abstract

The present invention provides a multiplex PCR detection primer composition for identifying the serotype of Streptococcus pasteurianus and its application, belonging to the field of biotechnology. The multiplex PCR detection primer composition comprises first to fifth primer pairs. The present invention also provides the application of the multiplex PCR detection primer composition in the preparation of a kit for identifying the serotype of Streptococcus pasteurianus. The primer composition of the present invention can be used to detect five main serotypes of Streptococcus pasteurianus through multiplex PCR. The detection method has the advantages of simple operation, strong specificity, high detection sensitivity, strong anti-interference and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial detection, and in particular relates to a multiplex PCR detection primer composition for identifying serotypes of Streptococcus pasteurianus and an application thereof. Background Art

[0002] Streptococcus pasteurianus ( Streptococcus pasteurianus ) is a new zoonotic pathogen. It can cause meningitis and sepsis in nine animals, including geese, turkeys and pigs. The bacteria can also infect humans and cause illness or death, causing endocarditis, urinary tract infection and intrauterine infection in pregnant women in newborns, the elderly and immunocompromised patients, and may also be associated with gastrointestinal malignancies in humans. Some researchers have reported that the bacteria can not only cause meningitis in pigs, but also show resistance to multiple antibiotics, which may be transmitted through horizontal transfer of mobile gene elements (MGEs). The capsule is a key virulence factor of the bacteria. In healthy pig samples, the infection rate of Streptococcus pasteurianus exceeded 9%, indicating that healthy pig herds may be a potential reservoir of the strain, increasing the potential risk of zoonotic disease transmission and outbreaks. Different Streptococcus pasteurianus have different tropism and pathogenicity to the host. In order to fully understand Streptococcus pasteurianus and develop drugs and vaccines against Streptococcus pasteurianus, it is urgent to accurately classify and serotype Streptococcus pasteurianus. However, there is no method for identifying the serotype of S. pasteurianum with high accuracy, high sensitivity and good specificity in the prior art. Summary of the invention

[0003] The invention provides a multiplex PCR detection primer composition for identifying serotypes of Streptococcus pasteurianus with high accuracy, good specificity, high sensitivity and short detection time.

[0004] The purpose of the present invention is achieved by the following technical solutions:

[0005] The invention discloses a multiplex PCR detection primer composition for identifying serotypes of Streptococcus pasteurianus, comprising first to fifth primer pairs, wherein the first primer pair comprises primers 1a-F and 1a-R, whose sequences are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively; the second primer pair comprises primers 1b-F and 1b-R, whose sequences are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively; the third primer pair comprises primers 2-F and 2-R, whose sequences are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively; the fourth primer pair comprises primers 3-F and 3-R, whose sequences are shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively; and the fifth primer pair comprises primers 4-F and 4-R, whose sequences are shown in SEQ ID NO: 9 and SEQ ID NO: 10, respectively.

[0006] In a preferred technical solution, the concentration of each primer in the composition is 0.8-1.2 μM.

[0007] The present invention also provides application of the multiplex PCR detection primer combination in preparing a kit for identifying serotypes of Streptococcus pasteurianus.

[0008] The present invention also provides a kit containing the multiplex PCR detection primer composition.

[0009] The present invention also provides a method for identifying the serotype of Streptococcus pasteurianus by using the multiplex PCR detection primer composition for non-diagnostic purposes, comprising the following steps: using the Streptococcus pasteurianus to be detected or its genomic DNA as a template, and using the multiplex PCR detection primer composition to perform multiplex PCR detection; subjecting the PCR product to agarose gel electrophoresis, if a band with a size of 238 bp appears, the sample to be detected contains the serotype 1a of Streptococcus pasteurianus; if a band with a size of 380 bp appears, the sample to be detected contains the serotype 1b of Streptococcus pasteurianus; if a band with a size of 834 bp appears, the sample to be detected contains the serotype 2 of Streptococcus pasteurianus; if a band with a size of 544 bp appears, the sample to be detected contains the serotype 3 of Streptococcus pasteurianus; if a band with a size of 1066 bp appears, the sample to be detected contains the serotype 4 of Streptococcus pasteurianus.

[0010] In the present invention, the multiplex PCR reaction system includes: 2×Green Taq Mix 12.5 μL, primer 1a-F 0.4 μL, primer 1a-R 0.4 μL, primer 1b-F 1.5 μL, primer 1b-R 1.5 μL, primer 2-F 0.6 μL, primer 2-R 0.6 μL, primer 3-F 0.15 μL, primer 3-R 0.15 μL, primer 4-F 0.3 μL, primer 4-R 0.3 μL, PCR template 1.0 μL and ultrapure water 5.6 μL.

[0011] In the present invention, the PCR reaction procedure is as follows: pre-denaturation at 95°C for 3 min; 95°C for 15 s, 52°C for 15 s, 72°C for 25 s, 35 cycles; 72°C for 5 min.

[0012] Beneficial effects: Currently, there is no serotype classification method for Streptococcus pasteurianus. The present invention performs serotyping on Streptococcus pasteurianus through agglutination reaction for the first time. However, traditional agglutination experiments require the preparation of antisera for various serotype strains and the need to immunize animals multiple times, which is time-consuming and costly. The multiplex PCR detection primer composition provided by the present invention is used to identify the serotype of Streptococcus pasteurianus. Only one-step PCR and electrophoresis technology is required to achieve high specificity, high sensitivity, and high accuracy in identifying its serotype. In addition, the detection method of the present invention is fast, easy to operate, low cost, and the results are clear and intuitive. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Homology analysis of the capsule gene cluster of Streptococcus pasteurianus.

[0014] Figure 2 It is the specific result of identifying Streptococcus pasteurianus by the multiplex PCR detection method of the present invention. (A) is the electrophoresis detection result of identifying serotypes 1a, 1b, 2, 3 and 4 of Streptococcus pasteurianus by the multiplex PCR detection method of the present invention. In the figure, M: Trans2K® DNA Marker 2K DNA, lane 1 is Streptococcus pasteurianus NCTC13784 (serotype 1a), lane 2 is Streptococcus pasteurianus WUSP067 (serotype 1b), lane 3 is Streptococcus pasteurianus WUSP070 (serotype 2), lane 4 is Streptococcus pasteurianus ATCC43144 (serotype 3), lane 5 is Streptococcus pasteurianus WUSP074 (serotype 4), lane 6 is a mixture of DNA of 5 serotypes of Streptococcus pasteurianus strains, and lane 7 is a negative control. (B) is the electrophoresis result diagram of the specific evaluation of the multiplex PCR detection method of the present invention for non-Streptococcus pasteurianus. In the figure, M: Trans2K® DNA Marker 2K DNA, lanes 1-10 are cultures of Streptococcus agalactiae, Streptococcus multizoans, Klebsiella pneumoniae, Streptococcus dysgalactiae, Enterococcus avium, Enterococcus faecium, R. romagnificus, Enterococcus caecum, Streptococcus vaginalis porcine and Streptococcus suis, lane 11 is a negative control, and lane 12 is a positive control (a mixture of DNA from five serotypes of Streptococcus pasteurianus strains).

[0015] Figure 3 The electrophoretic diagrams are obtained by using the multiplex PCR detection method of the present invention to detect different DNA concentrations of serotype 1a Streptococcus pasteurianus NCTC13784. Wherein M: Trans2K® DNA Marker 2K DNA. The genomic DNA concentrations of serotype 1a Streptococcus pasteurianus NCTC13784 corresponding to lanes 1 to 11 are 10.00 ng / μL, 5.00 ng / μL, 2.50 ng / μL, 1.25 ng / μL, 625 pg / μL, 313 pg / μL, 156 pg / μL, 78 pg / μL, 39 pg / μL, 20 pg / μL, and 10 pg / μL, respectively. Lane 12 is the product of PCR amplification using H2O instead of template.

[0016] Figure 4The electrophoretic diagrams are obtained by using the multiplex PCR detection method of the present invention to detect different DNA concentrations of serotype 1b Streptococcus pasteurianus WUSP067. Wherein M: Trans2K® DNA Marker 2K DNA. The WUSP067 genomic DNA concentrations corresponding to lanes 1 to 11 are 10.00 ng / μL, 5.00 ng / μL, 2.50 ng / μL, 1.25 ng / μL, 625 pg / μL, 313 pg / μL, 156 pg / μL, 78 pg / μL, 39 pg / μL, 20 pg / μL, and 10 pg / μL, respectively. Lane 12 is the product of PCR amplification using H2O instead of template.

[0017] Figure 5 The electrophoretic diagrams are obtained by using the multiplex PCR detection method of the present invention to detect different DNA concentrations of 2 serotypes of Streptococcus pasteurianus WUSP070. Wherein M: Trans2K® DNA Marker 2K DNA. The WUSP070 genomic DNA concentrations corresponding to lanes 1 to 11 are 10.00 ng / μL, 5.00 ng / μL, 2.50 ng / μL, 1.25 ng / μL, 625 pg / μL, 313 pg / μL, 156 pg / μL, 78 pg / μL, 39 pg / μL, 20 pg / μL, and 10 pg / μL, respectively. Lane 12 is the product of PCR amplification using H2O instead of template.

[0018] Figure 6 The electrophoretic diagrams are obtained by using the multiplex PCR detection method of the present invention to detect different concentrations of DNA of 3 serotypes of Streptococcus pasteurianus ATCC43144. Wherein M: Trans2K® DNA Marker 2K DNA. The concentrations of ATCC43144 genomic DNA corresponding to lanes 1 to 11 are 10.00 ng / μL, 5.00 ng / μL, 2.50 ng / μL, 1.25 ng / μL, 625pg / μL, 313 pg / μL, 156 pg / μL, 78 pg / μL, 39 pg / μL, 20 pg / μL, and 10 pg / μL, respectively. Lane 12 is the product of PCR amplification using H2O instead of template.

[0019] Figure 7The electrophoretic diagrams are obtained by using the multiplex PCR detection method of the present invention to detect different concentrations of DNA of 4 serotypes of Streptococcus pasteurianus WUSP074. Wherein M: Trans2K® DNA Marker 2K DNA. The WUSP074 genomic DNA concentrations corresponding to lanes 1 to 11 are 10.00 ng / μL, 5.00 ng / μL, 2.50 ng / μL, 1.25 ng / μL, 625 pg / μL, 313 pg / μL, 156 pg / μL, 78 pg / μL, 39 pg / μL, 20 pg / μL, and 10 pg / μL, respectively. Lane 12 is the product of PCR amplification using H2O instead of template.

[0020] Figure 8 The electrophoretic diagrams obtained by detecting a mixture of five serotypes of Streptococcus pasteurianus DNA at different concentrations using the multiplex PCR detection method of the present invention. Wherein M: Trans2K® DNA Marker 2K DNA. The concentrations of mixed genomic DNA corresponding to lanes 1 to 9 are 2.00 ng / μL, 1.00 ng / μL, 500 pg / μL, 250 pg / μL, 125 pg / μL, 62.5 pg / μL, 31.25 pg / μL, 15.60 pg / μL, and 7.80 pg / μL, respectively.

[0021] Fig. 9 The results of the test for the detection limit of NCTC13784 pure bacterial culture by the multiple PCR detection method of the present invention. Where M: Trans2K® DNA Marker 2K DNA. The concentrations of NCTC13784 pure bacterial culture corresponding to lanes 1 to 6 are 7×10 5 CFU / μL, 7×10 4 CFU / μL, 7×10 3 CFU / μL, 7×10 2 CFU / μL, 7×10 1 CFU / μL, 7×10 0 CFU / μL.

[0022] Fig.10 The results of the test for the detection limit of WUSP067 pure bacterial culture by the multiplex PCR detection method of the present invention. Where M: Trans2K® DNA Marker 2K DNA. The concentrations of WUSP067 pure bacterial culture corresponding to lanes 1 to 6 are 1.3×10 6 CFU / μL, 1.3×10 5 CFU / μL, 1.3×10 4 CFU / μL, 1.3×10 3 CFU / μL, 1.3×102 CFU / μL, 1.3×10 1 CFU / μL.

[0023] Fig.11 The results of the test for the detection limit of WUSP070 pure bacterial culture by the multiplex PCR detection method of the present invention. Where M: Trans2K® DNA Marker 2K DNA. The concentrations of WUSP070 pure bacterial culture corresponding to lanes 1 to 6 are 8×10 5 CFU / μL, 8×10 4 CFU / μL, 8×10 3 CFU / μL, 8×10 2 CFU / μL, 8×10 1 CFU / μL, 8×10 0 CFU / μL.

[0024] Fig.12 The results of the test for the detection limit of ATCC43144 pure bacterial culture by the multiplex PCR detection method of the present invention. M: Trans2K® DNA Marker 2K DNA. The concentrations of ATCC43144 pure bacterial culture corresponding to lanes 1 to 6 are 1.2×10 6 CFU / μL, 1.2×10 5 CFU / μL, 1.2×10 4 CFU / μL, 1.2×10 3 CFU / μL, 1.2×10 2 CFU / μL, 1.2×10 1 CFU / μL.

[0025] Fig.13 The results of the test for the detection limit of WUSP074 pure bacterial culture by the multiplex PCR detection method of the present invention. Where M: Trans2K® DNA Marker 2K DNA. The concentrations of WUSP074 pure bacterial culture corresponding to lanes 1 to 5 are 8×10 5 CFU / μL, 8×10 4 CFU / μL, 8×10 3 CFU / μL, 8×10 2 CFU / μL, 8×10 1 CFU / μL. DETAILED DESCRIPTION

[0026] The present invention is described and explained by way of examples and drawings below, but the present invention is not limited to the scope of the examples. In this example, all primers were delivered to Beijing Tsingke Biotechnology Co., Ltd. for synthesis, and the Trans2K® DNA Marker 2K DNA molecular weight standard used was purchased from Beijing TransGen Biotech Co., Ltd.

[0027] The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer. Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art.

[0028] Example 1 Preparation of standard antisera for five serotypes of Streptococcus pasteurianus

[0029] In order to identify the serotype of S. pasteurianus, the capsule gene clusters of 21 strains of S. pasteurianus were analyzed and it was found that the capsule gene clusters of these S. pasteurianus were divided into four categories: Figure 1 As shown. After further analysis of the nucleotide sequences of the strains, it was found that the capsule gene cluster of the NCTC13784 strain in category 1 showed a unique sequence of 915 bp, which was significantly different from the other five strains. The nucleotide sequence of the capsule gene cluster of the WUSP074 strain in category 4 was 120 bp more than that of the other strains. Therefore, the representative strains of these six different capsule gene clusters (NCTC13784, WUSP067, WUSP070, ATCC43144, WUSP074 and WUSP082) were selected to prepare antisera for these six strains. Through agglutination tests, NCTC13784 and WUSP067 were named serotype 1, WUSP070 was serotype 2, ATCC43144 was serotype 3, and WUSP074 and WUSP082 were serotype 4. Then, adsorption experiments were performed to further confirm whether the representative strains in the same serotype were different subtypes. Among them, NCTC13784 and WUSP067 are not the same subtype. NCTC13784 is named as subtype 1a and WUSP067 is named as subtype 1b; WUSP074 and WUSP082 are completely the same serotype.

[0030] Agglutination test

[0031] Table 1 Information on representative strains of each serotype

[0032]

[0033] In Table 1, BioSample ID is the number used to uniquely identify each biological sample record in the BioSample database of NCBI (National Center for Biotechnology Information).

[0034] Representative strains of each serotype of S. pasteurianus, NCTC13784, WUSP067, WUSP070, ATCC43144, WUSP074 and WUSP082 (for information, see Table 1 ), were cultured, and standard antisera of each strain were prepared. Then, agglutination test was used to serotype S. pasteurianus.

[0035] (1) Culture of Streptococcus pasteurianus

[0036] Glycerol-preserved Streptococcus pasteurianus NCTC13784, WUSP067, WUSP070, ATCC43144, WUSP074 and WUSP082 were inoculated on blood plates (THB solid medium containing 5% sheep blood) by streak inoculation method and cultured in a 37 ℃ incubator for 10 to 16 h. A single colony was picked from the blood plate, inoculated into THB liquid medium (purchased from Qingdao Haibo Biological), and cultured at 37 ℃ for 18 h, then placed in a 4 ℃ refrigerator overnight to obtain the first generation of bacterial liquid. 200 μL of the first generation of bacterial liquid was inoculated into 10 mL of THB liquid medium preheated to 37 ℃, and cultured at 37 ℃ for 6 hours on a shaking table, then placed in a 4 ℃ refrigerator overnight to obtain the second generation of bacterial liquid. Take 200 μL of the second-generation bacterial solution and inoculate it into 10 mL of preheated THB liquid medium, shake and culture it at 37 °C for 6 h, and place it in a 4 °C refrigerator overnight to obtain the third-generation bacterial solution. According to the same method, the third-generation bacterial solution was inoculated into THB liquid medium to prepare the fourth-generation bacterial solution, and then 5 mL of the fourth-generation bacterial solution was taken and injected into 50 mL of THB liquid medium preheated to 37 °C, shake and culture it at 37 °C for 2 hours to obtain the fifth-generation bacterial solution of each strain. The concentration of the fifth-generation bacterial solution was determined by the colony counting method.

[0037] (2) Preparation of antigens of various Streptococcus pasteurianus strains

[0038] The fifth generation bacterial solution of each strain was inactivated with formaldehyde, and the final concentration (volume percentage concentration) of formaldehyde was controlled at 0.5%, and the solution was shaken overnight at room temperature. A small amount of the inactivated bacterial solution was evenly spread on the blood plate and placed in a 37°C incubator to check the inactivation of the bacteria. After testing, all strains were completely inactivated. The inactivated bacterial solution was centrifuged at a speed of 10,000×g for 30 min, the supernatant was discarded, and the precipitate was collected. The precipitate was washed twice with PBS (phosphate buffer) at pH 7.4, and then the concentration of the bacterial solution was adjusted to the required concentration (see Table 2 for details) to obtain the antigens of each strain for animal immunization.

[0039] (3) Animal immunization method

[0040] Chinese white rabbits were selected as immunized animals. Chinese white rabbits were immunized with Streptococcus pasteurianus NCTC13784, WUSP067, WUSP070, ATCC43144, WUSP074 and WUSP082, respectively. The immunization procedures and doses were the same, as shown in Table 2. During immunization, the antigen was heated to 37°C and injected into the ear vein. There was a certain incubation period for the first contact of the animal with the antigen, so there was a 13-day interval between the first and second immunizations. Since the capsular polysaccharide can persist in the blood for 2 to 3 days, immunization was performed three times a week after the second immunization. A total of 10 immunizations were performed to achieve the ideal immunization effect.

[0041] Table 2 Immunization schedule and dosage of various strains

[0042]

[0043] Ten days after the last immunization, the antibody level in the Chinese white rabbit reached its peak. At this time, blood was collected from the heart and incubated at 37°C for 1 hour, 4°C overnight, and centrifuged at 3,000×g for 30 minutes. The supernatant was the antiserum.

[0044] (4) Determination of antiserum titer

[0045] Antigen-antibody binding test (slide agglutination reaction) was used as a method to detect the titer of animal serum after immunization with each strain. The specific steps are as follows: the serum to be tested was diluted with diluent (0.5% carbolic acid saline, i.e., an aqueous solution containing 0.5% carbolic acid and 0.85% NaCl) at a dilution ratio of 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, and 1:512. The negative serum (serum of Chinese white rabbits without immunization with antigens) and the blank control (PBS buffer) were diluted in multiples using the same dilution method. Take 10 μL of serum and add 10 μL of 4´10 9cfu / mL of the prepared antigen (in this example), the antigen and antibody were fully mixed, and the agglutination phenomenon was observed after 1 min and before 4 min. The maximum dilution at which accurate agglutination occurred was then taken as the titer of the antibody in the tested antiserum.

[0046] The amount of antiserum extracted for each serotype was more than 30 mL, and the slide agglutination reaction results showed that the antibody titer of the prepared antiserum was more than 1:32, proving that the antiserum was successfully prepared. The titers of each antiserum are shown in Table 3.

[0047] Table 3 Antiserum titers

[0048]

[0049] Note: In Table 3, “+” indicates that the slide agglutination result is positive, and “-” indicates that the slide agglutination result is negative.

[0050] (5) Agglutination test

[0051] The representative strains of each serotype of Streptococcus pasteurianus, NCTC13784, WUSP067, WUSP070, ATCC43144, WUSP074 and WUSP082, and the antisera of the six strains were subjected to plate agglutination experiments according to the agglutination reaction matrix in Table 4.

[0052] Table 4 Agglutination reaction matrix of 6 strains

[0053]

[0054] Note: In Table 4, "+" indicates a positive result of plate agglutination, and "-" indicates a negative result of plate agglutination.

[0055] The specific method of the plate agglutination test for Streptococcus pasteurianus is as follows: Plate agglutination antigen: Representative strains of each serotype of Streptococcus pasteurianus NCTC13784, WUSP067, WUSP070, ATCC43144, WUSP074 and WUSP082 were cultured in THB liquid medium until OD 600 1.0, washed twice with PBS buffer at pH 7.4, and resuspended in PBS buffer containing 5% crystal violet to make the bacterial solution 4´10 9cfu / mL, and obtain the plate agglutination antigen. Prepare diluent: 0.5% carbolic acid saline (aqueous solution containing 0.5% carbolic acid and 0.85% NaCl). A clean glass plate is divided into 3 cm × 3 cm areas per grid, and a small space is left under each grid for filling in the serum number. Add 10 μL of plate agglutination antigen to each grid of the glass plate, and add 10 μL of positive serum or negative serum of different serotypes respectively. Use a toothpick or pipette tip to stir the serum and antigen to mix them evenly to form a liquid area with a diameter of about 1 cm. Shake gently and observe the results after 1 min and before 4 min at room temperature. It is advisable to operate no more than 20 serum samples each time to avoid drying of the samples and making it difficult to observe the results. When the negative serum control (antibody mixed with serum from unimmunized Chinese white rabbits) does not show agglutination (-), and the positive serum control (undiluted antisera mixed with the corresponding antigen) shows agglutination (+), the test is established and can be judged. Otherwise, it should be repeated.

[0056] The results are shown in Table 4. Through the agglutination test, it was observed that the NCTC13784 antiserum could produce agglutination with both NCTC13784 and WUSP067 bacterial solutions, and the WUSP067 antiserum could also produce agglutination reactions with these two bacterial solutions. This result suggests that NCTC13784 and WUSP067 may belong to the same serotype. In addition, the WUSP074 antiserum only produced agglutination with WUSP074 and WUSP082 bacterial solutions, and the WUSP082 antiserum also only agglutinated with these two bacterial solutions. This result suggests that NCTC13784 and WUSP067 may belong to the same serotype. In contrast, the WUSP070 and ATCC43144 antisera only produced agglutination effects with their respective strains, and had no agglutination reactions with other strains, indicating that WUSP070 and ATCC43144 are two independent serotypes.

[0057] Based on the above findings and combined with the homology analysis of the capsular gene cluster, it is reasonable to name NCTC13784 and WUSP067 as serotype 1, WUSP070 as serotype 2, ATCC43144 as serotype 3, and WUSP074 and WUSP082 as serotype 4.

[0058] 2. Adsorption test to determine subtypes

[0059] (1) Verify whether NCTC13784 and WUSP067 are of the same serotype subtype

[0060] In order to further verify whether NCTC13784 and WUSP067 are of the same serotype subtype, the agglutination test was performed after the strains were adsorbed with serum. The specific operation steps are as follows: the formaldehyde-inactivated NCTC13784 and WUSP067 bacterial solutions were centrifuged at 10,000 × g for 15 min, resuspended twice with PBS buffer, the supernatant was removed under sterile conditions, and cross-mixed with two antisera with 9 times the volume of the bacterial solution. The specific combinations are: NCTC13784 bacteria and NCTC13784 antiserum; NCTC13784 bacteria and WUSP067 antiserum; WUSP067 bacteria and NCTC13784 antiserum; WUSP067 bacteria and WUSP067 antiserum. The mixed samples were incubated overnight at 4 °C. After incubation, the unbound antiserum was separated by centrifugation at 13,000 × g for 2 min, and the supernatant was the adsorbed antiserum. Repeat the above steps until the agglutination titer of the antiserum after strain adsorption against the adsorbed strain drops to <2. Subsequently, the adsorbed antiserum is filtered using a 0.22 um filter (biosharp catalog number: BS-PES25-22-S) to remove possible bacterial contamination. The agglutination titer of each antiserum before and after adsorption against each strain is recorded to evaluate whether different strains are of the same serotype.

[0061] The data in Table 5 indicate that NCTC13784 and WUSP067 may share some surface-exposed antigens, based on the fact that the agglutination titer of WUSP067 antiserum against NCTC13784 antiserum decreased to <2 after being adsorbed by WUSP067 bacteria. However, there are also different surface-exposed antigens between the two strains, as confirmed by the fact that the agglutination titer of NCTC13784 antiserum against NCTC13784 decreased less after being adsorbed by WUSP067 bacteria and did not drop to <2. Therefore, NCTC13784 and WUSP067 belong to different subtypes within serotype 1, and NCTC13784 is defined as subtype a within serotype 1, i.e., serotype 1a, while WUSP067 is defined as subtype b within serotype 1, i.e., serotype 1b.

[0062] Table 5 Agglutination titer after adsorption

[0063]

[0064] (2) Verify whether WUSP074 and WUSP082 are of the same serotype subtype

[0065] In order to further verify whether WUSP074 and WUSP082 are of the same serotype subtype, the agglutination test was performed after the strain was adsorbed with serum. The specific operation steps are as follows: the formaldehyde-inactivated WUSP074 and WUSP082 bacterial solutions were centrifuged at 10,000×g for 15 min, resuspended twice with PBS buffer, the supernatant was removed under sterile conditions, and cross-mixed with two antisera with 9 times the volume of the bacterial solution, respectively. The specific combinations are: WUSP074 bacteria and WUSP074 antiserum, WUSP074 bacteria and WUSP082 antiserum, WUSP082 bacteria and WUSP074 antiserum, WUSP082 bacteria and WUSP082 antiserum, and the mixed samples were incubated at 4°C overnight. After incubation, centrifuged at 13,000×g for 2 min to separate the unbound antiserum, and the supernatant was the antiserum after strain adsorption. Repeat the above steps until the agglutination titer of the antiserum after strain adsorption against the adsorbed strain drops to <2. Subsequently, the adsorbed antiserum is filtered using a 0.22 um filter (biosharp catalog number: BS-PES25-22-S) to remove possible bacterial contamination. The agglutination titer of each antiserum before and after adsorption against each strain is recorded to evaluate whether different strains are of the same serotype.

[0066] The data in Table 6 revealed that WUSP074 and WUSP082 had the same surface exposed antigens, because after the WUSP074 antiserum was adsorbed by the WUSP074 bacteria, the agglutination titer against WUSP082 was also reduced to <2; and after the WUSP082 antiserum was adsorbed by the WUSP082 bacteria, the agglutination titer against WUSP074 was also reduced to <2. Therefore, WUSP074 and WUSP082 are the same serotype, not different subtypes, that is, both are serotype 4.

[0067] Table 6 Agglutination titer after adsorption

[0068]

[0069] Note: “-” in Table 6 means none.

[0070] Example 2 Multiplex PCR detection primers and identification method for identifying serotypes of Streptococcus pasteurianus

[0071] Agglutination test has some limitations in identifying serotypes, including complicated operation process, low sensitivity, possible non-specific reaction and cross reaction, subjective influence on result judgment, high detection cost, high technical requirements and sensitivity to environmental factors. In order to quickly and accurately identify the serotype of Streptococcus pasteurianus, a large number of PCR detection primer compositions have been designed, and it is found that only the multiplex PCR detection primer composition for identifying the serotype of Streptococcus pasteurianus of the present invention has the characteristics of strong specificity, high detection sensitivity and strong anti-interference in identifying the serotype of Streptococcus pasteurianus.

[0072] 1. Multiplex PCR primer composition for identifying serotypes of Streptococcus pasteurianus according to the present invention

[0073] The multiplex PCR detection primer composition for identifying the serotype of Streptococcus pasteurianus (abbreviated as the primer composition of the present invention) of the present invention comprises the first to fifth primer pairs. The concentration of the upstream and downstream primers in each primer pair is 10 μM.

[0074] The sequence of the upstream primer 1a-F (SEQ ID NO: 1) of the first primer pair (for S. pasteurianus serotype 1a) is: 5'-TTGATGCAAATGGCTGAAGCAGAT-3', and the sequence of the downstream primer 1a-R (SEQ ID NO: 2) is: 5'-CAGACGTACAACTAGCTGATGTAA-3'. The detection characteristic fragment is 238 bp.

[0075] The sequence of the upstream primer 1b-F (SEQ ID NO: 3) of the second primer pair (for S. pasteurianus serotype 1b) is: 5'-GGCAGAATCAGAACTGTTAACT-3', and the sequence of the downstream primer 1b-R (SEQ ID NO: 4) is: 5'-TATTAATCTCAATAGCATCACCGGAT-3'. The detection characteristic fragment is 380 bp.

[0076] The sequence of the upstream primer 2-F (SEQ ID NO: 5) of the third primer pair (for S. pasteurianus serotype 2) is: 5'-ATTGAGGGCGTAGCTGAATTTAGA-3', and the sequence of the downstream primer 2-R (SEQ ID NO: 6) is: 5'-CTTCAGAAAAAGAAGAGCTATGGA-3'. The detection characteristic fragment is 834 bp.

[0077] The sequence of the upstream primer 3-F (SEQ ID NO: 7) of the fourth primer pair (for S. pasteurianus serotype 3) is: 5'-TTGACTGGTCTTCTAGGGGTATGG-3', and the sequence of the downstream primer 3-R (SEQ ID NO: 8) is: 5'-CTGGGGTAAAAGTCAACTCTGCAA-3'. The detection characteristic fragment is 544 bp.

[0078] The sequence of the upstream primer 4-F (SEQ ID NO: 9) of the fifth primer pair (for S. pasteurianus serotype 4) is: 5'-GTGGAGGACCTGTAACGAGTATTA-3', and the sequence of the downstream primer 4-R (SEQ ID NO: 10) is: 5'-CTCAAACATAGCTTTATGCCCTAC-3'. The detection characteristic fragment is 1066 bp.

[0079] 2. Multiplex PCR detection method for identifying serotypes of Streptococcus pasteurianus using the primer combination of the present invention

[0080] A multiplex PCR detection method for identifying serotypes of Streptococcus pasteurianus using the primer combination of the present invention (abbreviated as the multiplex PCR detection method of the present invention) comprises the following steps:

[0081] (1) Obtain PCR template for the bacteria to be detected

[0082] The strain of Streptococcus pasteurianus to be tested is inoculated into THB liquid culture medium and cultured overnight at 37°C to obtain bacterial culture solution, that is, bacterial solution that can be used as a PCR template. The obtained bacterial solution is used to extract genomic DNA according to the instructions of the Tiangen Bacterial Genomic DNA Extraction Kit, and the genomic DNA can also be used as a PCR template.

[0083] (2) PCR amplification reaction

[0084] The bacterial solution or genomic DNA in step (1) is used as a PCR template and the primer combination of the present invention is used for multiple PCR amplification. The concentration of each primer in the primer combination is 10 μM. PCR reaction system: 2×Green Taq Mix (Nanjing Novozyme Biotechnology Co., Ltd.) 12.5 μL, 10 μM primer 1a-F 0.4 μL, 10 μM primer 1a-R 0.4 μL, 10 μM primer 1b-F 1.5 μL, 10 μM primer 1b-R 1.5 μL, 10 μM primer 2-F 0.6 μL, 10 μM primer 2-R 0.6 μL, 10 μM primer 3-F 0.15 μL, 10 μM primer 3-R 0.15 μL, 10 μM primer 4-F 0.3 μL, 10 μM primer 4-R 0.3 μL, PCR template 1.0 μL, ultrapure water 5.6 μL, mix well to obtain 25 μL PCR reaction system.

[0085] The above PCR reaction system was subjected to PCR amplification reaction under predetermined PCR reaction parameter conditions to obtain PCR amplification products. The predetermined PCR reaction parameter conditions were: 95°C pre-denaturation for 3 min; 95°C for 15 s, 52°C for 15 s, 72°C for 25 s, 35 cycles; 72°C for 5 min.

[0086] (3) Electrophoresis detection

[0087] Take 10 μL of PCR amplification product, spot it in the well of 2.0% agarose gel electrophoresis plate, electrophoresed for 20 min at 160 V, and photographed under the gel imaging system. If a band of 238 bp appears, the sample to be tested contains serotype 1a of Streptococcus pasteurianus; if a band of 380 bp appears, the sample to be tested contains serotype 1b of Streptococcus pasteurianus; if a band of 834 bp appears, the sample to be tested contains serotype 2 of Streptococcus pasteurianus; if a band of 544 bp appears, the sample to be tested contains serotype 3 of Streptococcus pasteurianus; if a band of 1066 bp appears, the sample to be tested contains serotype 4 of Streptococcus pasteurianus. Among them, no band should appear in the negative control (the product after PCR amplification reaction with H2O instead of the test bacterial solution).

[0088] Among them, the size of the target fragment amplified by the first primer pair (including 1a-F and 1a-R) is 238 bp; the size of the target fragment amplified by the second primer pair (including 1b-F and 1b-R) is 380 bp; the size of the target fragment amplified by the third primer pair (including 2-F and 2-R) is 834 bp; the size of the target fragment amplified by the fourth primer pair (including 3-F and 3-R) is 544 bp; the size of the target fragment amplified by the fifth primer pair (including 4-F and 4-R) is 1066 bp.

[0089] 3. Control Method for Identification of S. Pasteurella Serotypes

[0090] In order to be able to quickly and accurately identify the serotype of Streptococcus pasteurianus, a large number of PCR detection primer combinations have been designed. Table 7 lists one set of control primer combinations, which also includes the first to fifth primer pairs. The five pairs of primers can be used to identify each serotype of Streptococcus pasteurianus. However, when the five pairs of primers are used in the same PCR reaction system to perform multiplex PCR to identify serotype 1a, serotype 1b, serotype 2, serotype 3 and serotype 4 of Streptococcus pasteurianus, the serotypes of Streptococcus pasteurianus cannot be clearly distinguished because the product sizes are too similar.

[0091] Table 7 Specific sequences of control primer combinations

[0092]

[0093] Example 3 Evaluation of the multiplex PCR detection method of the present invention for identifying serotypes of Streptococcus pasteurianus

[0094] In this example, the specificity and sensitivity of the multiplex PCR detection method of the present invention for identifying serotypes 1a, 1b, 2, 3 and 4 of Streptococcus pasteurianus were evaluated respectively.

[0095] 1. Specificity evaluation

[0096] The bacterial liquid of serotype 1a Streptococcus pasteurianus NCTC13784, the bacterial liquid of serotype 1b Streptococcus pasteurianus WUSP067, the bacterial liquid of serotype 2 Streptococcus pasteurianus WUSP070, the bacterial liquid of serotype 3 Streptococcus pasteurianus ATCC43144, the bacterial liquid of serotype 4 Streptococcus pasteurianus WUSP074, the mixed bacterial liquid of 5 strains, Streptococcus agalactiae, Streptococcus multizoans, Klebsiella pneumoniae, Streptococcus dysgalactiae, avian enterococci, enterococci, wandering cocci, cecal enterococci, porcine vaginal Streptococcus and Streptococcus suis were used as templates, and detection was performed according to the multiplex PCR detection method of the present invention, and the PCR products were identified by 2% agarose gel electrophoresis.

[0097] The results are as follows Figure 2As shown, the results show that the mixed bacterial liquid of 5 serotypes of Streptococcus pasteurianus, including serotype 1a, serotype 1b, serotype 2, serotype 3, serotype 4, and Streptococcus pasteurianus, can be amplified to specific bands respectively; while the PCR results of Streptococcus agalactiae, Streptococcus multi-animal, Klebsiella pneumoniae, Streptococcus dysgalactiae, Enterococcus avium, Enterococcus, Wandering coccus, Enterococcus caecum, Streptococcus vaginalis porcine and Streptococcus suis are all negative. The test results show that the multiplex PCR detection method of the present invention has strong specificity and can accurately, quickly and specifically identify serotypes 1a, 1b, 2, 3, and 4 of Streptococcus pasteurianus.

[0098] 2. Sensitivity evaluation

[0099] This example evaluates the sensitivity of the multiplex PCR detection method of the present invention in identifying serotypes 1a, 1b, 2, 3 and 4 of Streptococcus pasteurianus.

[0100] In this example, PCR amplification was performed using 2-fold diluted bacterial DNA and 10-fold diluted bacterial culture as templates.

[0101] (1) Sensitivity test using 2-fold diluted bacterial DNA as template

[0102] Serotype 1a Streptococcus pasteurianus NCTC13784, serotype 1b Streptococcus pasteurianus WUSP067, serotype 2 Streptococcus pasteurianus WUSP070, serotype 3 Streptococcus pasteurianus ATCC43144, and serotype 4 Streptococcus pasteurianus WUSP074 were inoculated into THB liquid medium, and cultured overnight at 37°C to obtain bacterial liquid. Then, genomic DNA was extracted according to the instructions of the Tiangen Bacterial Genomic DNA Extraction Kit, and the genomic DNA concentration of each strain was adjusted to 20 ng / μL. The genomic DNA of each strain was diluted in a gradient according to the following concentrations: 10 ng / μL, 5 ng / μL, 2.5 ng / μL, 1.25 ng / μL, 625 pg / μL, 313 pg / μL, 156 pg / μL, 78 pg / μL, 40 pg / μL, 20 pg / μL, and 10 pg / μL. The genomic DNA of each strain at each concentration was taken as a PCR template, and the serotype was identified by the multiple PCR detection method of the present invention. The negative control was the product of PCR amplification using H2O instead of template. The results showed (as shown in Table 8, Figure 3-7), the multiplex PCR detection method of the present invention can detect a minimum of 78 pg of serotype 1a Streptococcus pasteurianus, 20 pg of serotype 1b Streptococcus pasteurianus, 78 pg of serotype 2 Streptococcus pasteurianus, 39 pg of serotype 3 Streptococcus pasteurianus, and 78 pg of serotype 4 Streptococcus pasteurianus. In addition, 100 μL of the genomic DNA of the above five strains, all with a concentration of 10 ng / μL, were taken respectively, mixed, and then diluted to 7.80 pg / μL in a two-fold ratio. The mixed genomic DNA of each concentration was taken as a template, and the multiplex PCR detection method of the present invention was used to simultaneously identify the species serotypes. A minimum of 31.25 pg of each serotype of Streptococcus pasteurianus could be detected simultaneously. The results are as follows: Figure 8 .

[0103] Table 8 Evaluation results of the sensitivity of the multiplex PCR detection method of the present invention to bacterial genomic DNA

[0104]

[0105] Note: In Table 8, “+” indicates the presence of specific bands, and “-” indicates the absence of specific bands.

[0106] (2) Sensitivity test using 10-fold diluted bacterial culture as template

[0107] Serotype 1a Streptococcus pasteurianus NCTC13784, serotype 1b Streptococcus pasteurianus WUSP067, serotype 2 Streptococcus pasteurianus WUSP070, serotype 3 Streptococcus pasteurianus ATCC43144, and serotype 4 Streptococcus pasteurianus WUSP074 were inoculated into THB liquid culture medium, shaken at 37°C until the stationary phase to obtain bacterial solution, which was then diluted 10 times to obtain bacterial solutions of different concentrations in turn, as shown in Table 9. The bacterial solutions of different concentrations of each strain were used as PCR templates to identify the serotype using the multiplex PCR detection method of the present invention to examine the sensitivity of the multiplex PCR detection method of the present invention. The results show (as shown in Table 9) that the multiplex PCR detection method of the present invention can detect a minimum of 7×10 2 CFU of serotype 1a Streptococcus pasteurianus, 1.3×10 2 CFU of serotype 1b Streptococcus pasteurianus, 8×10 2 CFU of serotype 2 Streptococcus pasteurianus, 1.2×10 3 CFU of serotype 3 Streptococcus pasteurianus and 8×10 2 CFU of 4 serotypes of Streptococcus pasteurianus (electrophoresis diagram as shown in Figure 9-13 ).

[0108] Table 9 Evaluation results of the sensitivity of the bacterial solution for identifying the serotypes of Streptococcus pasteurianus by the multiplex PCR detection method of the present invention

[0109]

[0110] Note: In Table 9, “+”: specific bands appeared; “-”: specific bands did not appear.

[0111] 3. Application of the multiplex PCR detection method of the present invention

[0112] The multiplex PCR detection method of the present invention was used to identify the serotype of 64 isolates of Streptococcus pasteurianus isolated in the laboratory. The results showed that 2 strains of Streptococcus pasteurianus were serotype 1a, 22 strains of Streptococcus pasteurianus were serotype 1b, 3 strains of Streptococcus pasteurianus were serotype 2, 26 strains of Streptococcus pasteurianus were serotype 3, and 11 strains of Streptococcus pasteurianus were serotype 4. This result is consistent with the detection result of the traditional serum agglutination method, that is, the identification result of the multiplex PCR method established by the present invention is completely consistent with the traditional serotype identification result. Therefore, the multiplex PCR detection method of the present invention is used to identify Streptococcus pasteurianus with high accuracy.

Claims

1. A multiplex PCR primer composition for identifying serotypes of Streptococcus pasteurianus, characterized in that: It includes first to fifth primer pairs, the first primer pair includes primers 1a-F and 1a-R, and the sequences are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively; the second primer pair includes primers 1b-F and 1b-R, and the sequences are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively; the third primer pair includes primers 2-F and 2-R, and the sequences are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively; the fourth primer pair includes primers 3-F and 3-R, and the sequences are shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively; the fifth primer pair includes primers 4-F and 4-R, and the sequences are shown in SEQ ID NO: 9 and SEQ ID NO: 10, respectively.

2. The multiplex PCR detection primer composition according to claim 1, characterized in that: The concentration of each primer in the composition is 0.8-1.2 μM.

3. Use of the multiplex PCR detection primer composition according to claim 1 in preparing a kit for identifying serotypes of Streptococcus pasteurianus.

4. A kit comprising the multiplex PCR detection primer composition according to claim 1.

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