Bordetella pertussis ptxP1 / ptxP3 genetic typing detection kit and detection method

Abstract

The invention relates to the technical field of pertussis bordetella detection, in particular to a pertussis bordetella ptxP1 / ptxP3 genotyping detection kit which comprises a detection solution, a positive reference substance and a blank reference substance, the reaction solution comprises MgCl2, dNTP, DMSO, taq enzyme, bovine serum albumin, primers and probes, the positive reference substance is liquid containing target gene plasmids, and the blank reference substance is liquid containing target gene plasmids. The blank reference substance is sterile purified water and can be used for quickly and conveniently completing genotyping detection by taking bordetella pertussis DNA as a detection sample, and meanwhile, the invention also provides a corresponding detection method.

Description

Technical Field

[0001] The present invention relates to the technical field of Bordetella pertussis detection, and particularly to a kit and a detection method for genotyping Bordetella pertussis ptxP1 / ptxP3 genes. Background Art

[0002] Bordetella pertussis (Bp) is the pathogen that causes whooping cough, and the ptxP gene is an important virulence gene in Bordetella pertussis, which is closely related to the pathogenicity of bacteria and the ability to escape vaccines. Therefore, genotyping detection of the ptxP gene of Bordetella pertussis is of great significance in the epidemiological study of whooping cough, vaccine effect evaluation, and formulation of disease prevention and control strategies.

[0003] The ptxP gene is a key virulence gene in Bordetella pertussis, and the pertussis toxin encoded by it is one of the main pathogenic factors of whooping cough. The pertussis toxin can bind to receptors on host cells, enter the cells and interfere with their normal functions, thereby causing the clinical symptoms of whooping cough. There are multiple genotypes of the ptxP gene in Bordetella pertussis, and these genotypes are mainly based on differences in gene sequences. Common ptxP gene genotypes include ptxP1, ptxP2, and ptxP3, etc. Different genotypes may have different pathogenicity, transmission ability, and vaccine escape ability.

[0004] ptxP1 type: In the past, the ptxP1 type was widely prevalent globally, especially in some areas with high vaccine coverage rates. However, with the widespread use of vaccines, the prevalence rate of ptxP1 type strains has gradually decreased. ptxP3 type: In recent years, ptxP3 type strains have gradually become the mainstream in some areas. Compared with the ptxP1 type, ptxP3 type strains may have higher pathogenicity and vaccine escape ability. In addition, ptxP3 type strains may also be related to antibiotic resistance.

[0005] Therefore, when dealing with the condition of whooping cough, it is necessary to understand its specific genotyping situation in order to track the prevalence of Bordetella pertussis, understand its distribution and change trends in different regions and different time periods, which helps to formulate effective prevention and control strategies and reduce the transmission of whooping cough. Existing detection kits cannot meet the detection requirements. Summary of the Invention

[0006] In order to solve the above technical problems, the present invention provides a kit for genotyping Bordetella pertussis ptxP1 / ptxP3 genes, which can use Bordetella pertussis DNA as a detection sample to quickly and conveniently complete genotyping detection. At the same time, the present invention also provides a corresponding detection method.

[0007] Its technical solution is as follows: A Bordetella pertussis ptxP1 / ptxP3 genotyping detection kit, characterized in that it includes a detection solution, a positive control and a blank control. The reaction solution includes MgCl2, dNTP, DMSO, taq enzyme, bovine serum albumin, primers and probes. The positive control is a liquid containing the target gene plasmid, and the blank control is sterile purified water.

[0008] It is further characterized in that the primers and the probes are: Bordetella pertussis ptxP3 type amplification primers F1, R1 and probe P1, and Bordetella pertussis internal reference primers F2, R2 and probe P2; The 5' end of the probe P1 is labeled with the reporter fluorescent dye FAM, and the 3' end is labeled with the quenching fluorescent dye MGB; the 5' end of the probe P2 is labeled with the reporter fluorescent dye VIC, and the 3' end is labeled with the quenching fluorescent dye BHQ; The positive control is: the specific amplification fragment of Bordetella pertussis ptxP3 type is ligated to a pUC57 plasmid vector, and the amplification fragment of Bordetella pertussis internal reference gene is ligated to a pUC57 plasmid vector. The two plasmids are respectively diluted 10 5 times with sterile purified water, and then the two diluted plasmids are mixed in equal volume as the final positive control.

[0009] A Bordetella pertussis genotyping detection method, characterized in that it includes the following steps: (1) Using the Bordetella pertussis DNA solution as a template, and using the positive control and the blank control at the same time, perform fluorescence quantitative PCR detection with the above-mentioned Bordetella pertussis ptxP1 / ptxP3 genotyping detection kit; (2) Data processing: After the reaction is completed, adjust the baseline and threshold line, and judge the result through the Ct value of the sample.

[0010] After adopting the present invention, amplification primers, probes, internal reference primers and probes are designed for the genotyping of Bordetella pertussis. In the final judgment, by comparing the Ct values of the internal reference, the genotyping detection of different genes of different Bordetella pertussis can be realized. Description of the Drawings

[0011] Figure 1 It is the list of primers and probes of the present invention; Figure 2 It is the PCR amplification system table; Figure 3 It is the PCR amplification program table; Figure 4 It is the validity judgment standard table; Figure 5 It is the genotyping interpretation standard table; Figure 6 is the accuracy test result table; Figure 7 is the repeatability test result table; Figure 8 Concentration 10 9 Detection results of Bordetella pertussis ptxP1 type DNA at copies / mL; Figure 9 Concentration 10 7 Detection results of Bordetella pertussis ptxP1 type DNA at copies / mL; Figure 10 Concentration 10 4 Detection results of Bordetella pertussis ptxP1 type DNA at copies / mL; Figure 11 Concentration 10 3 Detection results of Bordetella pertussis ptxP1 type DNA at copies / mL; Figure 12 Concentration 10 9 Detection results of Bordetella pertussis ptxP3 type DNA at copies / mL; Figure 13 Concentration 10 7 Detection results of Bordetella pertussis ptxP3 type DNA at copies / mL; Figure 14 Concentration 10 4 Detection results of Bordetella pertussis ptxP3 type DNA at copies / mL; Figure 15 Concentration 10 3 Detection results of Bordetella pertussis ptxP3 type DNA at copies / mL. Detailed implementation manners

[0012] A Bordetella pertussis ptxP1 / ptxP3 genotyping detection kit includes reaction solution, positive control, and blank control. Among them, The reaction solution contains: MgCl2 (2 mM), dNTP (200 μM), DMSO (5%), taq enzyme (0.05 U / μl), bovine serum albumin (0.1 μg / μl), primers and probes. Among them, the primers and probes are: primers F1, R1 and probe P1 for amplifying Bordetella pertussis ptxP3 type; primers F2, R2 and probe P2 for the internal reference of Bordetella pertussis. Further, the concentrations of F1, R1, P1, F2, R2, and P2 are 0.4 mM, 0.4 mM, 0.3 mM, 0.3 mM, 0.3 mM, and 0.3 mM respectively. The 5' end of probe P1 is labeled with the reporter fluorescent dye FAM, and the 3' end is labeled with the quenching fluorescent dye MGB; the 5' end of probe P2 is labeled with the reporter fluorescent dye VIC, and the 3' end is labeled with the quenching fluorescent dye BHQ. The primer and probe sequences are as Figure 1 shown.

[0013] The positive control is: the specific amplification fragment of Bordetella pertussis ptxP3 type is ligated to a pUC57 plasmid vector, and the amplification fragment of the internal reference gene of Bordetella pertussis is ligated to a pUC57 plasmid vector. The two plasmids are respectively diluted 10 5 times with sterile purified water, and then the two diluted plasmids are mixed in equal volume as the final positive control.

[0014] The blank control is distilled water without any DNA fragments.

[0015] A method for genotyping Bordetella pertussis ptxP1 / ptxP3 genes, characterized in that it comprises the following steps: (1) Using the Bordetella pertussis DNA solution as a template, and simultaneously using the positive control and the blank control, perform fluorescence quantitative PCR detection with the above-mentioned kit for genotyping Bordetella pertussis ptxP1 / ptxP3 genes by fluorescence quantitative PCR. The amplification system of the fluorescence quantitative PCR is as Figure 2 shown, and the reaction conditions of the fluorescence quantitative PCR are as Figure 3 shown.

[0016] Data processing: After the reaction is completed, adjust the baseline and threshold line, and determine the result by the Ct value of the sample.

[0017] PCR amplification and result interpretation: A. Design of primers and probes: Design internal reference primers and probes according to the general DNA sequence of Bordetella pertussis, and design primers and probes for specifically detecting Bordetella pertussis ptxP3 type according to the differential DNA sequence of ptxP1 / ptxP3 type Bordetella pertussis. The primers and probes are as Figure 1 shown in.

[0018] B. Positive control: The specific amplified fragment of Bordetella pertussis ptxP3 type was connected to a pUC57 plasmid vector, and the amplified fragment of Bordetella pertussis internal reference gene was connected to a pUC57 plasmid vector. The two plasmids were respectively purified with sterile water for 10 5 The two diluted plasmids were then mixed in equal volumes as the final positive control.

[0019] C. Blank control: The blank control is distilled water and does not contain any DNA fragments.

[0020] D. Detection: Add 20 μl of reaction solution and 5 μl of nucleic acid sample to a total volume of 25 μl of fluorescent PCR amplification system. PCR amplification reaction conditions are: 95°C pre-denaturation for 10 min, followed by 45 cycles of denaturation at 95°C for 10 s, annealing and extension at 58°C for 45 s.

[0021] E. Data Processing: After the reaction is complete, adjust the Start value, End value, and threshold value of the FAM / VIC channel baseline according to actual conditions (the Start value is recommended to be set between 3 and 15, the End value is recommended to be set between 5 and 20, and the threshold value of both channels is adjusted to 100). Click Analyze to obtain the analysis results and obtain the Ct values of the FAM and VIC channels.

[0022] F. Validity judgment: Positive control and blank control must meet Figure 4 Otherwise, the experimental results will be invalid.

[0023] G. Interpretation of results: Based on Figure 5 Interpret the results.

[0024] Experimental results: The typing specificity test results of the nucleic acid sample of Bordetella pertussis ptxP1: The DNA sample of Bordetella pertussis ptxP1 was diluted to 10 9 , 10 7 , 10 4 , 10 3 The concentration of copies / mL was determined by the fluorescent PCR method established by the present invention. 9 , 10 7 , 10 4 , 10 3 The test results of samples with the concentration of copies / mL are shown in Figures 8 - 11 The results show that in the detection results of ptxP1 type pertussis B. pertussis DNA from high concentration to low concentration, the Ct difference between the FAM channel and the VIC channel is always greater than 5 (or the Ct value of the VIC channel is less than 35 while the Ct value of the FAM channel is not detected). Figure 5According to the genotyping judgment criteria in [specific context], for the Bordetella pertussis ptxP1 type DNA sample, the kit designed by the present invention can make an accurate genotyping judgment through the Ct difference between the FAM channel and the VIC channel.

[0025] Typing specific detection results of Bordetella pertussis ptxP3 type nucleic acid sample: Dilute the Bordetella pertussis ptxP3 type DNA sample to 10 9 、10 7 、10 4 、10 3 copies / mL concentration, and perform genotyping detection using the fluorescence PCR method established by the present invention. The detection results of the samples with concentrations of 10 9 、10 7 、10 4 、10 3 copies / mL are shown in Figures 8 - 11 . It can be seen from the results that in the detection results of Bordetella pertussis ptxP3 type DNA from high concentration to low concentration, the Ct difference between the FAM channel and the VIC channel is always less than 5. According to the genotyping judgment criteria in Table 5, for the Bordetella pertussis ptxP3 type DNA sample, the kit designed by the present invention can make an accurate genotyping judgment through the Ct difference between the FAM channel and the VIC channel.

[0026] Sensitivity test results: The amplification system of the present invention was used to amplify and detect the Bordetella pertussis ptxP1 and ptxP3 type DNA samples with a concentration of 10 3 copies / mL. The results are shown in Figures 12 - 15 . Both DNA samples of the two low concentrations of Bordetella pertussis can be detected, indicating that the sensitivity of the fluorescence PCR method involved in the present invention for detecting Bordetella pertussis ptxP1 and ptxP3 type DNA reaches 1000 copies / mL.

[0027] Accuracy test results: The amplification system of the present invention was used to perform genotyping detection on 20 known genotype Bordetella pertussis positive DNA samples, and the experimental results are shown in Figure 6 . The results indicate that the accuracy of the genotyping detection of the Bordetella pertussis ptxP1 / ptxP3 type samples by the present invention is as high as 100%.

[0028] Repeatability test results: The amplification system of the present invention was used to perform 5 repeated detections on 1 ptxP1 type positive sample and 1 ptxP3 type positive sample. The experimental results are shown in Figure 7As shown, the CV of the CT values of the detection results repeated 5 times is < 3% (CV = standard deviation of CT ÷ average CT value), where the CV value is the coefficient of variation, which is a statistic measuring the degree of variation of each observed value in the measurement index. The fluorescence PCR platform usually uses the CV value of the CT value to measure the detection repeatability, indicating that the detection repeatability of the fluorescence PCR method established according to the present invention in this example is good.

[0029] A Bordetella pertussis ptxP1 / ptxP3 genotyping detection kit related to the present invention, the detection principle of the kit is based on MGB probe qPCR. A minor groove binder (MGB) that can insert into the minor groove of DNA is added to the 3' end of the TaqMan probe, which can greatly increase the Tm value of the probe and enhance the specificity of the hybridization reaction. The 5' end of the MGB probe is labeled with a fluorescent group, and the 3' end is labeled with a non-fluorescent quenching group NFQ. The signal generated by the 5' end fluorescent reporter group is quenched by the NFQ group, avoiding the background generated by the traditional quenching group and reducing the fluorescence background signal. The MGB probe has stronger specificity: since the MGB part is added to the probe, the melting temperature (Tm) of the probe can be increased by 10-15°C. This means that the TaqMan MGB probe can be much shorter than the traditional probe, providing better sequence discrimination ability and flexibility, and can adapt to more targets, especially suitable for SNP genotyping detection. Due to its higher resolution, the MGB probe always maintains no amplification signal or a very low amplification signal in the case of as few as a single base mismatch. With the internal reference, good genotyping detection of single nucleotide polymorphisms can be performed.

Claims

1. A Bordetella pertussis ptxP1 / ptxP3 genotyping detection kit, characterized in that, It includes a detection solution, a positive control and a blank control. The reaction solution includes MgCl2, dNTP, DMSO, taq enzyme, bovine serum albumin, primers and probes. The positive control is a liquid containing a plasmid of the target gene, and the blank control is sterile purified water.

2. The Bordetella pertussis ptxP1 / ptxP3 genotyping detection kit according to claim 1, wherein The primers and the probes are: Bordetella pertussis ptxP3 type amplification primers F1, R1 and probe P1, and Bordetella pertussis internal reference primers F2, R2 and probe P2.

3. The Bordetella pertussis ptxP1 / ptxP3 genotyping detection kit according to claim 1, characterized in that The 5' end of the probe P1 is labeled with a reporter fluorescent dye FAM, and the 3' end is labeled with a quenching fluorescent dye MGB; the 5' end of the probe P2 is labeled with a reporter fluorescent dye VIC, and the 3' end is labeled with a quenching fluorescent dye BHQ.

4. The Bordetella pertussis ptxP1 / ptxP3 genotyping detection kit according to claim 1, wherein, The positive control was: a specific amplified fragment of Bordetella pertussis ptxP3 type connected to a pUC57 plasmid vector, and an amplified fragment of Bordetella pertussis internal reference gene connected to a pUC57 plasmid vector. The two plasmids were respectively purified with sterile water for 10 5 The two diluted plasmids were then mixed in equal volumes as the final positive control.

5. A method for genotyping detection of Bordetella pertussis, characterized in that, It includes the following steps: (1) Using the Bordetella pertussis DNA solution as a template, and simultaneously using the positive control and the blank control, perform fluorescence quantitative PCR detection with the above-mentioned Bordetella pertussis ptxP1 / ptxP3 genotyping detection kit; (2) Data processing: After the reaction ends, adjust the baseline and threshold line, and determine the result through the Ct value of the sample.

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