A kit and method for detecting Bordetella pertussis genotype and / or its immune antigen-related gene variation

Abstract

The present invention relates to the field of gene detection technology, and relates to a kit and method for detecting Bordetella pertussis genotype and / or its immune antigen-related gene variation. The primer set for detecting Bordetella pertussis genotype and / or its immune antigen-related gene variation provided by the present invention includes at least one group of the following primers, and the primer set includes primer set 1 to primer set 21; the Bordetella pertussis detection primer set is based on the design of multiple PCR primers for all vaccine antigen-related genes of Bordetella pertussis, and at the same time, specific primers are designed for special regions, which can perform targeted amplification of all vaccine antigen-related genes of Bordetella pertussis, perform sequencing and typing based on the amplified products, and efficiently obtain the results of Bordetella pertussis genotype or its immune antigen-related genotype changes, which can be applied to identify Bordetella pertussis, monitor Bordetella pertussis genotype changes, and monitor immune antigen variations for monitoring vaccine immune escape.

Description

Technical Field

[0001] The present invention relates to the technical field of gene detection, and in particular to a kit and method for detecting Bordetella pertussis genotypes and / or immune antigen-related gene variations. Background Art

[0002] Bordetella pertussis is a Gram-negative bacterium that primarily causes whooping cough, an acute respiratory illness. Typical cases are characterized by paroxysmal, spasmodic coughs accompanied by a crowing, inspiratory sound. These symptoms can last for two to three months, hence the name "whooping cough." Vaccines play a significant role in the prevention and treatment of whooping cough.

[0003] The main vaccines currently used are acellular vaccines (ACVs), which are composed of multiple purified pertussis antigens, including pertussis toxin A subunit (ptxA), pertussis toxin promoter (ptxP), pertussis adhesin (Prn), and fimbrial lectins (Fim) type 2 and type 3. Compared with whole-cell pertussis vaccines, they have higher safety and compliance, but the induction of immunity is slightly worse. In addition, they may be driven by the selective pressure of vaccines and antibiotics, leading to mutations, thereby reducing the preventive efficacy of vaccines and the development of antibiotic resistance. Therefore, it is very necessary to further monitor the bacterial mutation of Bordetella pertussis.

[0004] Multilocus sequence typing (MLST) is a bacterial typing method based on nucleic acid sequencing. It uses PCR to amplify multiple housekeeping gene fragments, determine their sequences, and analyze strain variation to perform typing. MLST can distinguish subtle differences between bacteria of the same species and classify them into multiple subtypes, which is very helpful for studying bacterial genetic diversity and tracking the transmission pathways and evolutionary relationships of pathogens. MLST is generally based on seven key guardian genes. In pertussis, genes such as adk, fumC, glyA, tyrB, icd, pepA, and pgm are commonly used as molecular typing markers. However, in practice, it can also be used to type genes related to immune antigens to assess changing trends in immune characteristics. It has been successfully used in epidemiological surveys and studies of various bacteria.

[0005] The allele types of pertussis vaccine strains Tohama Ⅰ and CS and strains isolated in the early 20th century are ptxA2 or ptxA4, while strains isolated in recent years are dominated by ptxA2. Researchers believe that vaccines containing ptxA2 may have weaker protection against strains containing the ptxA1 gene. A study on the changes in the sensitivity of Bordetella pertussis to erythromycin and the bacterial virulence genotype in China from 1970 to 2014 found that the pertussis strain prevalent in 1970 was ptxA2 / ptxC1 / ptxP1 / prn1 / fim2-1 / fim3-1 / tcfA2, which completely matched the vaccine strain genotype. 224 strains isolated from 2015 to 2016 were sequenced, of which 203 (90.6%) had the antigen genotype of ptxA1 / ptxC1 / ptxP1 / prn1 / fim2-1 / fim3-1 / tcfA2, which did not match the vaccine strain genotype, consistent with the trend of reduced immune protection of pertussis vaccine.

[0006] A 2018 study examined 50 isolates, finding that 31 (62%) had the genotype ptxA1 / ptxC2 / ptxP3 / prn2 / fim2-1 / fim3-1, demonstrating significant variation. The variation in prn2 was particularly significant. Internationally, PRN-deficient Bordetella pertussis strains have been reported in many countries using acellular diphtheria, pertussis, and tetanus vaccines. These strains have become dominant in some countries (accounting for >50%). Whole-genome sequencing of these strains revealed that insertions and deletions at the IS481 locus are the most common cause of PRN deficiency. Any mutation in PRN, PTX, or other acellular vaccine antigens could significantly impact the efficacy of current vaccines. Furthermore, it has been shown that the primary immunoprotective site within the PRN molecule is the GGXXP region. One of the dominant immunotopes identified using monoclonal antibodies is the (PQP)5 repeat sequence, and the number of repeats is also a key factor in PRN variation.

[0007] In addition to the aforementioned genes, filamentous hemagglutinin (FHA), fimbriae (FIM), and lipooligosaccharides (LOS) are also major virulence factors and have been used as vaccine antigens. The FHA gene is very large, comprising approximately 12,000 bases. Due to its large size, conventional sequencing of this gene has not yet been performed. Studies have suggested that FHA may inhibit immune responses by interacting with the nuclear factor (NF)-κB pathway. Furthermore, all ER-ptxP1s carry the fhaB3 isoform, while all ptxP3s carry the fhaB1 isoform, forming a strong correlation. When whole-cell pertussis vaccine efficacy was assessed, high levels of antibodies to PRN, FHA, and Fim were found in the serum of immunized mice, primarily in the IgG and IgM classes, demonstrating the importance of these antigens in specific humoral immunity.

[0008] Given the large number of molecular typing, virulence factor and vaccine-related genes of Bordetella pertussis, some of which are particularly large and accompanied by complex mutations, such as gene defects caused by large fragment insertions, short fragment repeat number variations, numerous high GC content regions and a large number of insertion sequences, these factors make detection and analysis quite challenging. Currently, routine testing is limited to the use of one or two gene fragments for species identification and does not cover in-depth genotyping and mutation analysis, so it is impossible to effectively monitor changes in immune antigen typing. As for the simplest and most traditional typing method for Bordetella pertussis - serotyping, since it can only distinguish three serotypes, its role in explaining the epidemiology of pertussis strains is extremely limited.

[0009] High-throughput sequencing technology has become a key tool for pathogen monitoring, and its high-throughput characteristics make it very suitable for large-scale multi-gene testing. Currently, there are two main methods for enriching target gene regions: one is capture probe-based enrichment technology, which can obtain a wide range of sequence information at a low cost, but this method is time-consuming and requires a high concentration of target bacterial DNA. Due to the special detoxification characteristics of Bordetella pertussis, it is difficult to obtain samples that meet the concentration requirements; the other method is PCR-based multiplex amplification technology. Although this method can obtain relatively limited target regions, it can quickly enrich low-concentration samples and is therefore more suitable. However, the currently commonly used multiplex PCR target capture technology can only amplify shorter fragments and has difficulty in obtaining genetic variation information such as large insertions and duplications. For example, there are difficulties in detecting short duplications and large deletions of PRN. Summary of the Invention

[0010] Therefore, the technical problem to be solved by the present invention is to provide a kit and method for detecting the genotype of Bordetella pertussis and / or its immune antigen-related gene variations, wherein the Bordetella pertussis detection primer set is a multiple PCR primer designed based on the MLST-related genes of Bordetella pertussis and all vaccine antigen-related genes, and specific primers are designed for special regions, which can detect the genotype of Bordetella pertussis and can also perform targeted amplification of all vaccine antigen-related genes of Bordetella pertussis, and perform sequencing and typing based on the amplified products, so as to efficiently obtain the results of the genotype changes related to the immune antigens of Bordetella pertussis.

[0011] To this end, the present invention provides the following technical solutions:

[0012] An embodiment of the present invention provides a primer set for detecting Bordetella pertussis genotypes and / or immune antigen-related gene variations, comprising at least one of the following primers:

[0013] Primer set 1: comprising a forward primer and a reverse primer, the nucleotide sequence of the forward primer being shown in SEQ ID NO. 1, and the nucleotide sequence of the reverse primer being shown in SEQ ID NO. 2;

[0014] Primer set 2: comprising a forward primer and a reverse primer, the nucleotide sequence of the forward primer being shown in SEQ ID NO. 3, and the nucleotide sequence of the reverse primer being shown in SEQ ID NO. 4;

[0015] Primer set 3: comprising a forward primer and a reverse primer, the nucleotide sequence of the forward primer being shown in SEQ ID NO. 5, and the nucleotide sequence of the reverse primer being shown in SEQ ID NO. 6;

[0016] Primer set 4: comprising a forward primer and a reverse primer, the nucleotide sequence of the forward primer being shown in SEQ ID NO. 7, and the nucleotide sequence of the reverse primer being shown in SEQ ID NO. 8;

[0017] Primer set 5: comprising a forward primer and a reverse primer, the nucleotide sequence of the forward primer being shown in SEQ ID NO. 9, and the nucleotide sequence of the reverse primer being shown in SEQ ID NO. 10;

[0018] Primer set 6: comprising a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is shown in SEQ ID NO. 11, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO. 12;

[0019] Primer set 7: comprising a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is shown in SEQ ID NO. 13, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO. 14;

[0020] Primer set 8: comprising a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is shown in SEQ ID NO. 15, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO. 16;

[0021] Primer set 9: comprising a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is shown in SEQ ID NO. 17, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO. 18;

[0022] Primer set 10: comprising a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is shown in SEQ ID NO. 19, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO. 20;

[0023] Primer set 11: comprising a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is shown in SEQ ID NO. 21, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO. 22;

[0024] Primer set 12: comprising a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is shown in SEQ ID NO. 23, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO. 24;

[0025] Primer set 13: comprising a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is shown in SEQ ID NO. 25, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO. 26;

[0026] Primer set 14: comprising a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is shown in SEQ ID NO. 27, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO. 28;

[0027] Primer set 15: includes a forward primer and a reverse primer, wherein the forward primer includes a first forward primer and a second forward primer, and the nucleotide sequences of the first forward primer and the second forward primer are shown in SEQ ID NO. 29 and SEQ ID NO. 31, respectively; and the reverse primer includes a first reverse primer and a second reverse primer, and the nucleotide sequences of the first reverse primer and the second reverse primer are shown in SEQ ID NO. 30 and SEQ ID NO. 32, respectively.

[0028] Primer set 16: comprising a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is shown in SEQ ID NO. 33, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO. 34;

[0029] Primer set 17: includes a forward primer and a reverse primer, wherein the forward primer includes a first forward primer and a second forward primer, and the nucleotide sequences of the first forward primer and the second forward primer are shown in SEQ ID NO. 35 and SEQ ID NO. 37, respectively; and the reverse primer includes a first reverse primer and a second reverse primer, and the nucleotide sequences of the first reverse primer and the second reverse primer are shown in SEQ ID NO. 36 and SEQ ID NO. 38, respectively.

[0030] Primer set 18: comprising a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is shown in SEQ ID NO. 39, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO. 40;

[0031] Primer set 19: comprising a forward primer and a reverse primer, wherein the forward primer comprises a first forward primer, a second forward primer, and a third forward primer, the nucleotide sequences of the first forward primer, the second forward primer, and the third forward primer being shown in SEQ ID NO. 41, SEQ ID NO. 43, and SEQ ID NO. 45, respectively; and the reverse primer comprises a first reverse primer, a second reverse primer, and a third reverse primer, the nucleotide sequences of the first reverse primer, the second reverse primer, and the third reverse primer being shown in SEQ ID NO. 42, SEQ ID NO. 44, and SEQ ID NO. 46, respectively.

[0032] Primer set 20: comprising a forward primer and a reverse primer, wherein the forward primer is shown in SEQ ID NO. 47, and the reverse primers include a first reverse primer, a second reverse primer, a third reverse primer, and a fourth reverse primer, wherein the nucleotide sequences of the first reverse primer, the second reverse primer, the third reverse primer, and the fourth reverse primer are shown in SEQ ID NO. 48, SEQ ID NO. 49, SEQ ID NO. 50, and SEQ ID NO. 51, respectively;

[0033] Primer set 21: includes a forward primer and a reverse primer, the forward primer includes a first forward primer, a second forward primer and a third forward primer, the nucleotide sequence of the first forward primer is shown in SEQ ID NO.52, the nucleotide sequence of the second forward primer is shown in SEQ ID NO.54, and the nucleotide sequence of the third forward primer is shown in SEQ ID NO.56; the reverse primer includes a first reverse primer, a second reverse primer and a third reverse primer, the nucleotide sequence of the first reverse primer is shown in SEQ ID NO.53, the nucleotide sequence of the second reverse primer is shown in SEQ ID NO.55, and the nucleotide sequence of the third reverse primer is shown in SEQ ID NO.57.

[0034] In the above embodiment, the primer set 1 is a primer designed for the adk gene;

[0035] Primer set 2 is primers designed for the fumC gene;

[0036] Primer set 3 is primers designed for the pgm gene;

[0037] Primer set 4 is a primer designed for the pepA gene;

[0038] Primer set 5 is a primer designed for the icd gene;

[0039] Primer set 6 is a primer designed for the tyrB gene;

[0040] Primer set 7 is a primer designed for the glyA gene;

[0041] Primer set 8 is a primer designed for the ptxP gene;

[0042] Primer set 9 is a primer designed for the ptxA gene;

[0043] Primer set 10 is a primer designed for the ptxB gene;

[0044] Primer set 11 is a primer designed for the ptxD+E gene;

[0045] Primer set 12 is a primer designed for the ptxB gene;

[0046] Primer set 13 is a primer designed for the Fim2 gene;

[0047] Primer set 14 is a primer designed for the Fim3 gene;

[0048] Primer set 15 is a primer designed for the bopB gene;

[0049] Primer set 16 is a primer designed for the bopD gene;

[0050] Primer set 17 is a primer designed for the bopN gene;

[0051] Primer set 18 is a primer designed for the bsp22 gene;

[0052] Primer set 19 is a primer designed for the bteA gene;

[0053] Primer set 20 is a primer designed for the prn gene;

[0054] Primer set 21 is designed for the fhaB gene;

[0055] In the above embodiment, the prn gene contains an insertion of an approximately 1077-base IS481 sequence and an insertion of an IS1002 sequence at position 1613 bp. The present invention designs a forward primer (SEQ ID NO. 47) at position 1450 bp, a downstream primer (SEQ ID NO. 48) at position 2131 bp of the prn gene, R1 (SEQ ID NO. 49) at position 407 bp of the IS481 sequence, R2 (SEQ ID NO. 50) at position 682 bp of the reverse direction of the IS481 sequence, and R3 (SEQ ID NO. 51) at position 421 bp of the IS1002 sequence. These four sequences are placed in the same primer pool, and different amplification sequences are obtained based on the different insertion states, thereby completing insertion detection.

[0056] An embodiment of the present invention provides a kit for detecting Bordetella pertussis genotypes and / or immune antigen-related gene variations, comprising: a primer set for detecting Bordetella pertussis genotypes and / or immune antigen-related gene variations;

[0057] and, PCR amplification reagents.

[0058] In some embodiments, the PCR amplification reagents include, but are not limited to, DNA polymerase, dNTPs, buffer, and / or magnesium ions.

[0059] In some embodiments, a PCR amplification system is included, based on 25 μL:

[0060] DNA template, 200 ng-400 ng, 2.5 μl;

[0061] Primers, each primer concentration range 0.1-0.5 μM, 1 μl;

[0062] 2x Phanta Max Master Mix, 12.5 μl;

[0063] ddH2O, 9μl.

[0064] An embodiment of the present invention provides a method for amplifying Bordetella pertussis genes and / or their immune antigen-related genes, comprising amplifying a sample to be tested using the primer set for detecting Bordetella pertussis genotypes and / or their immune antigen-related gene variations or the kit for detecting Bordetella pertussis genotypes and / or their immune antigen-related gene variations.

[0065] In some embodiments, the method comprises the steps of:

[0066] S1. Extracting nucleic acid from the sample to be tested;

[0067] S2. Using the nucleic acid obtained in step S1 as a template, perform PCR amplification using the primer set for detecting the genotype of Bordetella pertussis and / or its immune antigen-related gene variation or the kit for detecting the genotype of Bordetella pertussis and / or its immune antigen-related gene variation.

[0068] In some embodiments, the PCR amplification conditions are: 98°C for 30s; 98°C for 15s, 63°C for 5min, 5 cycles; 98°C for 15s, 65°C for 4min, 25 cycles; 72°C for 2min, 1 cycle.

[0069] The present invention provides a method for detecting Bordetella pertussis genotypes and / or immune antigen-related gene variant typing, comprising the following steps:

[0070] Based on the amplification method of a Bordetella pertussis gene and / or its immune antigen-related gene, an amplification product is obtained, and sequencing, data quality control, filtering, comparison analysis, and molecular typing are performed.

[0071] In some embodiments, the method further comprises an insertion sequence analysis step, which is located after the alignment analysis step and before the molecular typing step.

[0072] The present invention provides a system for detecting Bordetella pertussis genotypes and / or immune antigen-related gene variation typing, comprising:

[0073] A nucleic acid extraction unit, used for extracting nucleic acid from Bordetella pertussis;

[0074] an amplification unit, using the nucleic acid obtained in the nucleic acid extraction unit as a template, for amplifying Bordetella pertussis and / or its immune antigen-related genes using the primer set for detecting Bordetella pertussis genes and / or its immune antigen-related genes or the kit for detecting Bordetella pertussis genotypes and / or its immune antigen-related gene variations;

[0075] A sequencing unit, used for sequencing the amplification product obtained by the amplification unit;

[0076] A data quality control and filtering unit, used to perform quality control and filtering on the sequencing data obtained by the sequencing unit;

[0077] an alignment and analysis unit, for aligning and analyzing the sequencing data processed by the data quality control and filtering units with the reference genome of Bordetella pertussis;

[0078] The molecular typing unit is used to type the comparison and analysis results obtained by the comparison and analysis unit.

[0079] In some embodiments, the reference genome of Bordetella pertussis is based on the NCBI reference genome NZ_CP025371.1;

[0080] And / or, it also includes an insertion sequence analysis unit for analyzing the sequencing data processed by the data quality control and filtering unit and comparing it with the reference genome of Bordetella pertussis to analyze the insertion sequence; the insertion sequence analysis unit obtains the sequence based on the upstream sequence 1450-1613bp of the upstream primer of SEQ ID NO.47 as the anchor point, that is, SEQ ID NO.47 is compared with SEQ ID NO.48-SEQ ID Any sequence amplified by NO.51 was aligned with a reference sequence without insertion and a reference sequence with a different insertion sequence. If the alignment identity was greater than 99%, it was indicated as an insertion sequence. The insertion sequence included any of the following: a sequence with a greater than 99% identity to the NCBI reference sequence AB670737.1 was an IS481 sequence insertion; a sequence with a greater than 99% identity to the reverse complementary sequence of the NCBI reference sequence AB670737.1 was an IS481 sequence reverse insertion; a sequence with a greater than 99% identity to the NCBI reference sequence LS483258.1 was an IS1002 sequence insertion; a sequence with a greater than 99% identity to NZ_CP025371.1 was not an insertion; if there was an insertion, the corresponding insertion sequence was added to the alignment result;

[0081] And / or, in the molecular typing unit, based on the PubMLST Pasteur pertussis typing database, MLST software is used to perform MLST genotyping analysis, and a similarity greater than 95% and a coverage greater than 90% are used as the standard for the same typing to obtain the typing results of each gene.

[0082] Use of the primer set for detecting Bordetella pertussis genotype and immune antigen-related gene variation, the kit for detecting Bordetella pertussis genotype and immune antigen-related gene variation, or the detection system for typing Bordetella pertussis genotype and / or immune antigen-related gene variation in any of the following:

[0083] (1) Use in identifying Bordetella pertussis or in preparing a product for identifying Bordetella pertussis;

[0084] (2) Use in monitoring genotypic changes of Bordetella pertussis or in preparing a product for monitoring genotypic changes of Bordetella pertussis;

[0085] (3) Use in monitoring immune antigen variation or in preparing products for monitoring immune antigen variation.

[0086] Furthermore, the invention relates to a use in monitoring the variation of Bordetella pertussis immune antigens or a use in preparing a product for monitoring the variation of Bordetella pertussis immune antigens.

[0087] The technical solution of the present invention has the following advantages:

[0088] 1. The present invention provides a primer set for detecting the genotype of Bordetella pertussis and / or its immune antigen-related gene variation, comprising at least one group of the following primers, wherein the primer set includes primer set 1 to primer set 21; the primer set for detecting Bordetella pertussis is a multiplex PCR primer designed based on all immune antigen-related genes of Bordetella pertussis, and specific primers are designed for special regions. It can perform targeted amplification on all vaccine antigen-related genes of Bordetella pertussis, perform sequencing and typing based on the amplified products, and efficiently obtain the results of changes in the genotype of Bordetella pertussis or its immune antigen-related genotype. It can be used to identify Bordetella pertussis, monitor changes in the genotype of Bordetella pertussis, and monitor immune antigen variations for monitoring vaccine immune escape (immune early warning). BRIEF DESCRIPTION OF THE DRAWINGS

[0089] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0090] Figure 1 This is a flow chart of the method for detecting vaccine antigen-related genotyping of Bordetella pertussis in Example 5 of the present invention;

[0091] Figure 2 is the electrophoresis result of the amplified product of the sample in Example 4 of the present invention;

[0092] Figure 3 is the comparison analysis result in Example 4 of the present invention;

[0093] Figure 4 This is the electrophoresis result of the amplified products detected by conventional methods in Experimental Example 1 of the present invention. In the figure, lane 1 is a 0.1-10 kb DNA molecular weight standard; lane 2 is Prn; lane 3 is PtxA; lane 4 is Fim3; lane 5 is Fim2; lane 6 is PtxP; lane 7 is Pgm; lane 8 is IS481; lane 9 is IS1002; lane 10 is IS1001; lane 11 is a 100 kb DNA molecular weight standard.

[0094] Figure 5 This is the result of fluorescent PCR testing of samples using an existing kit in Experimental Example 1 of the present invention; in the figure, FAM is IS481 positive, which means Bordetella pertussis is positive; CY5 is IS1001 positive, which means Bordetella parapertussis, a closely related bacterium of Bordetella pertussis, is positive; VIC is an internal reference, and a positive result of either of the two is displayed; as shown in the figure, the result is Bordetella pertussis positive;

[0095] Figure 6 This is the gene coverage detection result in Experimental Example 1 of the present invention. DETAILED DESCRIPTION

[0096] The following examples are provided for a better understanding of the present invention and are not intended to limit the best mode of implementation. They do not limit the content and scope of protection of the present invention. Any product identical or similar to the present invention obtained by anyone under the guidance of the present invention or by combining the features of the present invention with other prior arts shall fall within the scope of protection of the present invention.

[0097] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in this field can be used. If the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional reagents.

[0098] The PCR amplification enzyme system 2x Phanta Max Master Mix Vazyme involved in the following examples was purchased from Nanjing Novozymes, product number P515.

[0099] Example 1 A primer set for detecting Bordetella pertussis genotypes and / or immune antigen-related gene mutations

[0100] This example provides a primer set for detecting Bordetella pertussis genotypes and / or immune antigen-related gene variations, including:

[0101] Table 1. Primer sets

[0102]

[0103] Table 1 (Continued)

[0104]

[0105] The prn gene contains an insertion of the approximately 1077-base IS481 sequence (NCBI sequence number M28220.1) and the IS1002 sequence (NCBI sequence number JX034762.1) at 1613 bp. The present invention designed a forward primer, SEQ ID NO. 47, at 1450 bp. A downstream primer, R (SEQ ID NO. 48), was designed at 2131 bp in the prn gene. Primer R1 (SEQ ID NO. 49) was designed at 407 bp in the IS481 sequence. Primer R2 (SEQ ID NO. 50) was designed at 682 bp in the reverse direction of the IS481 sequence. And primer R3 (SEQ ID NO. 51) was designed at 421 bp in the IS1002 sequence. These four sequences were placed in the same primer pool. Different amplification sequences were obtained based on the different insertion states, completing insertion detection.

[0106] Example 2

[0107] This embodiment provides a kit for detecting Bordetella pertussis genotypes and / or immune antigen-related gene variations, comprising:

[0108] The primer set for detecting Bordetella pertussis genotypes and / or immune antigen-related gene variations in Example 1.

[0109] Furthermore, a PCR amplification system is included, based on 25 μL:

[0110] DNA template, 200 ng-400 ng, 2.5 μL;

[0111] Primers: a mixture of primers SEQ ID NO. 1 to SEQ ID NO. 57 in Example 1, each primer at a concentration of 0.2 μM, 1 μL;

[0112] 2x Phanta Max Master Mix, 12.5 μL;

[0113] ddH2O, 9 μL.

[0114] Example 3

[0115] This embodiment provides a method for amplifying vaccine antigen-related genes of Bordetella pertussis, comprising the following steps:

[0116] S1. Extract and quantify nucleic acid from Bordetella pertussis samples. Use a bacterial extraction kit and an automated nucleic acid extractor to extract nucleic acid from Bordetella pertussis. Store the extracted product at -20°C until ready for use. Quantify the nucleic acid using a Qubit assay. A concentration above 100 ng / μl meets the requirements and is suitable for subsequent experimental analysis.

[0117] S2. Targeted amplification of the nucleic acid of Bordetella pertussis in step S1 is performed using a composite multiplex amplification kit and a PCR instrument to complete the targeted amplification. The targeted amplification is completed using a primer system of about 600 bp. The PCR amplification system is prepared according to the kit in Example 2:

[0118] PCR amplification system, based on 25 μL:

[0119] DNA template, 200 ng-400 ng, 2.5 μl;

[0120] Primers: a mixture of primers SEQ ID NO. 1 to SEQ ID NO. 57 in Example 1, each primer at a concentration of 0.2 μM, 1 μl;

[0121] 2x Phanta Max Master Mix, 12.5 μl;

[0122] ddH2O, 9 μl;

[0123] The PCR amplification reaction conditions were as follows: 98°C for 30 s; 98°C for 15 s, 63°C for 5 min, 5 cycles; 98°C for 15 s, 65°C for 4 min, 25 cycles; 72°C for 2 min, 1 cycle.

[0124] Example 4

[0125] This embodiment provides a method for detecting vaccine antigen-related genotyping of Bordetella pertussis, comprising the following steps:

[0126] S1. Extract and quantify nucleic acid from Bordetella pertussis samples. Use a bacterial extraction kit and an automated nucleic acid extractor to extract nucleic acid from Bordetella pertussis. Store the extracted product at -20°C until ready for use. Quantify the nucleic acid using a Qubit assay. A concentration above 100 ng / μl meets the requirements and is suitable for subsequent experimental analysis.

[0127] S2. Targeted amplification of the nucleic acid of Bordetella pertussis in step S1 is performed using a composite multiplex amplification kit and a PCR instrument to complete the targeted amplification. The targeted amplification is completed using a primer system of about 600 bp. The PCR amplification system is prepared according to the kit in Example 2:

[0128] PCR amplification system, based on 25 μL:

[0129] DNA template, 200 ng-400 ng, 2.5 μL;

[0130] Primers: a mixture of primers SEQ ID NO. 1 to SEQ ID NO. 57 in Example 1, each primer at a concentration of 0.2 μM, 1 μL;

[0131] 2x Phanta Max Master Mix, 12.5 μL;

[0132] ddH2O, 9 μL;

[0133] The PCR amplification reaction conditions were as follows: 98°C for 30 seconds; 98°C for 15 seconds, 63°C for 5 minutes, 5 cycles; 98°C for 15 seconds, 65°C for 4 minutes, 25 cycles; 72°C for 2 minutes, 1 cycle. The amplified products were subjected to electrophoresis, and the results were shown in Table 2 and Table 3. Figure 2 :

[0134] Table 2. Electrophoresis results

[0135]

[0136] S3. Sequencing the amplified product of Bordetella pertussis obtained in step S2, using a second-generation sequencing platform to complete gene sequence determination, and using a rapid barcode sequencing kit to complete library construction, mainly including DNA fragmentation and tagging, merging barcoded samples, connecting sequencing adapters, chip pretreatment and sample loading, etc. (operation is performed according to the rapid barcode sequencing kit). The specific steps for constructing the above library are as follows: (1) the starting DNA input amount is 400 ng; (2) the library is purified during the library construction process, specifically using AMPure XP magnetic beads for purification; (3) the library is quantified using a high-sensitivity quantification kit and a fluorimeter; (4) sequencing is completed using a sequencing chip and a sequencer. In this embodiment, a BGI sequencer BGIseq or an Illumina miSeq sequencer is used for double-end sequencing.

[0137] S4. Use fastQC software to perform quality control on the second-generation data obtained in step S3, and use fastp to filter out sequencing reads with an average sequencing data quality less than Q30.

[0138] S5. For the data obtained in step S4, the primer sequence of each read segment is obtained by comparison, and the primer sequence is cut at its end to ensure that the obtained sequence is the target gene sequence and to avoid chimeric concatenation between amplified sequences. Then, the primer sequence is compared with the reference genome of Bordetella pertussis (NCBI reference genome NZ_CP025371.1) for comparison analysis. The BAM file obtained after comparison can be viewed using software such as IGV (Integrative Genomics Viewer). An exemplary result is shown in FIG. Figure 3 As shown;

[0139] S6. Use MLST for genotyping analysis to obtain the typing results of each gene. Based on the PubMLST Pasteur Pertussis Typing Database (https: / / bigsdb.pasteur.fr / bordetella / ), use MLST software (https: / / github.com / tseemann / mlst) for MLST genotyping analysis. A similarity greater than 95% and a coverage greater than 90% are used as the standard for the same typing to obtain the typing results of each gene. Compare with the corresponding genotype of the vaccine. If different genotypes appear, immune escape from the vaccine may occur. If the similarity is greater than 95% and less than 100%, it means that the sample is not completely consistent with the MLST typing. Use bwa and bcftools to obtain the differential mutation information to obtain the variation results of the antigen-related genes.

[0140] S7. Comprehensively obtain the MLST typing information of each gene and the mutation results, and compare them with the genotypes corresponding to the corresponding vaccines. The presence of different genotypes indicates that the pertussis Bordetella may have immune escape from the vaccine and obtain the immune mutation situation.

[0141] Example 5

[0142] This embodiment provides a method for detecting vaccine antigen-related genotyping of Bordetella pertussis, such as Figure 1 As shown, the difference from Example 4 is that between steps S5 and S6, the following steps are further included: discovering the inserted sequence and the repeated mutation information status;

[0143] (1) The sequence amplified by pairing SEQ ID NO.47 of the prn gene with any one of SEQ ID NO.48-SEQ ID NO.51 was aligned to the genome sequence and the template sequence of the three cases where the insertion sequence existed, to find out whether there was an insertion sequence in the genome and what kind of insertion it was; including: sequences with a consistency greater than 99% with the NCBI reference sequence AB670737.1 were insertions of the IS481 sequence; sequences with a consistency greater than 99% with the reverse complementary sequence of the NCBI reference sequence AB670737.1 were insertions of the IS481 sequence; sequences with a consistency greater than 99% with the NCBI reference sequence LS483258.1 were insertions of the IS1002 sequence; sequences with a consistency greater than 99% with the NZ_CP025371.1 were insertions; if there was an insertion, the corresponding insertion sequence was added to the alignment result;

[0144] (2) For the repetitive regions of genes such as prn, NanoSTR is used to detect the number of repeats and verify them in combination with the amplification length data;

[0145] If an insertion or duplication mutation is detected, the genome is corrected (the inserted sequence and the duplication mutation sequence are integrated into the alignment result in step S5, that is, the inserted sequence or the duplication mutation sequence are inserted into the corresponding site in the alignment result sequence according to their respective insertion sites or mutation sites).

[0146] Experimental Example 1

[0147] Take a sample with known genotype (BP20240807XZ) and perform electrophoresis on the amplified product in step S2 according to Example 4. The results are as follows: Figure 2 As shown in Table 2, conventional single-plex PCR was performed on the target gene using the traditional method and electrophoresis was performed on some of the amplified products. Figure 4 As shown; the detection experiment was carried out using the Macromicro Tess Pertussis and Bordetella parapertussis Nucleic Acid Detection Kit (Fluorescence PCR Method), which is a commercial kit approved on the market. The results of the test samples are shown in Figure 5 The amplified products in step S2 were sequenced and aligned in steps S3-S5, and the gene coverage was obtained by alignment according to the method of Example 4 of the present invention. The statistical results are shown in Tables 3 and Figure 6 , take the purified amplification product of BP-1-1 for subsequent sequencing and typing:

[0148] Table 3. Gene coverage

[0149]

[0150] According to step S6 of Example 4, the purified amplification product of BP-1-1 was typed. The overall typing of the test sample of the present application was MLST_ST2 type. The specific typing of each gene is shown in Table 4. The results show that the method of the present invention is capable of detecting vaccine antigen-related gene typing variations of Bordetella pertussis, and is completely consistent with the results of the control method of whole genome sequencing of the sample, with high accuracy.

[0151] Table 4. Typing results

[0152]

[0153] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A primer set for detecting Bordetella pertussis genotypes and / or immune antigen-related gene variations, characterized in that: It consists of the following primers: Primer set 1: comprising a forward primer and a reverse primer, the nucleotide sequence of the forward primer being shown in SEQ ID NO. 1, and the nucleotide sequence of the reverse primer being shown in SEQ ID NO. 2; Primer set 2: comprising a forward primer and a reverse primer, the nucleotide sequence of the forward primer being shown in SEQ ID NO. 3, and the nucleotide sequence of the reverse primer being shown in SEQ ID NO. 4; Primer set 3: comprising a forward primer and a reverse primer, the nucleotide sequence of the forward primer being shown in SEQ ID NO. 5, and the nucleotide sequence of the reverse primer being shown in SEQ ID NO. 6; Primer set 4: comprising a forward primer and a reverse primer, the nucleotide sequence of the forward primer being shown in SEQ ID NO. 7, and the nucleotide sequence of the reverse primer being shown in SEQ ID NO. 8; Primer set 5: comprising a forward primer and a reverse primer, the nucleotide sequence of the forward primer being shown in SEQ ID NO. 9, and the nucleotide sequence of the reverse primer being shown in SEQ ID NO. 10; Primer set 6: comprising a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is shown in SEQ ID NO. 11, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO. 12; Primer set 7: comprising a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is shown in SEQ ID NO. 13, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO. 14; Primer set 8: comprising a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is shown in SEQ ID NO. 15, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO. 16; Primer set 9: comprising a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is shown in SEQ ID NO. 17, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO. 18; Primer set 10: comprising a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is shown in SEQ ID NO. 19, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO. 20; Primer set 11: comprising a forward primer and a reverse primer, the nucleotide sequence of the forward primer being shown in SEQ ID NO. 21, and the nucleotide sequence of the reverse primer being shown in SEQ ID NO. 22; Primer set 12: comprising a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is shown in SEQ ID NO. 23, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO. 24; Primer set 13: comprising a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is shown in SEQ ID NO. 25, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO. 26; Primer set 14: comprising a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is shown in SEQ ID NO. 27, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO. 28; Primer set 15: includes a forward primer and a reverse primer, wherein the forward primer includes a first forward primer and a second forward primer, and the nucleotide sequences of the first forward primer and the second forward primer are shown in SEQ ID NO. 29 and SEQ ID NO. 31, respectively; and the reverse primer includes a first reverse primer and a second reverse primer, and the nucleotide sequences of the first reverse primer and the second reverse primer are shown in SEQ ID NO. 30 and SEQ ID NO. 32, respectively. Primer set 16: comprising a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is shown in SEQ ID NO. 33, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO. 34; Primer set 17: includes a forward primer and a reverse primer, wherein the forward primer includes a first forward primer and a second forward primer, and the nucleotide sequences of the first forward primer and the second forward primer are shown in SEQ ID NO. 35 and SEQ ID NO. 37, respectively; and the reverse primer includes a first reverse primer and a second reverse primer, and the nucleotide sequences of the first reverse primer and the second reverse primer are shown in SEQ ID NO. 36 and SEQ ID NO. 38, respectively. Primer set 18: comprising a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is shown in SEQ ID NO. 39, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO. 40; Primer set 19: comprising a forward primer and a reverse primer, wherein the forward primer comprises a first forward primer, a second forward primer, and a third forward primer, the nucleotide sequences of the first forward primer, the second forward primer, and the third forward primer being shown in SEQ ID NO. 41, SEQ ID NO. 43, and SEQ ID NO. 45, respectively; and the reverse primer comprises a first reverse primer, a second reverse primer, and a third reverse primer, the nucleotide sequences of the first reverse primer, the second reverse primer, and the third reverse primer being shown in SEQ ID NO. 42, SEQ ID NO. 44, and SEQ ID NO. 46, respectively. Primer set 20: comprising a forward primer and a reverse primer, the nucleotide sequence of the forward primer being shown in SEQ ID NO. 47; the reverse primers comprising a first reverse primer, a second reverse primer, a third reverse primer, and a fourth reverse primer, the nucleotide sequences of the first reverse primer, the second reverse primer, the third reverse primer, and the fourth reverse primer being shown in SEQ ID NO. 48, SEQ ID NO. 49, SEQ ID NO. 50, and SEQ ID NO. 51, respectively; Primer set 21: includes a forward primer and a reverse primer, the forward primer includes a first forward primer, a second forward primer and a third forward primer, the nucleotide sequence of the first forward primer is shown in SEQ ID NO.52, the nucleotide sequence of the second forward primer is shown in SEQ ID NO.54, and the nucleotide sequence of the third forward primer is shown in SEQ ID NO.56; the reverse primer includes a first reverse primer, a second reverse primer and a third reverse primer, the nucleotide sequence of the first reverse primer is shown in SEQ ID NO.53, the nucleotide sequence of the second reverse primer is shown in SEQ ID NO.55, and the nucleotide sequence of the third reverse primer is shown in SEQ ID NO.

57.

2. A kit for detecting Bordetella pertussis genotypes and / or immune antigen-related gene variations, characterized in that: include: A primer set for detecting Bordetella pertussis genotypes and / or immune antigen-related gene variations according to claim 1; and, PCR amplification reagents.

3. A kit for detecting Bordetella pertussis genotypes and / or immune antigen-related gene variations according to claim 2, characterized in that: The PCR amplification reagents include but are not limited to DNA polymerase, dNTPs, buffer and magnesium ions.

4. The kit for detecting Bordetella pertussis genotype and / or immune antigen-related gene variation according to claim 2 or 3, characterized in that: Including PCR amplification system, based on 25 μL: DNA template, 200 ng-400 ng, 2.5 μl; Primer set, each primer concentration range 0.1-0.5 μM, 1 μl; 2x Phanta Max Master Mix, 12.5 μl; ddH2O, 9μl.

5. A method for amplifying Bordetella pertussis genes and / or immune antigen-related genes for non-disease diagnosis, characterized in that: The method comprises amplifying a sample to be tested using a primer set for detecting Bordetella pertussis genotype and / or its immune antigen-related gene variation according to claim 1 or a kit for detecting Bordetella pertussis genotype and / or its immune antigen-related gene variation according to any one of claims 2-4.

6. The method for amplifying Bordetella pertussis genes and / or immune antigen-related genes thereof for non-disease diagnosis according to claim 5, characterized in that: The steps include: S1. Extracting nucleic acid from the sample to be tested; S2. Using the nucleic acid obtained in step S1 as a template, perform PCR amplification using the primer set for detecting the genotype of Bordetella pertussis and / or its immune antigen-related gene variation according to claim 1 or the kit for detecting the genotype of Bordetella pertussis and / or its immune antigen-related gene variation according to any one of claims 2-4.

7. The method for amplifying Bordetella pertussis genes and / or immune antigen-related genes thereof for non-disease diagnosis according to claim 6, characterized in that: The PCR amplification conditions were as follows: 98° C. for 30 s; 98° C. for 15 s, 63° C. for 5 min, 5 cycles; 98° C. for 15 s, 65° C. for 4 min, 25 cycles; 72° C. for 2 min, 1 cycle.

8. A method for detecting Bordetella pertussis genotypes and / or immune antigen-related gene variation typing for non-disease diagnosis, characterized in that: The steps include: A method for amplifying the Bordetella pertussis gene and / or its immune antigen-related gene for non-disease diagnosis according to any one of claims 5-7 is used to obtain amplified products, and then sequence, perform data quality control, filter, compare and analyze, and perform molecular typing.

9. A detection system for Bordetella pertussis genotype and / or its immune antigen-related gene variation typing, characterized in that: include: A nucleic acid extraction unit, used for extracting nucleic acid from Bordetella pertussis; an amplification unit, using the nucleic acid obtained in the nucleic acid extraction unit as a template, for amplifying Bordetella pertussis and / or its immune antigen-related gene using the primer set for detecting Bordetella pertussis genotype and / or its immune antigen-related gene variation according to claim 1 or the kit for detecting Bordetella pertussis genotype and / or its immune antigen-related gene variation according to any one of claims 2 to 4; A sequencing unit, used for sequencing the amplification product obtained by the amplification unit; A data quality control and filtering unit, used to perform quality control and filtering on the sequencing data obtained by the sequencing unit; an alignment and analysis unit, for aligning and analyzing the sequencing data processed by the data quality control and filtering units with the reference genome of Bordetella pertussis; The molecular typing unit is used to type the comparison and analysis results obtained by the comparison and analysis unit.

10. A detection system for Bordetella pertussis genotype and / or its immune antigen-related gene variation typing according to claim 9, characterized in that: The reference genome of Bordetella pertussis is based on the NCBI reference genome NZ_CP025371.1; Or, it also includes an insertion sequence analysis unit for analyzing the sequencing data processed by the data quality control and filtering unit and comparing it with the reference genome of Bordetella pertussis to analyze the insertion sequence; the insertion sequence analysis unit obtains the sequence based on the upstream sequence 1450-1613bp of the upstream primer of SEQ ID NO.47 as the anchor point, that is, SEQ ID NO.47 is compared with SEQ ID NO.48-SEQ ID Any sequence amplified by NO.51 was aligned with a reference sequence without insertion and a reference sequence with a different insertion sequence. If the alignment identity was greater than 99%, it was indicated as an insertion sequence. The insertion sequence included any of the following: a sequence with a greater than 99% identity to the NCBI reference sequence AB670737.1 was an IS481 sequence insertion; a sequence with a greater than 99% identity to the reverse complementary sequence of the NCBI reference sequence AB670737.1 was an IS481 sequence reverse insertion; a sequence with a greater than 99% identity to the NCBI reference sequence LS483258.1 was an IS1002 sequence insertion; a sequence with a greater than 99% identity to NZ_CP025371.1 was not an insertion; if there was an insertion, the corresponding insertion sequence was added to the alignment result; Alternatively, in the molecular typing unit, MLST genotyping analysis is performed using MLST software based on the PubMLST Pasteur pertussis typing database, with a similarity greater than 95% and a coverage greater than 90% used as the standard for the same typing to obtain the typing results of each gene.

11. Use of the primer set for detecting Bordetella pertussis genotypes and / or immune antigen-related gene variations according to claim 1, the kit for detecting Bordetella pertussis genotypes and / or immune antigen-related gene variations according to any one of claims 2 to 4, or the detection system for detecting Bordetella pertussis genotypes and / or immune antigen-related gene variation typing according to claim 9 or 10 in any of the following: (1) Use in the preparation of products for identifying Bordetella pertussis; (2) Use in the preparation of products for monitoring genotypic changes of Bordetella pertussis; (3) Use in the preparation of products for monitoring immune antigen variation.

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