Kit and method for detecting pertussis bordetella genotype and / or immune antigen related gene variation thereof
By designing multiple PCR primer sets for Bauterus pertussis, they can effectively monitor their genotype and immune antigen-related gene mutations, solving problems such as difficult to detect large fragment insertion and duplicate regions in the prior art, and achieving efficient monitoring of Bauterus pertussis mutations and detection of vaccine immune escape.
Patent Information
- Application Number
- CN202510623241.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-15
- Publication Date
- 2025-06-10
- Estimated Expiration
- 2045-05-15
AI Technical Summary
The prior art is difficult to effectively monitor the genotype and immune antigen-related gene mutations of Bautista pertussis, especially when detecting genetic variant information such as large fragment insertion and duplicate regions.
A primer set was designed to detect the genotype of Bautista pertussis and/or its immunoantigen-related gene variants. Multiple PCR primers were designed based on MLST-related genes and vaccine antigen-related genes, and specific primers were designed for special regions. This could efficiently obtain the results of genotype changes through PCR amplification and sequencing.
It has achieved efficient detection of changes in the genotype and immune antigen-related genotype of Bauterus pertussis, which can monitor vaccine immune escape and improve the monitoring ability of Bauterus pertussis mutation.
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Figure CN120119018A_ABST
Abstract
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 genotype and / or immune antigen-related gene variation. Background Art
[0002] Bordetella pertussis is a Gram-negative bacterium that mainly causes whooping cough, an acute respiratory disease. The typical case is characterized by paroxysmal spasmodic coughing accompanied by cock-like inhalation sounds, which can last for 2-3 months, hence the name "whooping cough". Vaccines play a big role in preventing and treating whooping cough.
[0003] The main vaccines currently used are acellular vaccines (ACVs), which are composed of a variety of purified pertussis antigens, including pertussis toxin A subunit (ptxA), pertussis toxin promoter (ptxP), pertussis adhesin (Prn), fimbriae lectin (Fim) type 2 and type 3, etc. Compared with whole-cell pertussis vaccines, they have higher safety and compliance, but the induced immune effect is slightly worse, and mutations may be driven by the selection pressure of vaccines and antibiotics, thereby causing reduced vaccine preventive efficacy and the emergence of antibiotic resistance. Therefore, it is very necessary to further monitor the bacterial mutations of Bordetella pertussis.
[0004] Multilocus sequence typing (MLST) is a bacterial typing method based on nucleic acid sequence determination. It amplifies multiple housekeeping gene fragments through PCR, determines their sequences, and analyzes strain variation to perform typing. MLST can distinguish minute differences between bacteria of the same species and divide bacteria into more subtypes, which is very helpful for studying the genetic diversity of bacteria, tracking the transmission paths and evolutionary relationships of pathogens. The general MLST is based on seven key guardian genes. In pertussis, adk, fumC, glyA, tyrB, icd, pepA, pgm and other genes are commonly used as molecular typing markers; but in practice, it can also be used to type genes involving immune antigens to evaluate the changing trends of their immune characteristics. It has been successfully used in epidemiological surveys of various bacteria.
[0005] The allelic genotypes of the pertussis vaccine strains Tohama Ⅰ and CS, as well as the early 20th century isolates, are ptxA2 or ptxA4. However, the strains isolated in recent years are mainly ptxA2. Researchers believe that the vaccine containing ptxA2 may have a weaker protective effect against the strains containing the ptxA1 gene. A study on the erythromycin susceptibility and changes in bacterial virulence genotypes of Bordetella pertussis in China from 1970 to 2014 found that the epidemic strain of pertussis in 1970 was ptxA2 / ptxC1 / ptxP1 / prn1 / fim2-1 / fim3-1 / tcfA2, which was exactly matched with the genotype of the vaccine strain. Sequencing of 224 strains isolated from 2015 to 2016 showed that the antigen genotypes of 203 strains (90.6%) were ptxA1 / ptxC1 / ptxP1 / prn1 / fim2-1 / fim3-1 / tcfA2, showing a mismatch with the genotype of the vaccine strain, which was consistent with the trend of the reduced immune protection effect of the pertussis vaccine.
[0006] A study in 2018 detected 50 isolates, among which 31 strains (62%) had the genotype ptxA1 / ptxC2 / ptxP3 / prn2 / fim2-1 / fim3-1, showing significant changes. The change of prn2 was relatively crucial and significant. Internationally, many countries using acellular DTaP vaccines have reported PRN-deficient Bordetella pertussis. And these strains have become dominant in some countries (proportion > 50%). Through whole-genome sequencing of the strains, researchers found that the insertion and deletion of the nucleic acid fragment at the IS481 locus were the most common causes of PRN deficiency. Any mutation in PRN, PTX, or other acellular vaccine antigens may have a serious impact on the efficacy of the current vaccine. In addition, it has now been shown that the immune protection site in the PRN molecule is mainly the GGXXP region, and one of the immunodominant epitopes determined by monoclonal antibodies is the (PQP)5 repeat sequence, and the number of this repeat is also a key point of prn change.
[0007] In addition to the above genes, filamentous hemagglutinin (FHA), fimbriae (FIM), lipooligosaccharide (LOS), etc. are also major virulence factors and have also been used as vaccine antigens. Among them, the FHA gene is very large, containing approximately 12,000 bases. Due to its huge sequence, the conventional sequencing of this gene has not been carried out. Some research reports suggest that FHA can inhibit the immune response by interacting with the nuclear factor (NF)-κB pathway. At the same time, all ER-ptxP1 carry the fhaB3 subtype, while ptxP3 all carry the fhaB1 subtype, forming a very strong correlation. When measuring the efficacy of the whole-cell pertussis vaccine, it was found that in the sera of immunized mice, the antibodies against PRN, FHA, and Fim were high, and the main antibody types were IgG and IgM, indicating the importance of the above antigens in specific humoral immunity.
[0008] Given the large number of molecular typing, virulence factors, and vaccine-related genes of Bordetella pertussis, some of these genes are particularly large and are accompanied by complex mutation situations, 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 all make the detection and analysis work quite challenging. Currently, the conventional detection is limited to using one or two gene fragments for species identification and does not cover in-depth genotype and mutation analysis, so it is unable to effectively monitor the changes in immune antigen typing. As for the simplest and most traditional typing method of 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 detection. Currently, there are mainly two enrichment methods for obtaining the target regions of genes: one is the enrichment technology based on capture probes, which can obtain a wide range of sequence information at a relatively low cost, but this method takes a long time and requires a relatively 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 the multiplex amplification technology based on PCR. Although the target regions obtained by this method are relatively limited, it can quickly enrich low-concentration samples and is therefore more applicable. However, the commonly used multiplex PCR target capture technology at present can only amplify relatively short fragments and is difficult to obtain genetic variation information such as large fragment insertions and repeat regions. For example, it is difficult to detect the short fragment repeats and large fragment 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 the variation of its immune antigen-related genes. The Bordetella pertussis detection primer set is designed as a multiplex PCR primer based on the MLST-related genes of Bordetella pertussis and all vaccine antigen-related genes, and specific primers are designed for special regions. It can detect the genotype of Bordetella pertussis and can also target amplify all vaccine antigen-related genes of Bordetella pertussis. Sequencing and typing are performed based on the amplification products, and the results of the genotype changes related to the immune antigens of Bordetella pertussis can be efficiently obtained.
[0011] For this purpose, the present invention provides the following technical solutions: An embodiment of the present invention provides a primer set for detecting the genotype of Bordetella pertussis and / or the variation of its immune antigen-related genes, including at least one group of the following primers: Primer set 1: includes a forward primer and a reverse primer. The nucleotide sequence of the forward primer is as shown in SEQ ID NO.1, and the nucleotide sequence of the reverse primer is as shown in SEQ ID NO.2; Primer set 2: includes a forward primer and a reverse primer. The nucleotide sequence of the forward primer is as shown in SEQ ID NO.3, and the nucleotide sequence of the reverse primer is as shown in SEQ ID NO.4; Primer set 3: includes a forward primer and a reverse primer. The nucleotide sequence of the forward primer is as shown in SEQ ID NO.5, and the nucleotide sequence of the reverse primer is as shown in SEQ ID NO.6; Primer set 4: includes a forward primer and a reverse primer. The nucleotide sequence of the forward primer is as shown in SEQ ID NO.7, and the nucleotide sequence of the reverse primer is as shown in SEQ ID NO.8; Primer set 5: includes a forward primer and a reverse primer. The nucleotide sequence of the forward primer is as shown in SEQ ID NO.9, and the nucleotide sequence of the reverse primer is as shown in SEQ ID NO.10; Primer set 6: includes a forward primer and a reverse primer. The nucleotide sequence of the forward primer is as shown in SEQ ID NO.11, and the nucleotide sequence of the reverse primer is as shown in SEQ ID NO.12; Primer set 7: includes a forward primer and a reverse primer. The nucleotide sequence of the forward primer is as shown in SEQ ID NO.13, and the nucleotide sequence of the reverse primer is as shown in SEQ ID NO.14; Primer set 8: includes a forward primer and a reverse primer. The nucleotide sequence of the forward primer is as shown in SEQ ID NO.15, and the nucleotide sequence of the reverse primer is as shown in SEQ ID NO.16; Primer set 9: It includes a forward primer and a reverse primer. The nucleotide sequence of the forward primer is as shown in SEQ ID NO.17, and the nucleotide sequence of the reverse primer is as shown in SEQ ID NO.18; Primer set 10: It includes a forward primer and a reverse primer. The nucleotide sequence of the forward primer is as shown in SEQ ID NO.19, and the nucleotide sequence of the reverse primer is as shown in SEQ ID NO.20; Primer set 11: It includes a forward primer and a reverse primer. The nucleotide sequence of the forward primer is as shown in SEQ ID NO.21, and the nucleotide sequence of the reverse primer is as shown in SEQ ID NO.22; Primer set 12: It includes a forward primer and a reverse primer. The nucleotide sequence of the forward primer is as shown in SEQ ID NO.23, and the nucleotide sequence of the reverse primer is as shown in SEQ ID NO.24; Primer set 13: It includes a forward primer and a reverse primer. The nucleotide sequence of the forward primer is as shown in SEQ ID NO.25, and the nucleotide sequence of the reverse primer is as shown in SEQ ID NO.26; Primer set 14: It includes a forward primer and a reverse primer. The nucleotide sequence of the forward primer is as shown in SEQ ID NO.27, and the nucleotide sequence of the reverse primer is as shown in SEQ ID NO.28; Primer set 15: It includes a forward primer and a reverse primer. 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 as shown in SEQ ID NO.29 and SEQ ID NO.31 respectively. 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 as shown in SEQ ID NO.30 and SEQ ID NO.32 respectively; Primer set 16: It includes a forward primer and a reverse primer. The nucleotide sequence of the forward primer is as shown in SEQ ID NO.33, and the nucleotide sequence of the reverse primer is as shown in SEQ ID NO.34; Primer set 17: It includes a forward primer and a reverse primer. 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 as shown in SEQ ID NO.35 and SEQ ID NO.37 respectively. 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 as shown in SEQ ID NO.36 and SEQ ID NO.38 respectively; Primer set 18: It includes a forward primer and a reverse primer. The nucleotide sequence of the forward primer is as shown in SEQ ID NO.39, and the nucleotide sequence of the reverse primer is as shown in SEQ ID NO.40; Primer set 19: It 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 sequences of the first forward primer, the second forward primer, and the third forward primer are as shown in SEQ ID NO.41, SEQ ID NO.43, and SEQ ID NO.45 respectively. The reverse primer includes 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 are as shown in SEQ ID NO.42, SEQ ID NO.44, and SEQ ID NO.46 respectively; Primer set 20: It includes a forward primer and a reverse primer. The forward primer is as shown in SEQ ID NO.47. The reverse primer includes 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 are as shown in SEQ ID NO.48, SEQ ID NO.49, SEQ ID NO.50, and SEQ ID NO.51 respectively; Primer set 21: It 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 as shown in SEQ ID NO.52, the nucleotide sequence of the second forward primer is as shown in SEQ ID NO.54, and the nucleotide sequence of the third forward primer is as 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 as shown in SEQ ID NO.53, the nucleotide sequence of the second reverse primer is as shown in SEQ ID NO.55, and the nucleotide sequence of the third reverse primer is as shown in SEQ ID NO.57.
[0012] In the above embodiments, the primer set 1 is a primer designed for the adk gene; Primer set 2 is a primer designed for the fumC gene; Primer set 3 is a primer designed for the pgm gene; Primer set 4 is a primer designed for the pepA gene; Primer set 5 is a primer designed for the icd gene; Primer set 6 is a primer designed for the tyrB gene; Primer set 7 is a primer designed for the glyA gene; Primer set 8 is a primer designed for the ptxP gene; Primer set 9 is a primer designed for the ptxA gene; Primer set 10 is a primer designed for the ptxB gene; Primer set 11 is a primer designed for the ptxD+E gene; Primer set 12 is a primer designed for the ptxB gene; Primer set 13 is a primer designed for the Fim2 gene; Primer set 14 is a primer designed for the Fim3 gene; Primer set 15 is a primer designed for the bopB gene; Primer set 16 is a primer designed for the bopD gene; Primer set 17 is a primer designed for the bopN gene; Primer set 18 is a primer designed for the bsp22 gene; Primer set 19 is a primer designed for the bteA gene; Primer set 20 is a primer designed for the prn gene; Primer set 21 is a primer designed for the fhaB gene; In the above embodiments, there are insertions of an IS481 sequence of approximately 1077 bases and an IS1002 sequence at 1613 bp in the prn gene. The forward primer SEQ ID NO.47 designed by the present invention at 1450 bp, a downstream primer R (SEQ ID NO.48) is designed at 2131 bp of the prn gene, an R1 (SEQ ID NO.49) is designed at the 407 bp position of the IS481 sequence, a primer R2 (SEQ ID NO.50) is designed at the 682 bp position in the reverse direction of the IS481 sequence, and an R3 (SEQ ID NO.51) is designed at the 421 bp position of the IS1002. These four sequences are placed in the same primer pool. According to different insertion states, different result sequences will be obtained by amplification to complete the insertion detection.
[0013] The embodiments of the present invention provide a kit for detecting the genotype of Bordetella pertussis and / or the variation of its immune antigen-related genes, including: the primer set for detecting the genotype of Bordetella pertussis and / or the variation of its immune antigen-related genes as described above; And, PCR amplification reagents.
[0014] In some embodiments, the PCR amplification reagents include but are not limited to DNA polymerase, dNTPs, buffer solution, and / or magnesium ions.
[0015] In some embodiments, a PCR amplification system is included. Taking 25 μL as an example: DNA template, 200 ng - 400 ng, 2.5 μL; Primers, with the concentration range of each primer being 0.1 - 0.5 μM, 1 μL; 2x Phanta Max Master Mix enzyme system, 12.5 μL; ddH 2 O, 9 μL.
[0016] An embodiment of the present invention provides a method for amplifying Bordetella pertussis genes and / or genes related to its immune antigens, including using the primer set for detecting Bordetella pertussis genotype and / or variation of genes related to its immune antigens or the kit for detecting Bordetella pertussis genotype and / or variation of genes related to its immune antigens to perform amplification on a sample to be tested.
[0017] In some embodiments, the following steps are included: S1. Extract the nucleic acid of the sample to be tested; S2. Using the nucleic acid obtained in step S1 as a template, perform PCR amplification by using the primer set for detecting Bordetella pertussis genotype and / or variation of genes related to its immune antigens or the kit for detecting Bordetella pertussis genotype and / or variation of genes related to its immune antigens.
[0018] In some embodiments, the conditions for the PCR amplification are: maintain at 98 °C for 30 s; maintain at 98 °C for 15 s, maintain at 63 °C for 5 min, for 5 cycles; maintain at 98 °C for 15 s, maintain at 65 °C for 4 min, for 25 cycles; maintain at 72 °C for 2 min, for 1 cycle.
[0019] An embodiment of the present invention provides a method for detecting the genotyping of Bordetella pertussis genes and / or variation of genes related to its immune antigens, including the following steps: Based on the amplification product obtained by the method for amplifying Bordetella pertussis genes and / or genes related to its immune antigens, perform sequencing, data quality control, filtering, alignment analysis, and molecular typing.
[0020] In some embodiments, an insertion sequence analysis step is further included, and the insertion sequence analysis step is located after the alignment analysis step and before the molecular typing step.
[0021] An embodiment of the present invention provides a detection system for the genotyping of Bordetella pertussis genes and / or variation of genes related to its immune antigens, including: A nucleic acid extraction unit for extracting the nucleic acid of Bordetella pertussis; An amplification unit, using the nucleic acid obtained from the nucleic acid extraction unit as a template, for amplifying Bordetella pertussis and / or its immune antigen-related genes by using the primer set for detecting Bordetella pertussis genes and / or their immune antigen-related genes or the kit for detecting Bordetella pertussis genotype and / or variation of its immune antigen-related genes; A sequencing unit, for sequencing the amplification product obtained by the amplification unit; A data quality control and filtering unit, for performing quality control and filtering on the sequencing data obtained by the sequencing unit; An alignment and analysis unit, for performing alignment and analysis on the sequencing data processed by the data quality control and filtering unit with the reference genome of Bordetella pertussis; A molecular typing unit, for typing the alignment and analysis results obtained by the alignment and analysis unit.
[0022] In some embodiments, the reference genome of Bordetella pertussis is based on the NCBI reference genome NZ_CP025371.1; And / or, it further includes an insertion sequence analysis unit, for analyzing the insertion sequence by comparing the sequencing data processed by the data quality control and filtering unit with the reference genome of Bordetella pertussis; the insertion sequence analysis unit obtains a sequence with the upstream sequence of the upstream primer of SEQ ID NO.47 at 1450-1613bp as an anchor point, that is, any one of the sequences amplified by SEQ ID NO.47 and SEQ ID NO.48 - SEQ ID NO.51, and compares it with the reference sequence without insertion and the reference sequences of different insertion sequences. If the alignment consistency is greater than 99%, it indicates the situation of the insertion sequence. The situation of the insertion sequence includes any one of the following: a sequence with a consistency greater than 99% with the NCBI reference sequence AB670737.1 is an IS481 sequence insertion; a sequence with a consistency greater than 99% with the reverse complementary sequence of the NCBI reference sequence AB670737.1 is an IS481 sequence reverse insertion; a sequence with a consistency greater than 99% with the NCBI reference sequence LS483258.1 is an IS1002 sequence insertion; a sequence with a consistency greater than 99% with NZ_CP025371.1 has no insertion; if there is an insertion, the corresponding insertion sequence is added to the alignment result; And / or, in the molecular typing unit, based on the PubMLST Pasteurella pertussis typing database, the MLST software is used for MLST gene typing analysis. A similarity greater than 95% and a coverage greater than 90% are used as the criteria for the same typing to obtain the typing results of each gene.
[0023] Use of the primer set for detecting Bordetella pertussis genotype and mutations in genes related to immune antigens, the kit for detecting Bordetella pertussis genotype and mutations in genes related to immune antigens, or the detection system for genotyping Bordetella pertussis and / or mutations in its genes related to immune antigens in any of the following: (1) Use in differentiating Bordetella pertussis or use in preparing a product for differentiating Bordetella pertussis; (2) Use in monitoring changes in Bordetella pertussis genotype or use in preparing a product for monitoring changes in Bordetella pertussis genotype; (3) Use in monitoring mutations in immune antigens or use in preparing a product for monitoring mutations in immune antigens.
[0024] Further, use in monitoring mutations in Bordetella pertussis immune antigens or use in preparing a product for monitoring mutations in Bordetella pertussis immune antigens.
[0025] The technical solution of the present invention has the following advantages: 1. A primer set for detecting Bordetella pertussis genotype and / or mutations in its genes related to immune antigens provided by the present invention includes at least one group of the following primers, and the primer set includes primer sets 1 to 21; the primer set for detecting Bordetella pertussis is a multiplex PCR primer designed based on all genes related to immune antigens of Bordetella pertussis, and specific primers are designed for special regions at the same time, which can target amplify all genes related to vaccine antigens of Bordetella pertussis. Based on the amplified products for sequencing and genotyping, the results of Bordetella pertussis genotype or changes in its genes related to immune antigens can be efficiently obtained, and it can be applied to differentiate Bordetella pertussis, monitor changes in Bordetella pertussis genotype, and monitor mutations in immune antigens for application in monitoring vaccine immune escape (for immune early warning). BRIEF DESCRIPTION OF THE DRAWINGS
[0026] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.
[0027] Figure 1 It is a flow chart of the detection method for genotyping genes related to vaccine antigens of Bordetella pertussis in Example 5 of the present invention; Figure 2 It is the electrophoresis result of the amplified products of the samples in Example 4 of the present invention; Figure 3 It is the comparative analysis result in Example 4 of the present invention; Figure 4 It is the electrophoresis result of the amplification product obtained by detecting the sample by the conventional method in Experimental Example 1 of the present invention; in the figure, lane 1 is the 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 the 100 kb DNA molecular weight standard; Figure 5 It is the result of detecting the sample by fluorescence PCR method using the existing kit in Experimental Example 1 of the present invention; in the figure, FAM being positive for IS481 indicates that Bordetella pertussis is positive; CY5 being positive for IS1001 indicates that Bordetella parapertussis, a bacterium related to Bordetella pertussis, is positive; VIC is the internal reference, and if either of the two is positive, it shows; as shown in the figure, the result shows that Bordetella pertussis is positive; Figure 6 It is the gene coverage detection result in Experimental Example 1 of the present invention. Detailed implementation manners
[0028] The following embodiments are provided to better further understand the present invention. They are not limited to the described optimal implementation manners, and do not constitute a limitation to the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with those of other existing technologies falls within the protection scope of the present invention.
[0029] For those where specific experimental steps or conditions are not indicated in the embodiments, the operations or conditions of the conventional experimental steps described in the literature in this field can be followed. For the reagents or instruments whose manufacturers are not indicated, they are all conventional reagent products that can be obtained through commercial purchase.
[0030] The PCR amplification enzyme system 2x Phanta Max Master Mix Vazyme involved in the following embodiments is purchased from Nanjing Novoprotein, with the product number P515.
[0031] Experimental Example 1 A primer set for detecting the genotype of Bordetella pertussis and / or the variation of its immune antigen-related genes This embodiment provides a primer set for detecting the genotype of Bordetella pertussis and / or the variation of its immune antigen-related genes, including: Table 1. Primer set
[0032] Table 1 (continued)
[0033] Among them, there are insertions of an IS481 sequence (NCBI serial number M28220.1) approximately 1077 bases in length at 1613 bp of the prn gene and an insertion of an IS1002 sequence (NCBI serial number JX034762.1). The forward primer SEQ ID NO.47 designed by the present invention at 1450 bp, a downstream primer R (SEQ ID NO.48) is designed at 2131 bp of the prn gene, R1 (SEQ ID NO.49) is designed at the 407 bp position of the IS481 sequence, primer R2 (SEQ ID NO.50) is designed at the 682 bp position in the reverse direction of the IS481 sequence, and R3 (SEQ ID NO.51) is designed at the 421 bp position of the IS1002. These four sequences are placed in the same primer pool, and according to different insertion states, different resulting sequences will be amplified to complete the insertion detection.
[0034] Example 2 This example provides a kit for detecting the genotype of Bordetella pertussis and / or the variation of its immune antigen-related genes, including: The primer set for detecting the genotype of Bordetella pertussis and / or the variation of its immune antigen-related genes in Example 1.
[0035] Furthermore, it includes a PCR amplification system, taking 25 μL as an example: DNA template, 200 ng - 400 ng, 2.5 μL; Primer, a mixture of primers SEQ ID NO.1 - SEQ ID NO.57 in Example 1, with the concentration of each primer being 0.2 μM, 1 μL; 2x Phanta Max Master Mix enzyme mixture system, 12.5 μL; ddH 2 O, 9 μL.
[0036] Example 3 This example provides a method for amplifying vaccine antigen-related genes of Bordetella pertussis, including the following steps: S1. Extract and quantify the nucleic acid of the Bordetella pertussis sample. Use a bacterial extraction kit and an automatic nucleic acid extractor to extract the nucleic acid of Bordetella pertussis, and place the extracted product at a low temperature of -20 °C for storage for later use. And use Qubit for nucleic acid quantification. If the concentration is above 100 ng / μl, it meets the requirements and subsequent experimental analysis can be carried out.
[0037] S2. Targetedly amplify the nucleic acid of Bordetella pertussis in step S1. Use a composite multiplex amplification kit and a PCR instrument to complete the targeted amplification, and the targeted amplification is completed using a primer system of about 600 bp. Prepare the PCR amplification system according to the kit in Example 2: PCR amplification system, based on 25 μL: DNA template, 200 ng - 400 ng, 2.5 μl; Primers, a mixture of primers SEQ ID NO.1 to SEQ ID NO.57 in Example 1, with the concentration of each primer being 0.2 μM, 1 μl; 2x Phanta Max Master Mix enzyme mixture system, 12.5 μl; ddH 2 O, 9 μl; The PCR amplification reaction conditions are: hold at 98°C for 30 s; hold at 98°C for 15 s, hold at 63°C for 5 min, for 5 cycles; hold at 98°C for 15 s, hold at 65°C for 4 min, for 25 cycles; hold at 72°C for 2 min, for 1 cycle.
[0038] Example 4 This example provides a method for detecting the genotyping of vaccine antigen-related genes of Bordetella pertussis, including the following steps: S1. Extract and quantify the nucleic acid of the Bordetella pertussis sample. Use a bacterial extraction kit and an automatic nucleic acid extractor to extract the nucleic acid of Bordetella pertussis, and place the extracted product at a low temperature of -20°C for storage for later use. Use Qubit for nucleic acid quantification, and if the concentration is above 100 ng / μl, it meets the requirements and subsequent experimental analysis can be carried out.
[0039] S2. Targetedly amplify the nucleic acid of Bordetella pertussis in step S1. Use a composite multiplex amplification kit and a PCR instrument to complete the targeted amplification, and the targeted amplification is completed using a primer system of about 600 bp. Prepare the PCR amplification system according to the kit in Example 2: PCR amplification system, based on 25 μL: DNA template, 200 ng - 400 ng, 2.5 μL; Primers, a mixture of primers SEQ ID NO.1 to SEQ ID NO.57 in Example 1, with the concentration of each primer being 0.2 μM, 1 μL; 2x Phanta Max Master Mix enzyme mixture system, 12.5 μL; ddH 2 O, 9 μL; The PCR amplification reaction conditions were as follows: hold at 98°C for 30 s; hold at 98°C for 15 s, 63°C for 5 min, for 5 cycles; hold at 98°C for 15 s, 65°C for 4 min, for 25 cycles; hold at 72°C for 2 min, for 1 cycle. The amplified products were electrophoresed, and the results are shown in Table 2 below and Figure 2 : Table 2. Electrophoresis results
[0040] S3. Sequence the Bordetella pertussis amplification products obtained in step S2. The gene sequence determination work was completed using a second-generation sequencing platform, and the library construction was completed using a rapid barcode sequencing kit, mainly including DNA fragmentation and tagging, merging barcode-containing samples, ligating sequencing adapters, chip pretreatment and loading, etc. (operated according to the rapid barcode sequencing kit). The specific steps for constructing the above library were as follows: (1) The initial DNA input amount was 400 ng; (2) The library was purified during the library construction process, and AMPure XP magnetic beads were specifically used for purification; (3) A high-sensitivity quantification kit and a fluorescence spectrometer were used to quantify the library; (4) The sequence determination work was completed using a sequencing chip and a sequencer. In this example, the BGIseq sequencer of BGI or the Illumina miSeq sequencer was used for paired-end sequencing.
[0041] S4. Use the fastQC software to perform quality control on the second-generation data obtained in step S3, and use fastp to filter out the sequencing reads with an average sequencing data quality less than Q30.
[0042] For the data obtained in step S4, the primer sequences of each read were obtained by alignment, and they were cut at their ends to ensure that the obtained sequences were target gene sequences and to avoid chimeric concatenation between amplified sequences. Then, they were aligned with the Bordetella pertussis reference genome (NCBI reference genome NZ_CP025371.1). For the BAM file obtained after alignment, the results after alignment can be seen using software such as IGV (Integrative Genomics Viewer). Exemplary results are as Figure 3 shown; S6. Perform genotyping analysis using MLST to obtain the genotyping results of each gene. Based on the PubMLST Bordetella pertussis typing database (https: / / bigsdb.pasteur.fr / bordetella / ), use the MLST software (https: / / github.com / tseemann / mlst) to perform MLST genotyping analysis. Sequences with a similarity greater than 95% and a coverage greater than 90% are used as the criteria for the same genotype, and the genotyping results of each gene are obtained; compare with the genotype corresponding to the vaccine. If different genotypes appear, immune escape of the vaccine may occur; if the similarity is greater than 95% and less than 100%, it indicates that the sample shows a situation that is not completely consistent with the MLST typing. Use bwa and bcftools to obtain the differential mutation information therein to obtain the variation results of antigen-related genes.
[0043] S7. Comprehensively obtain the MLST genotyping information of each gene and the variation results, and compare with the genotype corresponding to the corresponding vaccine. If different genotypes appear, it indicates that Bordetella pertussis may have immune escape to this vaccine, and the immune variation situation is obtained.
[0044] Example 5 This example provides a method for detecting the genotyping of vaccine antigen-related genes of Bordetella pertussis. As Figure 1 shown, the difference from Example 4 is that between steps S5 and S6, the following steps are also included: discovering the insertion sequence and the status of repeat mutation information; (1) For the sequences amplified by pairing SEQ ID NO.47 of the prn gene with any one of SEQ ID NOs. 48 - 51, respectively align them to the genomic sequences and the template sequences in three cases where insertion sequences exist, and search for whether there are insertion sequences in the genome and what kind of insertion they are; including: sequences with a consistency greater than 99% with the NCBI reference sequence AB670737.1 are IS481 sequence insertions; sequences with a consistency greater than 99% with the reverse complementary sequence of the NCBI reference sequence AB670737.1 are reverse insertions of the IS481 sequence; sequences with a consistency greater than 99% with the NCBI reference sequence LS483258.1 are IS1002 sequence insertions; sequences with a consistency greater than 99% with NZ_CP025371.1 have no insertion; if there is an insertion, add the corresponding insertion sequence to the alignment result; (2) For the repetitive regions of genes such as prn, use NanoSTR to detect the number of repeats and verify it in combination with the amplification length data; If an insertion or duplication mutation is detected, the genome is corrected (integrate the inserted sequence and the sequence of the duplication mutation into the alignment result in step S5, that is, insert the inserted sequence or the sequence of the duplication mutation into the corresponding site in the alignment result sequence according to their respective insertion sites or mutation sites). Experimental Example 1 Take a sample with known genotyping (BP20240807XZ), and perform according to Example 4. Electrophoresis is performed on the amplification product in step S2, and the results are as Figure 2 shown in Table 2. The target gene is subjected to a conventional single PCR method using a traditional method, and electrophoresis is performed on some of the amplification products. The results are as Figure 4 shown; a detection experiment is performed using the Macro-Vetus Bordetella pertussis and Bordetella parapertussis nucleic acid detection kit (fluorescent PCR method) of a commercially available and approved kit on the market. The results of the detected samples are as Figure 5 shown. The amplification product in step S2 is sequenced and aligned through steps S3 - S5, and the gene coverage rate obtained by alignment according to the method of Example 4 of the present invention is statistically shown in Table 3 below and Figure 6 , and take the purified amplification product of BP-1-1 for subsequent sequencing and genotyping: Table 3. Gene coverage rate
[0045] According to step S6 of Example 4, the purified amplification product of BP-1-1 is genotyped. The total genotyping of the detection sample of the present application is MLST_ST2 type. The specific genotyping of each gene is shown in Table 4. The results show that the method of the present invention can detect the genotyping variation of the vaccine antigen-related genes of Bordetella pertussis, and is completely consistent with the results of the control method of whole genome sequencing of this sample, with high accuracy.
[0046] Table 4. Genotyping results
[0047] Obviously, the above embodiments are merely examples for clear illustration and not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.
Claims
1. A primer set for detecting the genotype of Bordetella pertussis and / or its immune antigen-related gene variation, characterized in that: Includes at least one of the following primers: Primer set 1: comprising a forward primer and a reverse primer, the nucleotide sequence of the forward primer is shown in SEQ ID NO.1, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.2; Primer set 2: comprising a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is shown in SEQ ID NO.3, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.4; Primer set 3: comprising a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is shown in SEQ ID NO.5, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.6; Primer set 4: comprising a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is shown in SEQ ID NO.7, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.8; Primer set 5: comprising a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is shown in SEQ ID NO.9, and the nucleotide sequence of the reverse primer is 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, 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; 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: comprising a forward primer and a reverse primer, wherein the forward primer comprises 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 comprises 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: comprising a forward primer and a reverse primer, wherein the forward primer comprises 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; the reverse primer comprises 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, and the nucleotide sequences of the first forward primer, the second forward primer and the third forward primer are shown in SEQ ID NO.41, SEQ ID NO.43 and SEQ ID NO.45, respectively; the reverse primer comprises a first reverse primer, a second reverse primer and a third reverse primer, and the nucleotide sequences of the first reverse primer, the second reverse primer and the third reverse primer are 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 primer 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 genotype and / or its immune antigen-related gene variation, characterized in that: include: A primer set for detecting the genotype of Bordetella pertussis and / or its immune antigen-related gene variation as described in claim 1; and, PCR amplification reagents.
3. A kit for detecting Bordetella pertussis genotype and / or its immune antigen-related gene variation 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. A kit for detecting Bordetella pertussis genotype and / or its immune antigen-related gene variation according to claim 2 or 3, characterized in that: Including PCR amplification system, based on 25μL: DNA template, 200ng-400ng, 2.5μl; Primer set, each primer concentration ranged from 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 thereof, characterized in that: The method comprises amplifying a sample to be tested using a primer set for detecting the genotype of Bordetella pertussis and / or its immune antigen-related gene variation as described in claim 1 or a kit for detecting the genotype of Bordetella pertussis and / or its immune antigen-related gene variation as described in any one of claims 2-4.
6. The method for amplifying a Bordetella pertussis gene and / or its immune antigen-related gene 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 a primer set for detecting the genotype of Bordetella pertussis and / or its immune antigen-related gene variation according to claim 1 or a 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 a Bordetella pertussis gene and / or its immune antigen-related gene according to claim 6, characterized in that: The conditions for the PCR amplification 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 genotype and / or its immune antigen-related gene variation typing, characterized in that: The steps include: The amplified product obtained by the method for amplifying a Bordetella pertussis gene and / or its immune antigen-related gene according to any one of claims 5 to 7 is sequenced, data quality controlled, filtered, compared and analyzed, and molecularly typed.
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 of 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 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 to 4; A sequencing unit, used for sequencing the amplification product obtained by the amplification unit; A data quality control and filtering unit, used for quality control and filtering of the sequencing data obtained by the sequencing unit; A comparison and analysis unit, used for comparing 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, which is used to analyze the sequencing data processed by the data quality control and filtering unit and compare 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 respectively compared with SEQ ID NO.48-SEQ ID Any sequence amplified by NO.51 is compared with a reference sequence without insertion and a reference sequence of a different insertion sequence. If the alignment consistency is greater than 99%, it indicates an insertion sequence. The insertion sequence includes any of the following: a sequence with a consistency greater than 99% with the NCBI reference sequence AB670737.1 is an IS481 sequence insertion; a sequence with a consistency greater than 99% with the reverse complementary sequence of the NCBI reference sequence AB670737.1 is an IS481 sequence reverse insertion; a sequence with a consistency greater than 99% with the NCBI reference sequence LS483258.1 is an IS1002 sequence insertion; a sequence with a consistency greater than 99% with NZ_CP025371.1 has no insertion; if there is an insertion, the corresponding insertion sequence is added to the alignment result; Alternatively, in the molecular typing unit, based on the PubMLST Pasteur pertussis typing database, MLST software is used to perform MLST genotyping analysis, and similarity greater than 95% and coverage greater than 90% are used as the standard for the same typing to obtain the typing results of each gene.
11. Use of a primer set for detecting Bordetella pertussis genotype and / or its immune antigen-related gene variation according to claim 1, a kit for detecting Bordetella pertussis genotype and / or its immune antigen-related gene variation according to any one of claims 2 to 4, or a detection system for typing Bordetella pertussis genotype and / or its immune antigen-related gene variation according to claim 9 or 10 in any of the following: (1) Use in identifying Bordetella pertussis or in preparing a product for identifying Bordetella pertussis; (2) Use in monitoring genotypic changes of Bordetella pertussis or in the preparation of products for monitoring genotypic changes of Bordetella pertussis; (3) Use in monitoring immune antigen variation or in the preparation of products for monitoring immune antigen variation.