Primer group for MLVA typing of bordetella pertussis and application of primer group
By providing primer sets for MLVA typing for Baptist pertussis and combining multiplex PCR amplification and NGS sequencing technologies, the problems of high cost, low throughput and long time in the existing methods are solved, and rapid and low-cost multiple sample detection is achieved.
Patent Information
- Application Number
- CN202510564283.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-30
- Publication Date
- 2025-06-20
AI Technical Summary
The existing MLVA typing method of Bauterus pertussis has problems such as high cost, low throughput and long time, and is difficult to be applicable to large-scale testing and screening.
A primer set for MLVA typing of Baptista pertussis can be directly used for the detection of clinically positive samples, and can achieve rapid and low-cost multiple samples detection through multiple PCR amplification and NGS sequencing technologies.
It has achieved rapid and low-cost detection of MLVA typing of Bauterus pertussis, shortened experimental cycles and increased throughput, and is suitable for large-scale testing and screening.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of biotechnology, and particularly to a primer set for multilocus variable-number tandem-repeat (MLVA) typing of Bordetella pertussis and its application. Background Art
[0002] Pertussis is an acute respiratory infectious disease caused by Bordetella pertussis (Bp), which can cause persistent coughing symptoms in children and adults. The genetic backgrounds of pertussis strains vary greatly, and there is a severe lack of data in the molecular epidemiology study of pertussis. In the field of public health, there is an urgent need for a set of efficient and low-cost methods for molecular data monitoring of pertussis epidemiology to predict and track pathogen outbreak events.
[0003] However, existing technical methods have many limitations, which hinder the progress of large-scale genetic background screening of pertussis strains: First, Bordetella pertussis is difficult to culture, requiring a special medium and a long culture period, usually 3-5 days; Second, with the development of high-throughput sequencing technology (NGS), although the whole genome sequencing of bacteria can be performed, this technology relies on cultured strains, resulting in a long detection period and high costs, and is not suitable for large-scale detection and screening; Third, the pathogen capture technology based on probe capture, although this method does not require sample culture and can be directly applied to clinical positive samples, is costly, has cumbersome experimental steps, and a long experimental period, and is also not suitable for large-scale sample detection and screening.
[0004] Multiple-locus variable-number tandem-repeat analysis (MLVA) typing method uses the differences in the number of tandem repeat sequences (Variable-number tandem-repeat, VNTR) at different loci in the genomes of different microorganisms for typing, and is widely used in the research of microorganisms, especially bacteria. This typing method is simple, rapid, has high resolution, can generate clear digital results, and has good repeatability, which is conducive to data exchange, sharing and result comparison among laboratories. By performing MLVA analysis on the VNTR1-6 region of Bordetella pertussis, the genetic differences of different strains can be distinguished, which helps in epidemiological research and pathogen outbreak event tracking.
[0005] However, the existing bacterial MLVA typing uses the first-generation sequencing capillary electrophoresis method for sequencing, which has the problems of high cost, low throughput, and long time consumption. To obtain accurate MLVA typing data, the traditional conventional method requires first isolating and culturing Bordetella pertussis strains on a culture medium, then designing corresponding primers, performing PCR amplification with a single set of primers and then electrophoresis or performing TA cloning and then first-generation Sanger sequencing. On the one hand, the reaction conditions of each primer sequence are different, and the detection process cannot be completed in the same reaction system, resulting in complex operations. On the other hand, the entire experimental cycle usually takes 5 - 7 days, which is time-consuming. In addition, limited by the method, only 1 sequence can be obtained per sequencing by first-generation sequencing, and the throughput is extremely low.
[0006] In view of this, the present invention is specifically proposed. Summary of the Invention
[0007] The object of the present invention is to provide a primer set for Bordetella pertussis MLVA typing and its application. Through the limitation of the primer set, the present invention can be directly used for clinical positive Bordetella pertussis patient samples, and can perform multiplex PCR amplification on Bordetella pertussis VNTR1 - 6, with short time consumption and low cost. Moreover, the NGS sequencing technology can be used to detect multiple samples simultaneously, with high detection efficiency.
[0008] To achieve the above object of the present invention, the following technical solutions are specifically adopted:
[0009] The first aspect of the present invention provides a primer set for Bordetella pertussis MLVA typing, and the primer set for Bordetella pertussis MLVA typing includes the following primer sequences for VNTR 1 - 6;
[0010] The upstream primer of VNTR1 is as shown in SEQ ID NO: 1, and the downstream primer is as shown in SEQ ID NO: 2;
[0011] The upstream primer of VNTR2 is as shown in SEQ ID NO: 3, and the downstream primer is as shown in SEQ ID NO: 4;
[0012] The upstream primer of VNTR3 is as shown in SEQ ID NO: 5, and the downstream primer is as shown in SEQ ID NO: 6;
[0013] The upstream primer of VNTR4 is as shown in SEQ ID NO: 7, and the downstream primer is as shown in SEQ ID NO: 8;
[0014] The upstream primer of VNTR5 is as shown in SEQ ID NO: 9, and the downstream primer is as shown in SEQ ID NO: 10;
[0015] The upstream primer of VNTR6 is shown as SEQ ID NO: 11, and the downstream primer is shown as SEQ ID NO: 12.
[0016] Preferably, the 5' ends of the primer sequences of VNTR 1-6 are respectively modified with phosphate groups.
[0017] The second aspect of the present invention provides an application of the above primer set for Bordetella pertussis MLVA typing in the preparation of a product for Bordetella pertussis MLVA typing.
[0018] Preferably, the product includes biological agents and kits.
[0019] The third aspect of the present invention provides a product for Bordetella pertussis MLVA typing, and the product includes the primer set for Bordetella pertussis MLVA typing.
[0020] The fourth aspect of the present invention provides a method for Bordetella pertussis MLVA typing, and the method for Bordetella pertussis MLVA typing includes the following steps:
[0021] (a) Obtain a sample positive for Bordetella pertussis and perform nucleic acid extraction to obtain a DNA template;
[0022] (b) Perform multiplex PCR amplification on the DNA template using the primer set for Bordetella pertussis MLVA typing described in claim 1 or 2;
[0023] (c) After the amplification is completed, construct a library, perform gene sequencing, and then determine the Bordetella pertussis MLVA typing result according to the sequencing result.
[0024] Preferably, in step (a), sputum, throat swab or bronchoalveolar lavage fluid of a pertussis-positive patient.
[0025] Preferably, in step (b), the multiplex PCR amplification system includes: PrimerSTAR GC buffer, dNTP, polymerase, the primer set for Bordetella pertussis MLVA typing described in claim 1 or 2, DNA template, ddH2O;
[0026] The reaction program is: 94°C for 1 min; 98°C for 10 s, 65°C for 5 s, 72°C for 30 s, 30 cycles; 72°C for 5 min.
[0027] Preferably, in step (c), the sequencing read length in gene sequencing is not less than 150 bp reads.
[0028] Preferably, in step (c), determining the Bordetella pertussis MLVA typing result according to the sequencing result includes:
[0029] Use the fastp software to perform quality control on the original off-machine data, remove adapters and low-quality sequences, and obtain clean_reads;
[0030] Use the K-mer Alignment Model (KMA) software to determine the number of repeat fragments in each VNTR in the clean_reads, and then use the largest repeat number with the highest depth in each VNTR as the final VNTR copy number result. Then, determine the MLVA typing result of Bordetella pertussis according to the copy number result.
[0031] Compared with the prior art, the beneficial effects of the present invention at least include:
[0032] (1) By selecting the primer set for MLVA typing of Bordetella pertussis, the present invention can be directly applied to clinically detect samples positive for pertussis. The sample types can be common clinical sample types such as sputum, throat swabs, and bronchoalveolar lavage fluid, without the need for isolation and culture.
[0033] (2) In the traditional MLVA typing method based on Sanger sequencing, each primer needs to be reacted separately, and only 1 sequence can be obtained by sequencing once, with extremely low throughput and long time consumption. Compared with the traditional MLVA typing based on the first-generation Sanger sequencing technology, the MLVA typing method of Bordetella pertussis in the present invention has a short experimental period, and a large amount of sequencing sequence data can be obtained at one time. Moreover, the 6 groups of primers designed in the present invention can be carried out in the same reaction tube, and the time consumption for one experiment is about 24 hours. Combined with the NGS sequencing technology, the sequencing results of multiple samples can be obtained, and the detection efficiency is high.
[0034] (3) When the typing method of the present invention is used for genetic data monitoring of pertussis MLVA typing, compared with other methods limited by high costs for large-scale applications, it has the advantage of low cost; specifically, to obtain the complete sequence information of a sample by the traditional MLVA method, it is necessary to perform TA cloning combined with Sanger sequencing after PCR amplification, and the detection cost for a single sample usually exceeds 100 yuan (about 100 yuan for TA cloning and usually 10 yuan for Sanger sequencing). The detection cost for a single sample of whole-genome sequencing based on culture usually exceeds 200 yuan. For the pathogen sequencing technology method based on probe capture, because it is necessary to design and synthesize specific probes, the detection cost for a single sample is about 500 yuan; however, for the typing method of the present invention for pertussis MLVA typing, after PCR amplification, directly construct a library and perform NGS sequencing, the detection cost for a single sample is less than 50 yuan (about 20 yuan for library construction and about 10 yuan for sequencing), and sequence information and digital MLVA typing results can be obtained, with good repeatability, which is conducive to data exchange, sharing, and result comparison among laboratories. Detailed implementation manners
[0035] The embodiments of the technical solution of the present invention will be described in detail below in conjunction with the embodiments. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, so they are only used as examples and cannot be used to limit the protection scope of the present invention.
[0036] It should be noted that unless otherwise specified, the technical terms or scientific terms used in this application should have the ordinary meaning understood by those skilled in the art to which the present invention belongs.
[0037] The embodiment of the present invention provides a primer set for the MLVA typing of Bordetella pertussis. The primer set for the MLVA typing of Bordetella pertussis includes the primer sequences of the following VNTR 1-6;
[0038] The upstream primer of VNTR1 is shown in SEQ ID NO: 1, specifically 5'-P-CACCACCACGTCTCCCGCTGCCAGG-3'; the downstream primer is shown in SEQ ID NO: 2, specifically 5'-P-ACAGAGTCAGCCCACATGCCGCAAAA-3';
[0039] The upstream primer of VNTR2 is shown in SEQ ID NO: 3, specifically 5'-P-CGCGCCGCCTACGACCGCTATGG-3'; the downstream primer is shown in SEQ ID NO: 4, specifically 5'-P-CCCGCGCCGAAGCCTGGCGAGATCGATAT-3';
[0040] The upstream primer of VNTR3 is shown in SEQ ID NO: 5, specifically 5'-P-GCCTCGCAATTTCGCCGCTGAAC-3'; the downstream primer is shown in SEQ ID NO: 6, specifically 5'-P-GCGGGCGAGGAAACGCCCGAGACC-3';
[0041] The upstream primer of VNTR4 is shown in SEQ ID NO: 7, specifically 5'-P-CGTGCCCTGCGCCTGGACCTG-3'; the downstream primer is shown in SEQ ID NO: 8, specifically 5'-P-GCCGCTGCTCGACGCCAGGGACAA-3' (P is a phosphorylation group);
[0042] The upstream primer of VNTR5 is shown in SEQ ID NO: 9, specifically 5'-P-CGGCCCACCCGAGCTCCAGGCTCTT-3'; the downstream primer is shown in SEQ ID NO: 10, specifically 5'-P-TGCCGGGTATCTCGATGGGATACG-3';
[0043] The upstream primer of VNTR6 is shown in SEQ ID NO: 11, specifically 5’-P-GTCGGTTACTTCAGGGCGCTGGTCAC-3’; the downstream primer is shown in SEQ ID NO: 12, specifically 5’-P-AACGGCGGTCTGCTGGGTGGTCTGCT-3’.
[0044] In one embodiment, the 5’ ends of the primer sequences of VNTR 1-6 are respectively modified with a phosphoryl group.
[0045] The modification of the 5’ end of the primer with a phosphoryl group enhances its affinity and stability, improves the sensitivity and specificity of the experiment, and can be directly applied to the ligation reaction in the next library construction step, shortening the experimental detection process; by adjusting the PCR amplification reaction system, including the annealing temperature and the ion concentration in the system, specific amplification of all primers (VNTR1-6) is carried out in the same reaction tube.
[0046] Another embodiment of the present invention provides an application of the above primer set for MLVA typing of Bordetella pertussis in the preparation of a product for MLVA typing of Bordetella pertussis.
[0047] In one embodiment, the product includes a biological agent and a kit.
[0048] Another embodiment of the present invention provides a product for MLVA typing of Bordetella pertussis, and the product includes the primer set for MLVA typing of Bordetella pertussis.
[0049] Another embodiment of the present invention provides a method for MLVA typing of Bordetella pertussis, and the method for MLVA typing of Bordetella pertussis includes the following steps:
[0050] (a) Obtain a sample positive for Bordetella pertussis and perform nucleic acid extraction to obtain a DNA template;
[0051] (b) Perform multiplex PCR amplification on the DNA template using the primer set for MLVA typing of Bordetella pertussis;
[0052] (c) After the amplification is completed, perform library construction and gene sequencing, and then determine the MLVA typing result of Bordetella pertussis according to the sequencing result.
[0053] In one embodiment, in the step (a), sputum, throat swab or bronchoalveolar lavage fluid of a pertussis-positive patient.
[0054] In one embodiment, in the step (b), the multiplex PCR amplification system includes: PrimerSTAR GC buffer, dNTP, polymerase, the primer set for Bordetella pertussis MLVA typing according to claim 1 or 2, DNA template, ddH2O;
[0055] The reaction procedure is: 94°C for 1 min; 98°C for 10 s, 65°C for 5 s, 72°C for 30 s, 30 cycles; 72°C for 5 min.
[0056] In one embodiment, in the step (c), the sequencing read length in gene sequencing is not less than 150 bp reads.
[0057] In one embodiment, in the step (c), determining the Bordetella pertussis MLVA typing result according to the sequencing result includes:
[0058] Using the fastp software to perform quality control on the original off-machine data, removing adapters and low-quality sequences to obtain clean_reads;
[0059] Using the K-mer Alignment Model (KMA) software to determine the number of repeat fragments in each VNTR in clean_reads, then taking the repeat number with the largest depth in each VNTR as the final VNTR copy number result, and then determining the Bordetella pertussis MLVA typing result according to the copy number result.
[0060] The technical solution of the present invention will be further described in detail below through specific examples.
[0061] Example 1
[0062] This example is for the verification of the Bordetella pertussis MLVA typing method (NG-MLVA) and its accuracy:
[0063] 1. Obtain sputum samples from 10 patients positive for Bordetella pertussis and perform nucleic acid extraction to obtain DNA templates;
[0064] 2. Use the primer set for Bordetella pertussis MLVA typing to perform multiplex PCR amplification and purification on the DNA template to obtain a multiplex PCR amplification product; the multiplex PCR amplification system is as follows:
[0065] The multiplex amplification reaction system is 50 μL: 12.5 μL of 2X PrimerSTAR GC buffer, 4 μL of dNTP, 1 μL of high-fidelity polymerase, 2 μL of 10 μmol / L primer set, 5 μL of template DNA, 13 μL of ddH2O;
[0066] Reaction procedure: 94°C for 1 min; 98°C for 10 s, 65°C for 5 s, 72°C for 30 s, 30 cycles; 72°C for 5 min;
[0067] The primer set for MLVA typing of Bordetella pertussis is the primer sequences of the above VNTR 1-6 of the present invention, and the 5' end is modified with a phosphoryl group;
[0068] 3. After the amplification is completed, library construction is carried out, specifically including: ligating adapters. The ligation reaction system is 70 μL: 25 μL of ligation buffer, 5 μL of ligase, 30 μL of adapter, and 10 μL of multiplex PCR amplification product; the ligation reaction procedure is: 20°C for 15 min; 75°C for 5 min;
[0069] Library purification: Add 40 μL of purified magnetic beads equilibrated at room temperature to the ligation product, mix well and incubate at room temperature for 5 min, place on a magnetic stand to aspirate and discard the supernatant, wash the magnetic beads twice with 200 μL of washing solution, and finally add 30 μL of elution solution. Incubate at room temperature for 5 min and then place on a magnetic stand, aspirate the supernatant into a new sterile tube to obtain the final second-generation library;
[0070] Library quantification and mixing: Use fluorescence quantitative PCR to dilute the library 10 times for quantification, and finally mix the libraries in equal mass for the on-machine library;
[0071] Then, gene sequencing is carried out. Specifically, after the library is denatured and renatured, a sequencing strategy with a sequencing read length of not less than 150 bp reads is adopted for sequencing; then the sequencing results are analyzed. The analysis methods include:
[0072] First, perform quality control QC on the raw data. Use the fastp software to perform quality control on the raw off-machine data, and remove adapters and low-quality sequences. Use the K-mer Alignment Model (KMA) software to determine the number of repeat fragments in each VNTR for the obtained clean_reads, with all parameters being default parameters. Then, use the largest depth of repeat in each VNTR as the final VNTR copy number result, and then determine the MLVA typing result of Bordetella pertussis based on the copy number result;
[0073] Verification is carried out using the sequences obtained by first-generation Sanger sequencing. Specifically, the primer sequences of VNTR1-6 are amplified with single pairs of primers according to the amplification method in the invention content. After the amplification product is purified, TA cloning is carried out, and the PCR product after picking single colonies is verified by the first-generation Sanger DNA sequencing method using M13Primers.
[0074] 4. Results and analysis
[0075] The results of NG-MLVA typing and Sanger sequencing MLVA typing are shown in Table 1 and Table 2 below; the repeat fragment types of each VNTR region are shown in Table 3, and the specific sequencing sequences are shown in Table 4;
[0076] Table 1 MLVA typing results of Bordetella pertussis by NG-MLVA method
[0077]
[0078]
[0079] Table 2 MLVA typing results of Bordetella pertussis verified by the first-generation Sanger DNA sequencing method
[0080]
[0081] Table 3 Repeat fragment types of each VNTR region
[0082] VNTR Name Base Number Repeat Type VNTR1 15bp
GAACCCGCCAAGCAG
CCGCCCATGCCG
CTGGC
CAAGGACAAGGG
TGGTGC
CGAGCCGCC
[0083] Table 4 Sanger sequencing sequences and NGS sequencing sequences of the example sample C12
[0084]
[0085]
[0086]
[0087] As shown by the results in Tables 1 to 4:
[0088] The results obtained by the NG-MLVA typing data analysis method of the present invention are 100% consistent with the results of the first-generation Sanger sequencing, proving that the NG-MLVA method can accurately perform MLVA typing on Bordetella pertussis.
[0089] Example 2
[0090] This example is a high-throughput sequencing typing analysis of 297 clinical samples (clinical Bordetella pertussis throat swabs, sputum and bronchoalveolar lavage fluid samples) using the typing method of the present invention;
[0091] Experimental results and analysis: The MLVA typing results of 297 samples are shown in Table 5, and the NGS sequencing depths corresponding to each VNTR region of the specimens are shown in Table 6;
[0092] Table 5 NG-MLVA typing results of 297 clinical Bordetella pertussis specimens
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[0096]
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[0102] Note: The unknown prompt may indicate the potential emergence of new MLVA typing;
[0103] Table 6 NGS sequencing depths corresponding to each VNTR region of 297 clinical pertussis specimens
[0104]
[0105]
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[0107]
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[0110]
[0111]
[0112] It can be seen from Table 5 and Table 6 that:
[0113] In one experiment, the NG-MLVA method of the present invention successfully obtained the MLVA typing results of 297 samples in only about 24 hours. The amplification depth of the VNTR1-6 amplification sites of Bordetella pertussis can reach a high amplification efficiency of 500X, and the throughput is significantly higher than that of the traditional first-generation sequencing capillary electrophoresis method. In the same time, the traditional method can only complete the detection of about 10 samples.
[0114] In terms of cost accounting, this method is used for pertussis MLVA typing, and the average detection cost for a single sample is 50 yuan. Due to the large amount of base sequencing, complex data processing and analysis processes involved in whole-genome sequencing technology, the detection cost for a single sample is about 200 yuan; for the pathogen capture technology based on probe capture, because it requires the design and synthesis of specific probes, the cost is relatively high, and the detection cost for a single sample is about 500 yuan. In comparison, the method of the present invention has obvious cost advantages.
[0115] In the analysis of the results of this MLVA typing, some unique VNTR copy number combinations were found and temporarily named unknown. After preliminary comparison, unknown does not match any of the known VNTR copy number patterns of Bordetella pertussis in the database. If further research is carried out on the occurrence of this combination in combination with sample information, more valuable information can be obtained. This cannot be obtained by Sanger sequencing.
[0116] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be covered by the scope of the claims and the specification of the present invention.
Claims
1. A primer set for MLVA typing of Bordetella pertussis, characterized in that: The primer set for MLVA typing of Bordetella pertussis includes the following primer sequences of VNTR 1 to 6; The upstream primer of VNTR1 is shown in SEQ ID NO: 1, and the downstream primer is shown in SEQ ID NO: 2; The upstream primer of VNTR2 is shown in SEQ ID NO: 3, and the downstream primer is shown in SEQ ID NO: 4; The upstream primer of VNTR3 is shown in SEQ ID NO: 5, and the downstream primer is shown in SEQ ID NO: 6; The upstream primer of VNTR4 is shown in SEQ ID NO: 7, and the downstream primer is shown in SEQ ID NO: 8; The upstream primer of VNTR5 is shown in SEQ ID NO: 9, and the downstream primer is shown in SEQ ID NO: 10; The upstream primer of VNTR6 is shown in SEQ ID NO:11, and the downstream primer is shown in SEQ ID NO:
12.
2. The primer set for MLVA typing of Bordetella pertussis according to claim 1, characterized in that: The 5' ends of the primer sequences of VNTR1 to 6 are respectively modified with phosphorylation groups.
3. Use of the primer set for MLVA typing of Bordetella pertussis according to claim 1 or 2 in the preparation of a product for MLVA typing of Bordetella pertussis.
4. The use according to claim 3, characterized in that: The products include biologics and kits.
5. A product for MLVA typing of Bordetella pertussis, characterized in that: The product includes the primer set for MLVA typing of Bordetella pertussis according to claim 1 or 2.
6. A method for MLVA typing of Bordetella pertussis, characterized in that: The Bordetella pertussis MLVA typing method comprises the following steps: (a) obtaining a sample positive for Bordetella pertussis and performing nucleic acid extraction to obtain a DNA template; (b) performing multiple PCR amplification on a DNA template using the primer set for MLVA typing of Bordetella pertussis according to claim 1 or 2; (c) After the amplification is completed, library construction and gene sequencing are performed, and then the MLVA typing results of Bordetella pertussis are determined based on the sequencing results.
7. The method for MLVA typing of Bordetella pertussis according to claim 6, characterized in that: In the step (a), the sputum, throat swab or bronchoalveolar lavage fluid of a pertussis-positive patient is used.
8. The MLVA typing method for Bordetella pertussis according to claim 1, characterized in that: In the step (b), the multiplex PCR amplification system comprises: PrimerSTAR GC buffer, dNTP, polymerase, the primer set for MLVA typing of Bordetella pertussis according to claim 1 or 2, a DNA template, and ddH2O; The reaction program was: 94°C for 1 min; 98°C for 10 s, 65°C for 5 s, 72°C for 30 s, 30 cycles; 72°C for 5 min.
9. The method for MLVA typing of Bordetella pertussis according to claim 1, characterized in that: In the step (c), the sequencing read length in the gene sequencing is not less than 150 bp reads.
10. The MLVA typing method for Bordetella pertussis according to claim 1, characterized in that: In the step (c), determining the MLVA typing result of Bordetella pertussis according to the sequencing result comprises: Use fastp software to perform quality control on the original data, remove the adapters and low-quality sequences, and obtain clean_reads; The K-mer Alignment Model (KMA) software was used to determine the number of repeat fragments in each VNTR in clean_reads, and the number of repeats with the largest depth in each VNTR was used as the final VNTR copy number result, and the MLVA typing result of Bordetella pertussis was determined based on the copy number result.