Primer combination and kit for real-time fluorescent quantitative PCR detection of pertussis bordetella 23S rRNA drug-resistant site variation

Abstract

The invention discloses a primer combination and a kit for real-time fluorescent quantitative PCR detection of Bordetella pertussis 23S rRNA drug-resistant site variation, and the primer combination comprises a target point amplification primer group, a wild type fluorescent probe and a mutant type fluorescent probe, and can also comprise an internal reference amplification primer group and an internal reference detection fluorescent probe, the kit disclosed by the invention can be used for real-time fluorescent quantitative PCR detection of drug-resistant site variation of 23S rRNA of the bordetella pertussis, so that a detection result about whether the bordetella pertussis exists or not and the drug resistance of erythromycin can be obtained. The detection result has the advantages of high accuracy, high sensitivity, short detection window period and the like, can be applied to the fields of clinical diagnosis, public health monitoring, scientific research and the like, improves the detection efficiency and drug resistance detection level of pertussis infectious diseases, provides a scientific basis for prevention, control and treatment of pertussis infection, and has a wide application prospect. The method has important application value and wide market prospect.

Description

Technical Field

[0001] The invention relates to the fields of life science and biotechnology, and in particular to a kit for detecting the A2047G mutation of the erythromycin resistance site of the 23SrRNA gene of Bordetella pertussis. Background Art

[0002] Whooping cough is a regional acute respiratory infectious disease with an epidemic cycle of 2 to 5 years. It infects humans through cough droplets and is a Class B statutory infectious disease that is strictly managed in my country. Bordetella pertussis (Bp) is the main pathogen that causes whooping cough. People are generally susceptible to it, but it mainly causes respiratory diseases in children. The younger the age, the more serious the disease. It is easy to develop complications such as suffocation, convulsions, pneumonia, whooping encephalopathy, cardiovascular system disorders, and malnutrition that seriously threaten children's life and health. Infants under 6 months old are at high risk. Timely diagnosis and treatment of the source of infection and vaccination to protect susceptible people are the most effective ways to block the spread of the epidemic.

[0003] The incubation period of pertussis infection is generally 7 to 10 days, or even up to 21 days. The course of the disease can be divided into the catarrhal stage, the attack stage, and the recovery stage. For patients with typical symptoms, clinical diagnosis can be made directly, which is fast and simple. However, for vaccinated, adolescents and adults, most of them are atypical cases, and they still need to complete laboratory related tests before they can be confirmed. Laboratory tests mainly include etiology, serology and other methodologies, which are the most important means and guarantee for the diagnosis and differential diagnosis of pertussis infection, especially for patients with atypical clinical symptoms.

[0004] In recent years, the incidence of pertussis in China and even in the world has increased significantly. Macrolide antibiotics represented by erythromycin are the first choice antibiotics for the treatment of pertussis and preventive medication. Therefore, the resistance of Bordetella pertussis Bp to erythromycin has also attracted more and more attention. A large number of studies have confirmed that the A2047G mutation of the 23S rRNA gene of Bordetella pertussis is related to its resistance to erythromycin, that is, the wild-type strain (sensitive strain) is the A2047 site, and the mutant strain (resistant strain) is the 2047G site. Gene sequencing technology can directly detect whether the resistance site is mutated, which is also the recognized gold standard in the industry, but the technology is relatively expensive and time-consuming, and is not suitable for ordinary microbiological laboratories, especially in economically underdeveloped areas. Therefore, it is necessary to establish a simple and rapid kit detection method for detecting the variation of the 23S rRNA resistance site of Bordetella pertussis, which can conveniently and quickly help the laboratory to judge the erythromycin resistance of Bordetella pertussis, and buy time for early diagnosis, early guidance of medication, and early treatment of the infectious disease. Summary of the invention

[0005] In view of the fact that nucleic acid detection directly detects pathogen nucleic acid, it has the advantages of high sensitivity, high accuracy and short detection window period, and can buy time for early diagnosis, early guidance of medication and early treatment of infectious diseases. Therefore, the present invention provides a primer combination and a kit for detecting 23S rRNA resistance site variants of Bordetella pertussis using real-time fluorescence quantitative PCR, which can quickly detect whether Bordetella pertussis is resistant to erythromycin.

[0006] The technical solution adopted by the present invention is:

[0007] The primer combination for detecting the variation of the drug resistance site of Bordetella pertussis 23S rRNA by real-time fluorescence quantitative PCR comprises a target amplification primer set, a wild-type fluorescent probe, and a mutant fluorescent probe, and the sequence is as follows:

[0008] Target amplification primer set:

[0009] Bp-F:GTTAAGTGATGGGGTGCAAGCT

[0010] Bp-R:TGGCGACTCGAGTTCTGCGC;

[0011] Wild-type fluorescent probe Bp-probe-W: CTAGACGGAAAGACCCCA

[0012] Mutant fluorescent probe Bp-probe-T:TAGACGGGAAGACCCCAT.

[0013] Furthermore, the primer combination also includes an internal reference amplification primer set and an internal reference detection fluorescent probe, the sequences of which are as follows:

[0014] Internal reference amplification primer set

[0015] GAPDH-F:GGCTGCTCACATATTCTG

[0016] GAPDH-R:AAGAGACAAGAGGCAAGA

[0017] Internal reference detection fluorescent probe GAPDH-probe: AGCCTCCCCTCCTCATGCC.

[0018] The 5' end of the wild-type fluorescent probe, mutant fluorescent probe and internal reference detection fluorescent probe is labeled with a fluorescent reporter group, and the 3' end is labeled with a fluorescent quencher group; and the fluorescent reporter groups of the wild-type fluorescent probe, mutant fluorescent probe and internal reference detection fluorescent probe are different.

[0019] Furthermore, the fluorescent reporter group is one of FAM, HEX, CY5, TET, JOE or TAMRA, and the fluorescent quencher group is one of BHQ1, BHQ2, BHQ3 or MGB.

[0020] Preferably, the 5' end of the wild-type fluorescent probe is labeled with a fluorescent reporter group FAM, and the 3' end is labeled with a fluorescent quencher group MGB, and the sequence is Bp-probe-W:

[0021] FAM-CTAGACGGAAAGACCCCA-MGB.

[0022] Preferably, the 5' end of the mutant fluorescent probe is labeled with a fluorescent reporter group HEX, and the 3' end is labeled with a fluorescent quencher group MGB, and the sequence is Bp-probe-T: HEX-TAGACGGGAAGACCCCAT-MGB.

[0023] Preferably, the 5' end of the internal reference detection fluorescent probe is labeled with a fluorescent reporter group CY5, and the 3' end is labeled with a fluorescent quencher group BHQ2, and the sequence is GAPDH-probe:

[0024] CY5-AGCCTCCCCTCCTCATGCC-BHQ2.

[0025] The present invention also provides a kit for detecting the variation of 23S rRNA drug resistance sites of Bordetella pertussis by real-time fluorescence quantitative PCR. The kit comprises a target amplification primer set, a wild-type fluorescent probe, a mutant fluorescent probe, an internal reference amplification primer set and an internal reference detection fluorescent probe.

[0026] The kit for detecting the drug resistance site variation of Bordetella pertussis 23S rRNA by real-time fluorescence quantitative PCR can also include a real-time fluorescence quantitative PCR amplification reaction reagent, which includes an amplification enzyme, a buffer, etc., and a commercial real-time fluorescence quantitative PCR detection reagent can be used.

[0027] The kit may also include quality control products, including Bordetella pertussis resistance positive quality control products, Bordetella pertussis resistance negative quality control products and Bordetella pertussis negative quality control products.

[0028] The drug-resistant positive quality control product of Bordetella pertussis is a DNA plasmid containing the sequence shown in SEQ NO 1 and the internal reference sequence shown in SEQ NO 3.

[0029] The Bordetella pertussis drug-resistant negative control product is a DNA plasmid containing the sequence shown in SEQ NO 2 and the internal reference sequence shown in SEQ NO 3.

[0030] The Bordetella pertussis negative control product is a DNA plasmid that does not contain Bordetella pertussis DNA and contains the internal reference sequence shown in SEQ NO3.

[0031] SEQ NO 1 is a sequence fragment containing the 23S rRNA resistance site A2047G mutation:

[0032] AAGGTTAAGTGATGGGGTGCAAGCTCTTGATCGAAGCCCCGGTAAACGGCGGCCGTAACTATAACGGTCCTAAGGTAGCGAAATTCCTTGTCGGGTAAGTTCCGACCTGCACGAATGGCGTAACGATGGCCACACTGTCTCCTCCTGAG ACTCAGCGAAGTTGAAGTGTTTGTGATGATGCAATCTACCCGCGGCTAGACGGGAAGACCCCATGAACCTTTACTGTAGCTTTGCATTGGACTGTGAACCGGCCTGTGTAGGATAGGTGGGAGGCGCAGAACTCGAGTCGCCAGATTCGA

[0033] Pertussis drug-resistant negative control product, the plasmid includes the following sequence shown in SEQ NO 2:

[0034] SEQ NO 2:

[0035] AAGGTTAAGTGATGGGGTGCAAGCTCTTGATCGAAGCCCCGGTAAACGGCGGCCGTAACTATAACGGTCCTAAGGTAGCGAAATTCCTTGTCGGGTAAGTTCCGACCTGCACGAATGGCGTAACGATGGCCACACTGTCTCCTCCTGAG ACTCAGCGAAGTTGAAGTGTTTGTGATGATGCAATCTACCCGCGGCTAGACGGAAAGACCCCATGAACCTTTACTGTAGCTTTGCATTGGACTGTGAACCGGCCTGTGTAGGATAGGTGGGAGGCGCAGAACTCGAGTCGCCAGATTCGA

[0036] SEQ NO 3, internal reference sequence:

[0037] GGCTGCTCACATATTCTGGAGGAGCCTCCCCTCCTCATGCCTTCTTGCC TCTTGTCTCTT.

[0038] The present invention also provides a method for detecting the drug resistance site variation of Bordetella pertussis 23S rRNA by real-time fluorescence quantitative PCR. The method adopts the kit for detecting the drug resistance site variation of Bordetella pertussis 23S rRNA by real-time fluorescence quantitative PCR, performs real-time fluorescence quantitative PCR detection on a sample to be tested, and obtains the drug resistance of Bordetella pertussis in the sample to be tested. The method comprises the following steps:

[0039] (1) Extracting nucleic acid from the sample to be tested;

[0040] (2) Using the extracted nucleic acid as template DNA, a fluorescence quantitative PCR detection system is prepared, wherein the fluorescence quantitative PCR detection system includes a target amplification primer set, a wild-type fluorescent probe, a mutant fluorescent probe, an internal reference amplification primer set and an internal reference detection fluorescent probe, and fluorescence quantitative PCR detection is performed. According to the amplification curves of three different fluorescent channels, it is analyzed to determine whether the sample to be tested contains Bordetella pertussis and the drug resistance of Bordetella pertussis.

[0041] Furthermore, in step (2), the fluorescent quantitative PCR detection system includes a target amplification primer set, a wild-type fluorescent probe, a mutant fluorescent probe, an internal reference amplification primer set and an internal reference detection fluorescent probe, qPCR Mix, 50×ROX and sterile water.

[0042] The concentrations of the target amplification primer set, the wild-type fluorescent probe, the mutant fluorescent probe, the internal reference amplification primer set and the internal reference detection fluorescent probe are each 1 to 10 μM, preferably 10 μM.

[0043] Preferably, the fluorescent quantitative PCR detection system is a 20 μL system, comprising the following components: 10 μL qPCR Mix, 0.32 μL wild-type fluorescent probe Bp-probe-W, 0.32 μL mutant fluorescent probe Bp-probe-T, 0.32 μL internal reference detection fluorescent probe GAPDH-probe, 0.64 μL target amplification primer Bp-F, 0.64 μL target amplification primer Bp-R, 0.64 μL internal reference amplification primer GAPDH-F, 0.64 μL internal reference amplification primer GAPDH-R, 0.4 μL 50×ROX, 1.8 μL template DNA, 4.28 μL ddH 2 O.

[0044] The reaction program of real-time fluorescence quantitative PCR was as follows: pre-denaturation at 95°C for 1 min; denaturation at 95°C for 10 s, annealing and extension at 58°C for 35 s, and 40 cycles.

[0045] Furthermore, in step (2), analysis is performed based on the amplification curves of three different fluorescence channels, and the specific steps are as follows:

[0046] A) Quality control products determine the validity of test results:

[0047] ① The HEX channel and CY5 channel of the pertussis Bordetella pertussis resistance positive control product both produce amplification curves, the FAM channel has no amplification curve, and the Ct value is ≤38;

[0048] ② The FAM channel and CY5 channel of the pertussis Bordetella pertussis resistant negative control both produced amplification curves, and the Ct value was ≤38, and there was no amplification curve in the HEX channel;

[0049] ③ The negative control product of Bordetella pertussis has an amplification curve in the CY5 channel, and the Ct value is ≤38, and there is no amplification curve in the FAM channel and the HEX channel;

[0050] If the above three points are met, the experimental results are considered reliable and valid; if any of the above items do not meet the requirements, the experimental results are invalid;

[0051] B) If the experimental results of condition A) are valid, the judgment is made as follows:

[0052] 1) If both the FAM channel and the CY5 channel of the sample to be tested produce amplification curves, and the Ct value is ≤38, and there is no amplification curve in the HEX channel, the sample is judged to be pertussis positive and pertussis resistance negative;

[0053] 2) If both the HEX channel and the CY5 channel of the sample to be tested produce an amplification curve and the Ct value is ≤38, and there is no amplification curve in the FAM channel, the sample is judged to be pertussis positive and pertussis resistance positive;

[0054] 3) If there is no amplification curve in the FAM channel and HEX channel of the sample to be tested, but there is an amplification curve in the CY5 channel and the Ct value is ≤38, the sample is judged to be pertussis negative;

[0055] 4) If there is no curve in the CY5 channel of the sample to be tested, the sample is judged to be unqualified;

[0056] In the real-time fluorescence quantitative PCR detection method of the present invention, three probes are respectively labeled with different fluorescent markers (FAM, HEX, CY5), and one sample can simultaneously detect the pertussis Bordetella pertussis 23S rRNA gene and the human internal reference GAPDH gene in a one-tube PCR reaction system, and can simultaneously detect the A2047 resistant wild-type strain (sensitive strain) whose erythromycin resistance site A2047G has not been mutated and the mutated 2047G resistant mutant (resistant strain) of Bordetella pertussis.

[0057] The beneficial effects of the present invention are as follows: by using the kit of the present invention, real-time fluorescence quantitative PCR is performed on the DNA of the extracted nasopharyngeal swab sample, so as to detect whether the nucleic acid of Bordetella pertussis exists in the sample and whether the 23S rRNA resistance site is mutated, thereby obtaining the detection results of Bordetella pertussis and erythromycin resistance. The detection results of the present invention have the advantages of high accuracy, high sensitivity and short detection window period, and can be applied to clinical diagnosis, public health monitoring and scientific research and other fields, improve the detection efficiency of pertussis infectious diseases and the level of drug resistance detection, contribute to early diagnosis, early guidance of medication, early treatment, provide a scientific basis for the prevention, control and treatment of pertussis infection, and have important application value and broad market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 This is the result of fluorescence quantitative PCR detection under FAM channel.

[0059] Figure 2 This is the fluorescence quantitative PCR detection result diagram under the HEX channel.

[0060] Figure 3 This is the fluorescence quantitative PCR detection result under the CY5 channel.

[0061] Figure 4 This is the first-generation sequencing test result of pertussis resistance sites of samples A and B. DETAILED DESCRIPTION

[0062] The present invention is further described below in conjunction with specific examples. It should be noted that conventional conditions and methods not described in the examples are usually in accordance with conventional methods used by experimenters in the relevant field: for example, the fourth edition of the "Compendium of Molecular Biology Experiment Guide" edited by Osborne and Kingston, or the steps and conditions recommended by the manufacturer.

[0063] Example 1

[0064] The kit for detecting the 23S rRNA drug resistance site variation of Bordetella pertussis includes:

[0065] Real-time fluorescence quantitative PCR reagents: qPCR Mix, 50×ROX, 2 pairs of primers and corresponding probes for amplifying sample cDNA, sterile water, including:

[0066] Primers and probes for detecting the A2047G mutation in the 23S rRNA resistance site of Bordetella pertussis:

[0067] Bp-F:GTTAAGTGATGGGGTGCAAGCT

[0068] Bp-R:TGGCGACTCGAGTTCTGCGC

[0069] Bp-probe-W:FAM-CTAGACGGAAAGACCCCA-MGB

[0070] Bp-probe-T:HEX-TAGACGGGAAGACCCCAT-MGB

[0071] Primers and probes for detecting human endogenous reference gene GAPDH:

[0072] GAPDH-F:GGCTGCTCACATATTCTG

[0073] GAPDH-R:AAGAGACAAGAGGCAAGA

[0074] GAPDH-probe:CY5-AGCCTCCCCTCCTCATGCC-BHQ2

[0075] Quality control products: pertussis resistance positive quality control products, pertussis resistance negative quality control products, pertussis negative quality control products, including:

[0076] Pertussis resistance positive quality control product, the plasmid includes the following sequence shown in SEQ NO 1:

[0077] SEQ NO 1:

[0078] AAGGTTAAGTGATGGGGTGCAAGCTCTTGATCGAAGCCCCGGTAAACGGCGGCCGTAACTATAACGGTCCTAAGGTAGCGAAATTCCTTGTCGGGTAAGTTCCGACCTGCACGAATGGCGTAACGATGGCCACACTGTCTCCTCCTGAG ACTCAGCGAAGTTGAAGTGTTTGTGATGATGCAATCTACCCGCGGCTAGACGGGAAGACCCCATGAACCTTTACTGTAGCTTTGCATTGGACTGTGAACCGGCCTGTGTAGGATAGGTGGGAGGCGCAGAACTCGAGTCGCCAGATTCGA

[0079] Pertussis drug-resistant negative control product, the plasmid includes the following sequence shown in SEQ NO 2:

[0080] SEQ NO 2:

[0081] AAGGTTAAGTGATGGGGTGCAAGCTCTTGATCGAAGCCCCGGTAAACGGCGGCCGTAACTATAACGGTCCTAAGGTAGCGAAATTCCTTGTCGGGTAAGTTCCGACCTGCACGAATGGCGTAACGATGGCCACACTGTCTCCTCCTGAG ACTCAGCGAAGTTGAAGTGTTTGTGATGATGCAATCTACCCGCGGCTAGACGGAAAGACCCCATGAACCTTTACTGTAGCTTTGCATTGGACTGTGAACCGGCCTGTGTAGGATAGGTGGGAGGCGCAGAACTCGAGTCGCCAGATTCGA

[0082] SEQ NO 3, internal reference sequence:

[0083] GGCTGCTCACATATTCTGGAGGAGCCTCCCCTCCTCATGCCTTCTTGCC TCTTGTCTCTT

[0084] The pertussis negative control product is a DNA plasmid that does not contain Bordetella pertussis DNA and contains the internal reference sequence shown in SEQ NO3.

[0085] The method for using the kit comprises the following steps:

[0086] In the real-time fluorescence quantitative PCR detection method, three probes with different fluorescent labels (FAM, HEX, and CY5) are used to simultaneously detect the 23S rRNA gene of Bordetella pertussis and the human internal reference GAPDH gene in a one-tube PCR reaction system for one sample. The A2047-resistant wild-type strain (sensitive strain) with the erythromycin resistance site A2047G that has not mutated (sensitive strain) and the mutated 2047G-resistant mutant (resistant strain) of Bordetella pertussis can be detected simultaneously.

[0087] According to the real-time fluorescence quantitative PCR test results, it is possible to identify whether the test sample is infected with Bordetella pertussis and whether the infected Bordetella pertussis is resistant to erythromycin. The specific method is:

[0088] A) Quality control products determine the validity of test results:

[0089] ① The HEX channel and CY5 channel of the pertussis Bordetella pertussis resistance positive control product both produce amplification curves, the FAM channel has no amplification curve, and the Ct value is ≤38;

[0090] ② The FAM channel and CY5 channel of the pertussis Bordetella pertussis resistant negative control both produced amplification curves, and the Ct value was ≤38, and there was no amplification curve in the HEX channel;

[0091] ③ The negative control product of Bordetella pertussis has an amplification curve in the CY5 channel, and the Ct value is ≤38, and there is no amplification curve in the FAM channel and the HEX channel;

[0092] B) If the experimental results of condition A) are valid, the judgment is made as follows:

[0093] In a one-tube PCR reaction system for testing samples: 1) If both the FAM channel and the CY5 channel produce amplification curves, and the Ct value is ≤38, and the HEX channel has no amplification curve, the sample is judged to be pertussis positive and pertussis resistance negative; 2) If both the HEX channel and the CY5 channel produce amplification curves, and the Ct value is ≤38, and the FAM channel has no amplification curve, the sample is judged to be pertussis positive and pertussis resistance positive; 3) If both the FAM and HEX channels have no curves, the CY5 channel has an amplification curve and the Ct value is ≤38, the sample is judged to be pertussis negative; 4) If there is no curve in the CY5 channel, the sample is judged to be unqualified.

[0094] Example 2: Detection method

[0095] 1. Extract nucleic acid from the nasopharyngeal swab sample to be tested as template DNA;

[0096] 2. Use two pairs of primers and corresponding probes to perform real-time fluorescence quantitative PCR detection on template DNA in a single-tube PCR reaction system:

[0097] The PCR detection system was 20 μL, including 10 μL qPCR Mix, 0.32 μL of each 10uM probe (the probes were Bp-probe-W, Bp-probe-T, and GAPDH-probe); 0.64 μL of each 10uM forward primer (the forward primers were Bp-F and GAPDH-F); 0.64 μL of each 10uM reverse primer (the reverse primers were Bp-R and GAPDH-R); 0.4 μL of 50×ROX, 1.8 μL of DNA, and ddH 2 O 4.28μL; real-time fluorescence quantitative PCR reaction program: 95℃1min pre-denaturation; 95℃10sec, 58℃35sec, 40 cycles. qPCR Mix was purchased from ToYoBo's THUNDERBIRD Probe qPCR Mix kit, catalog number QPS-101T.

[0098] 3. Result analysis: The specific method is as follows: Under the premise of meeting the following conditions: ① The HEX channel and CY5 channel of the pertussis Bordetella pertussis resistant positive control product both produce amplification curves, the FAM channel has no amplification curve, and the Ct value is ≤38;

[0099] ② The FAM channel and CY5 channel of the pertussis Bordetella pertussis resistant negative control both produced amplification curves, and the Ct value was ≤38, and there was no amplification curve in the HEX channel;

[0100] ③ The negative control product of Bordetella pertussis has an amplification curve in the CY5 channel, and the Ct value is ≤38, and there is no amplification curve in the FAM channel and the HEX channel;

[0101] In a one-tube PCR reaction system for testing samples: 1) If both the FAM channel and the CY5 channel produce amplification curves, and the Ct value is ≤38, and the HEX channel has no amplification curve, the sample is judged to be pertussis positive and pertussis resistance negative; 2) If both the HEX channel and the CY5 channel produce amplification curves, and the Ct value is ≤38, and the FAM channel has no amplification curve, the sample is judged to be pertussis positive and pertussis resistance positive; 3) If both the FAM and HEX channels have no curves, the CY5 channel has an amplification curve and the Ct value is ≤38, the sample is judged to be pertussis negative; 4) If there is no curve in the CY5 channel, the sample is judged to be unqualified.

[0102] The test results of three samples A, B, and C and three quality control products: Bordetella pertussis resistance positive quality control product, Bordetella pertussis resistance negative quality control product and Bordetella pertussis negative quality control product are as follows Figure 1 , 2 , 3, Figure 1 The results of fluorescence quantitative PCR detection under FAM channel.

[0103] Figure 2 The results of fluorescence quantitative PCR detection in HEX channel.

[0104] Figure 3 This is the result of fluorescence quantitative PCR detection under CY5 channel.

[0105] Combining Figures 1, 2, and 3, we can see that:

[0106] Sample A: Both the FAM channel and the CY5 channel produced amplification curves, but the HEX channel did not produce an amplification curve, so sample A was determined to be pertussis positive and pertussis resistance negative;

[0107] Sample B: Both the HEX channel and the CY5 channel produced amplification curves, but the FAM channel did not produce an amplification curve, so sample B was determined to be pertussis positive and pertussis resistance positive;

[0108] Sample C: There are no curves in the FAM and HEX channels, but there is an amplification curve in the CY5 channel, so sample C is judged to be negative for pertussis;

[0109] Quality control: The pertussis resistance positive quality control produces an amplification curve in the HEX and CY5 channels, the pertussis resistance negative quality control produces an amplification curve in the FAM and CY5 channels, and the pertussis negative quality control only produces an amplification curve in the CY5 channel. The results of this experiment are valid.

[0110] Example 3

[0111] To further verify the accuracy of the fluorescence quantitative PCR results, Sanger sequencing was performed on the area near the drug resistance site A2047G of samples A and B. Figure 4 These are the first-generation sequencing results of pertussis resistance loci for samples A and B. Figure 4 The results showed that the sequencing result of sample A was 5'-GCTAGACGGAAAGACCCCAT-3', and the drug resistance site was A2047; the sequencing result of sample B was 5'-GCTAGACGGGAAGACCCCAT-3', and the drug resistance site was 2074G. In summary, the verification results were consistent with the results of fluorescence quantitative PCR, and the results of fluorescence quantitative PCR were reliable.

Claims

1. A primer combination for detecting the variation of the 23S rRNA resistance site of Bordetella pertussis by real-time fluorescence quantitative PCR, characterized in that The primer combination includes a target amplification primer set, a wild-type fluorescent probe, and a mutant fluorescent probe, and the sequence is as follows: Target amplification primer set: Bp-F:GTTAAGTGATGGGGTGCAAGCT; Bp-R:TGGCGACTCGAGTTCTGCGC; Wild-type fluorescent probe Bp-probe-W: CTAGACGGAAAGACCCCA Mutant fluorescent probe Bp-probe-T:TAGACGGGAAGACCCCAT.

2. The primer combination according to claim 1, characterized in that The primer combination also includes an internal reference amplification primer set and an internal reference detection fluorescent probe, the sequences of which are as follows: Internal reference amplification primer set GAPDH-F:GGCTGCTCACATATTCTG GAPDH-R:AAGAGACAAGAGGCAAGA Internal reference detection fluorescent probe GAPDH-probe: AGCCTCCCCTCCTCATGCC.

3. The primer combination according to claim 2, characterized in that The 5' end of the wild-type fluorescent probe, mutant fluorescent probe and internal reference detection fluorescent probe is labeled with a fluorescent reporter group, and the 3' end is labeled with a fluorescent quencher group; and the fluorescent reporter groups of the wild-type fluorescent probe, mutant fluorescent probe and internal reference detection fluorescent probe are different.

4. The primer combination according to claim 3, characterized in that The 5' end of the wild-type fluorescent probe is labeled with a fluorescent reporter group FAM, and the 3' end is labeled with a fluorescent quencher group MGB, and the sequence is Bp-probe-W: FAM-CTAGACGGAAAGACCCCA-MGB; The 5' end of the mutant fluorescent probe is labeled with a fluorescent reporter group HEX, and the 3' end is labeled with a fluorescent quencher group MGB, and the sequence is Bp-probe-T: HEX-TAGACGGGAAGACCCCAT-MGB; The 5' end of the internal reference detection fluorescent probe is labeled with a fluorescent reporter group CY5, and the 3' end is labeled with a fluorescent quencher group BHQ2, and the sequence is GAPDH-probe: CY5-AGCCTCCCCTCCTCATGCC-BHQ2.

5. A kit for detecting 23S rRNA drug resistance site variation of Bordetella pertussis by real-time fluorescence quantitative PCR, the kit comprising the primer combination according to claim 4.

6. The kit as claimed in claim 5, characterized in that The kit also includes quality control products, which include Bordetella pertussis resistance positive quality control products, Bordetella pertussis resistance negative quality control products and Bordetella pertussis negative quality control products.

7. A method for detecting the drug resistance site variation of Bordetella pertussis 23S rRNA by real-time fluorescence quantitative PCR, the method using the kit for detecting the drug resistance site variation of Bordetella pertussis 23S rRNA by real-time fluorescence quantitative PCR as claimed in claim 6, performing real-time fluorescence quantitative PCR detection on a sample to be tested, and obtaining the drug resistance of Bordetella pertussis in the sample to be tested, the method comprising the following steps: (1) Extracting nucleic acid from the sample to be tested; (2) Using the extracted nucleic acid as template DNA, a fluorescence quantitative PCR detection system is prepared, wherein the fluorescence quantitative PCR detection system includes a target amplification primer set, a wild-type fluorescent probe, a mutant fluorescent probe, an internal reference amplification primer set and an internal reference detection fluorescent probe, and fluorescence quantitative PCR detection is performed. According to the amplification curves of three different fluorescent channels, it is analyzed to determine whether the sample to be tested contains Bordetella pertussis and the drug resistance of Bordetella pertussis.

8. The method according to claim 7, characterized in that In the step (2), the fluorescent quantitative PCR detection system includes a target amplification primer set, a wild-type fluorescent probe, a mutant fluorescent probe, an internal reference amplification primer set and an internal reference detection fluorescent probe, qPCR Mix, 50×ROX and sterile water.

9. The method according to claim 8, characterized in that In the step (2), the fluorescent quantitative PCR detection system is a 20 μL system, comprising the following components: 10 μL qPCRMix, 0.32 μL wild-type fluorescent probe Bp-probe-W, 0.32 μL mutant fluorescent probe Bp-probe-T, 0.32 μL internal reference detection fluorescent probe GAPDH-probe, 0.64 μL target amplification primer Bp-F, 0.64 μL target amplification primer Bp-R, 0.64 μL internal reference amplification primer GAPDH-F, 0.64 μL internal reference amplification primer GAPDH-R, 0.4 μL 50×ROX, 1.8 μL template DNA, 4.28 μL ddH2O; The reaction program of real-time fluorescence quantitative PCR was as follows: pre-denaturation at 95°C for 1 min; denaturation at 95°C for 10 s, annealing and extension at 58°C for 35 s, and 40 cycles.

10. The method according to claim 7, characterized in that In step (2), analysis is performed based on the amplification curves of three different fluorescence channels, and the specific steps are: A) Quality control products determine the validity of test results: ① The HEX channel and CY5 channel of the pertussis Bordetella pertussis resistance positive control product both produce amplification curves, the FAM channel has no amplification curve, and the Ct value is ≤38; ② The FAM channel and CY5 channel of the pertussis Bordetella pertussis resistant negative control both produced amplification curves, and the Ct value was ≤38, and there was no amplification curve in the HEX channel; ③ The negative control product of Bordetella pertussis has an amplification curve in the CY5 channel, and the Ct value is ≤38, and there is no amplification curve in the FAM channel and the HEX channel; If the above three points are met, the experimental results are considered reliable and valid; if any of the above items do not meet the requirements, the experimental results are invalid; B) If the experimental results of condition A) are valid, the judgment is made as follows: 1) If both the FAM channel and the CY5 channel of the sample to be tested produce an amplification curve and the Ct value is ≤38, and there is no amplification curve in the HEX channel, the sample is judged to be pertussis positive and pertussis resistance negative; 2) If both the HEX channel and the CY5 channel of the sample to be tested produce an amplification curve and the Ct value is ≤38, and there is no amplification curve in the FAM channel, the sample is judged to be pertussis positive and pertussis resistance positive; 3) If there is no amplification curve in the FAM channel and HEX channel of the sample to be tested, but there is an amplification curve in the CY5 channel and the Ct value is ≤38, the sample is judged to be pertussis negative; 4) If there is no curve in the CY5 channel of the sample to be tested, the sample is judged to be unqualified.

Images