Method for simultaneously detecting bordetella pertussis and drug-resistant mutation sites thereof and kit

A drug-resistant mutation site and drug-resistant mutation technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., to achieve the effects of good specificity, prevention of false positives, and good precision

Pending Publication Date: 2021-07-09
GUANGDONG HECIN SCI INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] In the prior art, CN 103667512A discloses a primer and a detection method for rapid detection of erythromycin resistance of Bordetella pertussis from specimens. This method cannot directly determine whether there is a mutation of B. pertussis. Other methods to determine whether it is B. pertussis. On this basis, design primers for the variable regions on both sides of the 23S rRNA gene of Bordetella pertussis, including the 2047 site, in order to realize the detection of erythromycin resistance to B. pertussis
Although this method is also designed for the pertussis drug-resistant 23s rRNA region, it uses a sequencing method that needs to be amplified by ordinary PCR, followed by a first-generation sequencing method, and finally judge whether a mutation has occurred based on the sequencing peak map and comparison with the sequence of the standard strain

Method used

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  • Method for simultaneously detecting bordetella pertussis and drug-resistant mutation sites thereof and kit
  • Method for simultaneously detecting bordetella pertussis and drug-resistant mutation sites thereof and kit
  • Method for simultaneously detecting bordetella pertussis and drug-resistant mutation sites thereof and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1: a preferred embodiment that kit of the present invention forms

[0048] Using the Blast tool to analyze the genome sequence of B. pertussis nucleic acid and drug-resistant mutation sites in Genbank and domestic and foreign literature, select the gene promoter region unique to B. pertussis toxin and the 23S rRNA variable region A2047G of the B. pertussis drug-resistant region. The base mutation site was used as the detection target sequence, and multiple sets of primers and probes were designed and synthesized for the two detection target sequences, and specific primers and probes were designed for the conserved region of the positive quality control product human β-globin gene. The results are shown in Table 1. The primers and probes were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd., including the detection probe of B. pertussis (the 5' end is labeled with FAM fluorescent reporter group, and the 3' end is labeled with BHQ1 fluorescence quenche...

Embodiment 2

[0069] Embodiment 2: the second preferred embodiment that kit of the present invention forms

[0070] On the basis of Example 1, the Bacillus pertussis nucleic acid and drug-resistant mutation site detection kit (Taqman fluorescent probe method) of the present embodiment consists of the following table:

[0071]

[0072] This kit can also be matched with a quantitative standard kit as required.

Embodiment 3

[0073] Embodiment 3: A preferred embodiment of the PCR detection method of B. pertussis nucleic acid and drug-resistant mutation site established by the present invention

[0074] A preferred embodiment of the Bacillus pertussis nucleic acid and drug-resistant mutation site PCR detection method established by the present invention is as follows:

[0075] 1. Genomic DNA extraction from samples to be tested

[0076] Take 50 μL of the oropharyngeal swab sample to be tested and the negative control (Hep-2), centrifuge briefly and take the supernatant, and use the nucleic acid extraction or purification reagent produced by Guangdong Hexin Health Technology Co., Ltd. , operate in strict accordance with the instructions.

[0077] 2. Preparation of 20 μL of B. pertussis and drug resistance detection amplification reaction solution

[0078] Prepare 20 μL of amplification reaction solution for B. pertussis and drug resistance detection according to the following composition:

[0079]...

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Abstract

The invention discloses a fluorescent PCR detection method for simultaneously detecting bordetella pertussis DNA and a drug-resistant mutation site of bordetella pertussis, which comprises the following steps of: adding the DNA of a sample to be detected and an internal standard into a fluorescent PCR reaction solution for carrying out amplification reaction, interpreting a reaction result according to a Ct value of a fluorescent signal detection channel, and distinguishing whether the sample to be detected is bordetella pertussis or drug-resistant bordetella pertussis, therefore, clinical correct use of antibiotics is guided, and abuse of antibiotics is prevented. The invention also provides a kit for the detection method of the bordetella pertussis and the drug-resistant mutation site thereof. The kit comprises a fluorescent PCR reaction liquid for detecting the bordetella pertussis and an internal standard. The method and the kit provided by the invention can save detection time and cost, and have the advantages of high detection accuracy, high sensitivity, good specificity and good precision.

Description

technical field [0001] The invention belongs to the field of molecular biology detection, and more specifically relates to a method and a kit for simultaneously detecting Bacillus pertussis and its drug-resistant mutation sites. Background technique [0002] Pertussis is a severe acute respiratory infectious disease caused by Bacillus pertussis. Pertussis is characterized by a typical spasmodic cough accompanied by crowing echoes. Although the widespread vaccination of whooping cough vaccine has significantly reduced the incidence of the disease, the antibodies produced by the vaccination decline with age, and the antibodies in pregnant women are rarely transmitted to the fetus, so the resistance of young babies to whooping cough is still weak , or have not reached the age of vaccination, the incidence of whooping cough in infants younger than 6 months is significantly higher than that of other age groups. In addition, the decline of antibodies produced by vaccination with ...

Claims

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Application Information

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Patent Type & AuthorityApplications(China)
IPC IPC(8): C12Q1/689C12Q1/6858C12Q1/04C12N15/11
CPCC12Q1/689C12Q1/6858C12Q2600/166C12Q2531/113C12Q2563/107C12Q2545/101
Inventor李小锋李晨阳刘小翠刁礼福
OwnerGUANGDONG HECIN SCI INC