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Primers, probe, method and kit for detecting mycoplasma pneumoniae nucleic acid and drug-resistant mutation through real-time fluorescent PCR

A Mycoplasma pneumoniae, real-time fluorescence technology, applied in the field of molecular biology, can solve the problems of Mycoplasma pneumoniae, such as long time-consuming, inaccurate detection results, harsh growth conditions, etc., and achieve high accuracy, high sensitivity, and strong specificity

Inactive Publication Date: 2016-12-07
宁波基内生物技术有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The present invention provides a primer, probe, method and kit for real-time fluorescent PCR detection of Mycoplasma pneumoniae nucleic acid and drug-resistant mutations, and solves the problem of the prior art culture method that takes a long time to detect Mycoplasma pneumoniae, has harsh growth conditions, and has inaccurate detection results. technical problem

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  • Primers, probe, method and kit for detecting mycoplasma pneumoniae nucleic acid and drug-resistant mutation through real-time fluorescent PCR
  • Primers, probe, method and kit for detecting mycoplasma pneumoniae nucleic acid and drug-resistant mutation through real-time fluorescent PCR
  • Primers, probe, method and kit for detecting mycoplasma pneumoniae nucleic acid and drug-resistant mutation through real-time fluorescent PCR

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Embodiment 1

[0024] The embodiment of the present invention provides a primer and probe for real-time fluorescent PCR detection of Mycoplasma pneumoniae nucleic acid and drug resistance mutation, and the nucleotide sequence of the primer for detection of Mycoplasma pneumoniae-specific P1 gene is shown in SEQ ID NOs: 2-3, to detect Mycoplasma pneumoniae The nucleotide sequence of the Taqman probe specific for the P1 gene is shown in SEQ ID NO: 4; the nucleotide sequence of the primers for detecting the resistance mutation sites 2063 (A / G) and 2064 (A / G) genes is shown in SEQ ID NO: 6 ~7, the nucleotide sequence of the Taqman probe for detecting the 2063 (A / G) gene of the drug resistance mutation site is shown in SEQ ID NO: 8; the Taqman probe for detecting the 2064 (A / G) gene of the drug resistance mutation site The nucleotide sequence of the needle is shown in SEQ ID NO: 10; the nucleotide sequence of the primer for detecting the internal standard gene int is shown in SEQ ID NO: 12-13, and ...

Embodiment 2

[0044] The embodiment of the present invention also provides a real-time fluorescent PCR method for detecting Mycoplasma pneumoniae nucleic acid and drug resistance mutation, comprising:

[0045] Step 101, sample nucleic acid preparation to obtain a nucleic acid template;

[0046] Step 102: Design specific primers and fluorescently labeled probes for the Mycoplasma pneumoniae-specific P1 gene, 2063 (A / G) gene, 2064 (A / G) gene, and internal standard gene int, respectively;

[0047] Among them, the nucleotide sequences of primers for detecting Mycoplasma pneumoniae-specific P1 gene are shown in SEQ ID NOs: 2-3, and the nucleotide sequences of Taqman probes for detecting Mycoplasma pneumoniae-specific P1 gene are shown in SEQ ID NO: 4; detection of drug resistance The nucleotide sequences of the primers of the mutation sites 2063 (A / G) and 2064 (A / G) genes are shown in SEQ ID NOs: 6-7, and the Taqman probes of the 2063 (A / G) gene at the drug resistance mutation site are detected....

Embodiment 3

[0059] The embodiment of the present invention provides a kit for real-time fluorescent PCR detection of Mycoplasma pneumoniae nucleic acid and drug resistance mutation, including nucleic acid extraction solution, a first primer-probe mixture, a second primer-probe mixture, and a third primer-probe Mixed solution, PCR reaction enzyme system, internal standard, negative control substance, positive control substance, and packaging boxes for separating and centrally packaging these reagent bottles or tubes, wherein the first primer-probe mixture is composed of deoxyribonucleoside triphosphate dN(U)TP, the upstream and downstream primers of the P1 gene and a fluorescently labeled probe are composed of the upstream and downstream primers of the internal standard gene and a fluorescently labeled probe; the second primer-probe mixture is composed of deoxyribose nuclei The glucoside triphosphate dN(U)TP, the upstream and downstream primers of the 2063(A / G) gene and a fluorescently labe...

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Abstract

The invention relates to the technical field of molecular biology, and discloses primers, probe, method and kit for detecting mycoplasma pneumoniae nucleic acid and drug-resistant mutation through a real-time fluorescent PCR. By means of a Taqman-MGB probe real-time fluorescent PCR method, the primers and the fluorescent marking probe are designed for a mycoplasma pneumoniae specificity P1 gene, 23S rRNA gene drug resistance mutation genes (2063A / G and 2064A / G) and interior label (int) nucleic acids, the detection result is judged through the combination of an amplification curve and a CT value through PCR reaction detection fluorescent signals, and a target gene is detected through the change of the fluorescent signals. The primers, probe, method and kit have the advantages of being high in accuracy, specificity and sensitivity, and mycoplasma pneumoniae and drug resistance mutation sites in a throat swab sample can be rapidly and accurately detected.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a primer, a probe, a method and a kit for real-time fluorescent PCR detection of Mycoplasma pneumoniae nucleic acid and drug resistance mutation. Background technique [0002] Mycoplasma pneumoniae (Mycoplasma Pneumoniae, MP) is the main pathogen causing respiratory tract infection in children and the elderly, and is also a common pathogen causing community-acquired pneumonia. It mainly causes pneumonia, bronchitis, bronchitis, asthma and other clinical symptoms and some serious clinical complications. Macrolide antibiotics are currently the drug of choice for the treatment of Mycoplasma pneumoniae pneumonia, and the abuse of antibiotics has led to increasing drug resistance of MP. [0003] Culture is the gold standard for the detection of Mycoplasma pneumoniae, once detected, the diagnosis can be made. However, its disadvantage is that it takes a long time. Generally...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/35
CPCC12Q1/686C12Q1/689C12Q2600/106C12Q2600/156C12Q2600/166C12Q2561/113C12Q2561/101
Inventor 高华山王伟建倪剑锋翁毅
Owner 宁波基内生物技术有限公司
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