Collaborative multiplex PCR detection method for nucleic acids of nine respiratory tract pathogens

A pathogen and respiratory technology, applied in recombinant DNA technology, microbial assay/test, DNA/RNA fragments, etc., can solve the problems of low efficiency of accurate diagnosis, unclear pathogens, and inability to distinguish the result curve clearly.

Active Publication Date: 2020-04-28
苏州华益美生物科技有限公司
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  • Abstract
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Problems solved by technology

The clinical symptoms of upper respiratory tract infection and lower respiratory tract infection caused by these pathogens are similar, especially for young children and children who have difficulty describing their condition accurately, and accurate diagnosis is even less effective
[0003] For influenza A virus (Influenza A virus, FluA), influenza B virus (Influenza Bvirus, FluB), parainfluenza virus (Parainfluenza virus, PIV), respiratory Syncytial virus (Respiratorysyncytial virus, RSV), Mycoplasma pneumoniae (Mycoplasma pneumoniae, MP), Chlamydia pneumoniae (Chlamydia pneumoniae, Cpn) Cpn, Adenovirus (Adenovirus, ADV), Human rhinovirus (Humanrhinovirus, HRV), Human metapneumovirus (Human metapneumovirus, hMPV) these 9 kinds of pathogens and more pathogens, Chinese patent application CN110468234A discloses the multiplex fluorescent quantita

Method used

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  • Collaborative multiplex PCR detection method for nucleic acids of nine respiratory tract pathogens
  • Collaborative multiplex PCR detection method for nucleic acids of nine respiratory tract pathogens
  • Collaborative multiplex PCR detection method for nucleic acids of nine respiratory tract pathogens

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Embodiment 1

[0176] The extraction of embodiment 1 nucleic acid

[0177] Influenza A virus (FluA), Influenza B virus (FluB), Parainfluenza virus (PIV), Respiratory syncytial virus (RSV), Mycoplasma pneumoniae (MP), Chlamydia pneumoniae (Cpn), Adenovirus (ADV), human Rhinovirus (HRV) and human metapneumovirus (hMPV) can be purchased from the Clinical Inspection Center of the Ministry of Health under license conditions, and are standard products for nucleic acid extraction.

[0178] The extraction of nucleic acid is carried out according to the conventional magnetic bead extraction method. In order to adapt to the nucleic acid extraction of the above four viruses at the same time, the improvement is as follows: add 90uL nucleic acid extraction solution to 1uL standard (the formula and final concentration are: guanidine isothiocyanate 1.2M, Sodium edetate (pH8.0) 10mM, Tween-20 2% (W / W), sodium perchlorate 1M, ethanol 40% (V / V), Tris-HCl (pH8.0) 10mM) , incubate at 42°C for 10min, then add 1...

Embodiment 2

[0180] Example 2 3 PCR reaction tubes coordinated quadruple real-time PCR test

[0181] 1. Primer and probe sequences and grouping

[0182] Commission the synthesis of the following primer pairs and probes and group them according to the research (each group targets 3 pathogens):

[0183] Group 1:

[0184] Primer pairs for detection of influenza A virus (FluA):

[0185] FluA L: CATGGAATGGCTAAAGACA

[0186] FluA R: GCGTGAACACAAATCCTA

[0187] Probes for detection of influenza A virus (FluA):

[0188] FluA P:ACCAATCTTGTCACCTCTGACTAAGG,

[0189] Primer pairs for detection of influenza B virus (FluB):

[0190] FluB L: TGGTCTCAGCTATGAACAC

[0191] FluB R: GTTGCTTTGCAGCTCTTC

[0192] Probes for detection of influenza B virus (FluB):

[0193] FluB P: CGTCTTTCTCCTTTTCCCATTCCATTCA,

[0194] Primer pairs for detection of Mycoplasma pneumoniae (MP):

[0195] MP L:CCTCGATCCTGATTCTGTAC

[0196] MP R:GCCTTTCAGTCCCCAAAAA

[0197] Probes for the detection of Mycoplasma pneumoniae...

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Abstract

The invention discloses a collaborative quadruple real-time fluorescence PCR detection method for simultaneously detecting target nucleic acids of nine pathogens by using three PCR reaction tubes. Themethod is used for detecting influenza A virus (FluA), influenza B virus (FluB), parainfluenza virus (PIV), respiratory syncytial virus (RSV), mycoplasma pneumoniae (MP), chlamydia pneumoniae (Cpn),adenovirus (ADV), human rhinovirus (HRV) and human metapneumovirus (hMPV) in samples. The method is rapid to operate, can be completed by one-step cooperation and has high sensitivity. In addition, the invention also relates to a reagent involved in the PCR method, a detection kit used for the method, preparation and application of the detection kit and the like.

Description

technical field [0001] The invention belongs to the technical field of nucleic acid detection. Specifically, the invention relates to a method capable of simultaneously detecting influenza A virus (FluA), influenza B virus (FluB), parainfluenza virus (PIV), respiratory syncytial virus (RSV), pneumonia Mycoplasma (MP), Chlamydia pneumoniae (Cpn), adenovirus (ADV), human rhinovirus (HRV), human metapneumovirus (hMPV), housekeeping gene (β-actin) nine respiratory pathogens and one internal reference gene for detection A multiplex real-time PCR method that is fast, ultrasensitive and complete in one step. In addition, the present invention also relates to the reagents involved in the above PCR method, such as primers, probes, etc. that are irrelevant to each other, as well as the detection kit used in the above method and the preparation and application of the corresponding detection kit. Background technique [0002] Influenza virus (Flu) includes three types: A, B, and C. Typ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/689C12Q1/686C12Q1/04C12N15/11C12R1/93C12R1/35C12R1/01
CPCC12Q1/701C12Q1/689C12Q1/686C12Q2600/16C12Q2537/143C12Q2563/107Y02A50/30
Inventor 王川杨晓娟杨学敏张必新李会娜李振勇
Owner 苏州华益美生物科技有限公司
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