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

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...

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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 quantitative PCR kit that is used for 19 kinds of human respiratory virus detections, yet from the detection map of its PCR, The resulting curves cannot be clearly distinguished; Chinese patent application CN109355437A discloses a multiple detection kit for respiratory pathogens, which detects 16 respiratory pathogens at the same time. However, this method relies on color compensation and is difficult to use on the existing fluorescent PCR detection platform. Update equipment; CN107365876A discloses a kit for detecting 10 respiratory tract infection pathogens and its use method, wherein no primer and probe ratio is fully disclosed, although it claims to be able to simultaneously detect 10 common respiratory tract infection pathogens, but provided In the attached figure, at most 3 pathogens with unknown pathogen types can be detected at the same time

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