A multiplex reverse transcription quantitative PCR kit for joint detection of four respiratory viruses

A respiratory and reverse transcription technology, applied in the field of biotechnology, can solve the problems of long detection time, easy missed diagnosis, sensitivity and specificity to be improved

Active Publication Date: 2019-08-06
HANGZHOU FIRST PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Before the development of molecular diagnostic technology, virus detection methods mainly include virus culture, serological detection, etc., but the requirements for virus culture are very high, generally used in laboratories, and the detection time is too long, serologi...

Method used

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  • A multiplex reverse transcription quantitative PCR kit for joint detection of four respiratory viruses
  • A multiplex reverse transcription quantitative PCR kit for joint detection of four respiratory viruses
  • A multiplex reverse transcription quantitative PCR kit for joint detection of four respiratory viruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] A multiple reverse transcription quantitative PCR kit for rapid joint detection of four respiratory viruses, consisting of nucleic acid co-extraction solution, quadruple RT-qPCR reaction solution, negative quality control, strong positive quality control, weak positive quality control, five Concentration of positive quantitative standards, in which the quadruple fluorescent PCR reaction solution is a mixture of four sets of primers, probes, RT-qPCR reaction buffer, Hot Start TaqDNA polymerase, PrimeScript reverse transcriptase, and the negative quality control product is Sterile water for injection, strong and weak positive quality control products are positive plasmid samples mixed with equal amounts of RSV, INF, HMPV, and ADV (ADV is the target gene plasmid cloning carrier, RSV, INF, and HMPV are based on gene plasmid cloning. RNA fragments from the source, the same below), in which the concentration of strong positive quality control substance was 10 6 Copies / μl, wea...

Embodiment 2

[0111] The detection method of the multiple reverse transcription quantitative PCR kit of four kinds of respiratory viruses comprises the following steps:

[0112] (1) Primer design: Respiratory syncytial virus (RSV), influenza A virus (INF), human metapneumovirus (HMPV), and adenovirus (ADV) specific conserved genes were retrieved from GenBank, and sequence comparisons were performed , determine their respective highly conserved sequence regions, and design specific primers as the specific nucleic acid sequences of the target genes to be detected;

[0113] (2) Fluorescent probe design: design fluorescent probes according to the specific nucleic acid sequence of the target gene to be detected of four kinds of viruses in step (1);

[0114] (3) Construction of standard products: according to the specific primers determined in step (1), four kinds of standard plasmid molecules containing RSV, INF, HMPV, and ADV highly conserved sequence regions were constructed using gene cloning...

Embodiment 3

[0125] 1. Construction of four kinds of viruses (RSV, INF, HMPV, ADV) target gene plasmid cloning vector

[0126] Using the T-A vector cloning scheme, the PCR products of the four target genes were electrophoresed to confirm the molecular weight of the amplified fragments, and the amplified fragments were recovered and purified by 2% agarose gel, cloned into the pUC57 plasmid, and then the ligated products were transformed into competent large intestine In Escherichia coli, screen positive colonies on LB / Amp / X-Gal / IPTG plates, recover, extract, and purify recombinant plasmids, quantify the verified and correct target plasmids with a UV spectrophotometer, and use 1× TE (pH8.0) buffer diluted to 10 10 copies / μl as a stock solution, and stored at –20°C for future use.

[0127] 2. Three RNA viruses (RSV, INF, HMPV) target gene plasmid cloning vectors are transcribed into RNA fragments

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Abstract

The invention provides a multiplex reverse transcription PCR kit for detecting four respiratory viruses. The kit is composed of a nucleic acid co-extract, a quadruple RT-qPCR reaction liquid, a negative quality control, a strong positive quality control, a weak positive quality control, and five positive quantitative standards with different concentrations. The PCR kit can conveniently, rapidly, and parallelly detect the infection of four common children respiratory viruses (RSV, INF, HMPV, ADV) in a reaction tube at the same time. A single tube PCR can detect nucleic acids of four different viruses at the same time. Moreover, the detection is quantitative, the operation is simple and quick, the sensitivity is high, the specificity and repeatability are good, the results are accurate and reliable; according to the detected number of copies of nucleic acids of viruses, definite diagnosis, prevention, and treatment can be provided for the patients in the early stage; the clinical curative effect can be monitored; and in clinic, the PCR kit can also be used to cut off the infection source, reduce virus infection, and monitor or identify mixed infection caused by multiple viruses.

Description

technical field [0001] The invention belongs to biotechnology, and relates to a multiple reverse transcription quantitative PCR (Reverse transcription quantitative PCR, RT-qPCR) kit for rapid joint detection of four respiratory viruses, in particular to the detection of respiratory syncytial virus (Respiratory syncytial virus, RSV), influenza The quadruple reverse transcription quantitative PCR kit of virus (Influenza virus, INF), human metapneumovirus (Human metapneumovirus, HMPV), adenovirus (Human adenovirus, ADV), specifically, the present invention relates to application quadruple RT-qPCR Technology A kit for rapid and parallel simultaneous detection of RNA viruses and DNA viruses (RSV, INF, HMPV, ADV) in one PCR reaction tube. Background technique [0002] In recent years, the prevalence of various influenza viruses has made people pay more attention to respiratory viral infectious diseases. Due to immature immune function, children have become a high-risk group of ac...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851C12R1/93
CPCC12Q1/6851C12Q2531/113C12Q2537/143C12Q2545/113C12Q2563/107
Inventor 余道军何慧陈懿
Owner HANGZHOU FIRST PEOPLES HOSPITAL
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