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Method and kit for simultaneously detecting human type A and type B respiratory syncytial viruses and human metapneumoviruses

A technology of human metapneumovirus and syncytial virus, applied in the field of biotechnology applications

Active Publication Date: 2014-09-03
JIANGSU UNINOVO BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

We have successfully designed and synthesized specific primers and probes for the corresponding N genes, L genes and L genes of human A and B respiratory syncytial virus and human metapneumovirus

Method used

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  • Method and kit for simultaneously detecting human type A and type B respiratory syncytial viruses and human metapneumoviruses
  • Method and kit for simultaneously detecting human type A and type B respiratory syncytial viruses and human metapneumoviruses
  • Method and kit for simultaneously detecting human type A and type B respiratory syncytial viruses and human metapneumoviruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1. Design and synthesis of human A, B type respiratory syncytial virus and human metapneumovirus subtypes N gene, L gene and L gene specific primers and probes

[0032] 1.1 Selection of corresponding target genes N gene, L gene and L gene and determination of conserved regions of human A, B respiratory syncytial virus and representative strains of human metapneumovirus

[0033] The sequences used in this study were selected from NCBI GenBank (http: / / www.ncbi.nlm.nih.gov), the main selection basis is: (a) strains with a relatively recent age; (b) full-length sequences and main sequences (c) Select a representative strain after comparing the sequence with the subtype. The corresponding N gene, L gene and L gene information of selected representative strains of human type A, type B respiratory syncytial virus and human metapneumovirus are shown in Tables 1-1, 1-2 and 1-3.

[0034] In this study, the corresponding N genes, L genes, and L genes of the selected repre...

Embodiment 2

[0047] Example 2. Establishment of a one-step multiplex fluorescent RT-PCR detection system for human type A, type B respiratory syncytial virus and human metapneumovirus

[0048] 2.1 According to Invitrogen's nucleic acid extraction kit ( viralRNA Mini Kit) to extract sample nucleic acid.

[0049] 2.2 Preparation of human type A, type B respiratory syncytial virus and human metapneumovirus one-step multiplex fluorescent RT-PCR detection system The preparation reagents used AgPath-IDTM One-step RT-PCR Kit from Ambion Company, the system is 50 μl:

[0050] 2×RT-PCR buffer 25μl

[0051] 25×RT-PCEnzyme 2μl

[0052] Detection Enhancer 3μl

[0053] Add 0.5 μl of primers shown in SEQ NO: 1, 2, 4, 5, 7, 8, 10, and 11 at the same time for primers and probes, and the final concentration of primers is 300 nM; add primers such as SEQ NO: 3, 6, 9, 0.5 μl each of the probe primers indicated in 12, the final concentration is 150 nM

[0054] Template 6 μl (add 2 μl each of the three po...

Embodiment 3

[0063] Example 3. Specific identification of human type A, type B respiratory syncytial virus and human metapneumovirus one-step multiple fluorescent RT-PCR detection method

[0064] The multiple fluorescent RT-PCR reaction system established in Example 2 was used for human A respiratory syncytial virus, human B respiratory syncytial virus, human metapneumovirus, influenza A virus, influenza B virus, and influenza C virus respectively. , parainfluenza virus, enterovirus, adenovirus, bocavirus positive nucleic acid detection, the results showed that only human type A, type B respiratory syncytial virus and human metapneumovirus were detected in the FAM, JOE, ROX detection channels Corresponding specific fluorescence amplification curves appeared, and there was no cross-reaction, while the other 7 virus strains had no amplification curves except for the internal quality control CY5 channel, and the other three channels had no amplification curves, indicating that this method has ...

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Abstract

The invention belongs to the field of biotech applications and relates to a multiplex fluorescence RT-PCR (reverse transcription-polymerase chain reaction) method including internal quality control and a kit for simultaneously detecting human type A and type B respiratory syncytial viruses and human metapneumoviruses. Specific primers and probes are designed for N genes, L genes and conserved sequences of the L genes of representive strains of the human type A and type B respiratory syncytial viruses and the human metapneumoviruses, the one-step multiplex fluorescence RT-PCR quick detection method including the internal quality control is simple and quick to operate, the tediousness of a conventional single fluorescence RT-PCR single-hole single-detection method is avoided, the operation program is simplified, the test cost is reduced, and strong technical support is provided for aspects of field detection, hygienic evaluation, clinical diagnosis and the like of the viruses by virtue of relatively high specificity, sensitivity, high efficiency and stability of the method.

Description

technical field [0001] The invention relates to the application field of biotechnology, especially for the simultaneous detection and identification of human type A and type B respiratory syncytial virus and human metapneumovirus. Background technique [0002] Human respiratory syncytial virus (Human respiratory syncytial virus, hRSV) is one of the most common and important pathogens that cause lower respiratory tract infection in winter and spring in infants, elderly and infirm, and immunocompromised persons. RSV belongs to the genus Pneumovirus in the Paramyxoviridae family. Its genome is a single-stranded negative-sense RNA consisting of 15,222 nucleotides and encoding 11 proteins, of which 3 are transmembrane envelope glycoproteins F protein (fusion protein) and G protein. (attachment protein) and SH protein (small hydrophobic protein), according to the difference in G protein antigenicity, RSV is divided into two types, A and B, and the type A RSV is prevalent in my coun...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/686C12Q1/70C12Q2537/143C12Q2545/101C12Q2563/107
Inventor 邓瑛崔淑娟石伟先王海滨张玉松李爱华于霞丽
Owner JIANGSU UNINOVO BIOLOGICAL TECH
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