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Avian influenza virus NA subtype multi-probe combination fluorescent quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) typing method

An avian influenza virus and RT-PCR technology is applied in the field of avian influenza virus NA subtype multiple probe combination fluorescence quantitative RT-PCR typing, and achieves the effects of intuitive experimental results, time-saving experimental procedures, and improved specificity.

Inactive Publication Date: 2017-06-09
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Moreover, multiple experiments with different combinations are required to finally determine the NA subtype, and the results cannot be obtained quickly and intuitively through one experiment.

Method used

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  • Avian influenza virus NA subtype multi-probe combination fluorescent quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) typing method
  • Avian influenza virus NA subtype multi-probe combination fluorescent quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) typing method
  • Avian influenza virus NA subtype multi-probe combination fluorescent quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) typing method

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Experimental program
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Effect test

Embodiment 1

[0030] Embodiment 1 RT-PCR multiple detection of avian influenza virus NA subtype

[0031] (1) Design of primers

[0032] According to the purpose of the experiment, nine different NA gene sequences of popular avian influenza viruses were selected, including the sequences of strains of different animal origin and different ages. Then, by comparing the sequence homology of each NA subtype, select its conserved sequence, design type-specific primers and probes labeled with different fluorescent groups. Primers, probe sequences and product sizes are shown in Table 1:

[0033] Table 1 9 kinds of AIV NA subtyping primers and probes

[0034]

[0035] Merged bases: R=A / G, Y=C / T, S=G / C., H=A / C / T, V=A / G / C

[0036] (2) Extraction of viral nucleic acid

[0037] 1. Add 20μl Proteinase K to a sterile 1.5ml centrifuge tube.

[0038] 2. The sample is chicken embryo allantoic fluid, which comes from the positive avian influenza virus isolated clinically, and the avian influenza virus ...

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Abstract

The invention provides an avian influenza virus NA subtype RT-PCR (Reverse Transcription-Polymerase Chain Reaction) single / multi detection primer group, an avian influenza virus NA subtype RT-PCR multi-detection primer group and avian influenza virus NA subtype RT-PCR multi-detection kit. The avian influenza virus NA subtype RT-PCR multi-detection primer group comprises 3 avian influenza virus NA subtype RT-PCR detection primer groups, wherein each avian influenza virus NA subtype RT-PCR detection primer group comprises a specific primer pair and a probe marked by fluorescent groups. Different components of the three multi-probe fluorescent quantitative combination RT-PCR systems provided by the invention are not interfered by one another, and 3 different NA subtypes can be specifically detected. The NA subtypes can be directly judged according to CT (Computed Tomography) values by only performing fluorogenic quantitative PCR of the three systems, the testing process can be relatively time-saving and convenient, and testing results can be relatively visible and credible.

Description

technical field [0001] The invention belongs to the field of biological detection, and in particular relates to a multi-probe combination fluorescent quantitative RT-PCR typing method for avian influenza virus NA subtype. Background technique [0002] Avian influenza viruses are divided into 16 HA subtypes and 9 NA subtypes, which can be combined into different HA and NA subtypes, such as H5N1, H5N2, H5N5, H5N5, H5N8, etc. The NA subtype needs to be determined by neuraminidase inhibition test, and can also be sequenced and typed by RT-PCR amplification. The former requires specific NA antiserum, while the latter takes a long time, and after RT-PCR amplification, it is usually necessary to use agarose gel electrophoresis to display the size of the target band, and compare it with the expected product size to obtain the result. It takes a long time; or use the common SYBR Green method, that is, judge the negative and positive by the TM value of the melting curve in each combi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/6851C12Q1/701C12Q2521/107C12Q2531/113C12Q2563/107C12Q2561/101C12Q2537/143
Inventor 彭大新孙志豪陈素娟秦涛孟菲菲王宵刘秀梵顾敏
Owner YANGZHOU UNIV