Influenza A virus molecular detection kit and preparation method thereof

A technology of influenza A virus and detection kit, which is applied in the direction of biochemical equipment and methods, microbiological determination/inspection, etc., can solve the problems of poor PCR sensitivity, difficulty in realizing multiple detection, unsuitable detection, etc., and achieves an easy-to-operate Effect

Inactive Publication Date: 2016-02-03
JIANGSU PROVINCIAL CENT FOR DISEASE PREVENTION & CONTROL
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Real-timetimePCR method and LAMP method have good specificity and sensitivity, but the former relies on expensive instruments and reagents; while the latter method is difficult to achie...

Method used

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  • Influenza A virus molecular detection kit and preparation method thereof
  • Influenza A virus molecular detection kit and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Embodiment 1: Target gene plasmid construction

[0017] Design specific primers for the M segment of influenza A virus, amplify the 1027bp gene fragment by PCR method, recover and purify it and connect it with the T vector to obtain the positive plasmid of the target gene, which is named Puc-AM.

Embodiment 2

[0018] Embodiment 2: Primer probe design and screening

[0019] Through sequence analysis software analysis, using the relatively conservative M segment sequence of influenza A virus as a template, a total of 8 forward primers and 12 reverse primers were designed, and the length of the primers was 30-35bp; at the same time, 1 exo probe (named M-exo-probe) with a length of 53bp, in which the 33rd base T marks the fluorescent substance FAM, the 36th base is replaced by tetrahydrofuran, the 38th base marks the fluorescent substance BHQ, and the 3' terminal base is blocked at the same time.

[0020] Using TwistAmpBasickit (TwistDx Company, UK), the target gene cloning plasmid Puc-AM was used as a template, and agarose gel electrophoresis was used as a result indicating means to screen the primers designed above. The evaluation indicators of primer quality include specificity, sensitivity, amplification efficiency and primer noise. Take 29.5 μL of reaction buffer and add the follo...

Embodiment 3

[0021] Example 3: Sensitivity of RT-RPA

[0022] Dilute the target gene cloning plasmid Puc-AM 100 times, and then dilute it into 10 times by 10 times -2 -10 -9 A total of eight concentrations were used as templates for RT-RPA reactions: using the TwistAmpRTexos kit (TwistDx, UK). Take 29.5 μL reaction buffer and add the following components respectively: 2.1 μL each of primer L-rpaF (10 mM) and L-rpaR (10 mM), 2.6 μL RPAexo probe (10 mM), 1 μL RNase inhibitor (5 U), 7.2 μL supernatant Pure water, 5 μL sample RNA template. Vortex and centrifuge briefly. Another 2.5 μL of magnesium acetate (280 mM) was added. After mixing and centrifuging, put them into a Twista real-time fluorescence detector, the reaction temperature is 40°C, and the reaction time is 20 minutes.

[0023] The RT-RPA method established in this experiment can detect the lower limit is 15copies / μL, which has good sensitivity.

[0024] Example 3: Specificity of RT-RPA

[0025] Using the nucleic acid of infl...

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Abstract

The invention belongs to the technical field of biology, and particularly relates to an influenza A virus reverse transcription-recombinase polymerase amplification (RT-RPA) detection kit and a preparation method thereof. The detection kit consists of an RT-RPA reaction system, an RNA enzyme inhibitor, influenza A virus M-segment positive plasmids (Puc-AM), a negative quality control product, an M-segment RPA primer and an exo probe. By using the specificity primer and probe, preparing the RT-RPA reaction system and embedding a Twista real-time fluorescent detector to carry out real-time fluorescent detection, a rapid, sensitive and specific influenza A virus constant-temperature real-time fluorescent nucleic acid detection method is established. The invention relates to an application of a specific RPA primer and an exo probe in the influenza A virus infection clinical identification diagnosis and virus separation strain identification.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an influenza A virus reverse transcription-recombinase polymerase amplification detection kit and a preparation method thereof. Background technique [0002] Influenza A virus belongs to Orthomyxoviridae, and its genome consists of 8 negative-sense single-stranded RNA segments, encoding at least 11 proteins. The host of influenza A virus is widely distributed and can infect birds, humans, pigs, horses, dogs, cats, marine mammals (seals, whales, etc.) and the like. In the entire ecological distribution of influenza A virus, wild waterfowl and coastal birds are the main natural storage hosts and gene pools. Most of the influenza A virus subtypes (16 H subtypes and 9 N subtypes) found so far are found in wild waterfowl. Humans experienced three influenza pandemics in the 20th century, namely the Spanish flu (H1N1) in 1918, the Asian flu (H3N2) in 1957, and the Hong Kong fl...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68
CPCC12Q1/701C12Q1/6844C12Q2521/507C12Q2545/113
Inventor 祁贤宋勇春焦永军张洪英汤奋扬鲍倡俊
Owner JIANGSU PROVINCIAL CENT FOR DISEASE PREVENTION & CONTROL
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