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Non-diagnostic method for detecting flavivirus and alphavirus through double TaqMan probe real-time fluorescence RT-PCR

A RT-PCR, real-time fluorescence technology, applied in biochemical equipment and methods, fluorescence/phosphorescence, microbial measurement/inspection, etc., can solve the problems of time-consuming, cumbersome operation, and high requirements for experimental conditions, so as to improve biological safety , Simplify the detection process, and the effect of long test time

Inactive Publication Date: 2013-10-23
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the gold standard for arbovirus identification is virus isolation, but it is difficult to be popularized and applied in practical work due to its cumbersome operation, high technical difficulty, time-consuming, high requirements for experimental conditions, and serious biosafety problems.

Method used

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  • Non-diagnostic method for detecting flavivirus and alphavirus through double TaqMan probe real-time fluorescence RT-PCR
  • Non-diagnostic method for detecting flavivirus and alphavirus through double TaqMan probe real-time fluorescence RT-PCR
  • Non-diagnostic method for detecting flavivirus and alphavirus through double TaqMan probe real-time fluorescence RT-PCR

Examples

Experimental program
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Effect test

Embodiment 1

[0036] 1. The sample to be tested is dengue virus liquid, the positive control is a mixed template containing JE virus RNA and Getta virus RNA, and the negative control is RNase-Free Water.

[0037] 2. Dengue virus RNA extraction: According to the QIAamp Viral RNA Mini Kit instruction manual to extract the sample RNA, the specific steps are as follows:

[0038] ①According to the number of samples, divide the lysate AVL, 560μl per tube;

[0039] ② Take 140μl of virus solution and add 560μl aliquoted AVL, vortex for 15s, mix well, and incubate at room temperature for 10 minutes;

[0040] ③Add 560μl of absolute ethanol (96%-100%) to each tube, shake for 15 seconds to mix thoroughly, and centrifuge quickly;

[0041] ④ Take out the 2ml collection tube with filter column from the kit, open the package, and mark it. Take 630μl of the mixed solution in step ③ into the collection tube, centrifuge at 8000rpm for 1 minute;

[0042] ⑤Put the filter column into a new 2ml collection tube, suck all th...

Embodiment 2

[0059] 1. Extraction of chikungunya virus RNA: use QIAamp Viral RNA extraction kit and follow the instructions.

[0060] The specific steps are as follows:

[0061] ①According to the number of samples, divide the lysate AVL, 560μl per tube;

[0062] ② Take 140μl of virus solution and add it to 560μl aliquoted AVL, vortex for 15s, mix well, and incubate at room temperature for 10 minutes;

[0063] ③Add 560μl of absolute ethanol (96%-100%) to each tube, shake for 15 seconds to mix thoroughly, and centrifuge quickly;

[0064] ④ Take out the 2ml collection tube with filter column from the kit, open the package, and mark it. Take 630μl of the mixed solution in step ③ into the collection tube, centrifuge at 8000rpm for 1 minute;

[0065] ⑤Put the filter column into a new 2ml collection tube, suck all the remaining mixture into the filter column, and centrifuge at 8000rpm for 1 minute;

[0066] ⑥Take a clean 2ml collection tube, move the filter column to a new collection tube, add 500μl of elue...

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Abstract

The invention discloses a non-diagnostic method for detecting flavivirus and alphavirus through double TaqMan probe real-time fluorescence RT-PCR. The non-diagnostic method comprises real-time and quantitative detection carried out on a sample to be detected through designing complement sequences for an NS5 gene in the flavivirus sequence and an NS1 gene in the alphavirus sequence, and technologies of conventional retro-transcript PCR amplicaiton and real-time quantitative RT-PCR are adopted; therefore, the arbovirus detection process is simplified, the virus isolating technology with defects of complex operation, great technical difficulty and long experiment time is replaced, and the biosecurity during detection is improved. According to the invention, the high-flux, quick and sensitive method for detecting and screening flavivirus and alphavirus is established and plays an important role in detecting the arboviuses.

Description

Technical field [0001] The invention relates to a non-diagnostic method for detecting flavivirus and alphavirus in mosquito specimens by double TaqMan probe real-time fluorescent RT-PCR. Background technique [0002] Arbovirus (arbovirus) refers to a group of viruses that are naturally sourced diseases and zoonotic diseases caused by blood-sucking arthropods biting sensitive vertebrates. It is mainly concentrated in the Flaviviridae, Flaviviridae, Togaviridae Alphavirus, Reoviridae and Bunyaviridae. Flaviviruses are the most harmful to humans. Humans and animals are infected by bites of arthropods carrying the virus, which not only causes human diseases and deaths, but also causes a large number of livestock diseases and even deaths, causing huge economic losses and becoming a public health problem for all countries. [0003] At present, the gold standard for arbovirus identification is virus isolation, but due to its cumbersome operation, technical difficulty, time-consuming, hig...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68G01N21/64
Inventor 孙肖红郭金金燕清丽姚李四刘丽娟杨宇王旺
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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