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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, technical difficulty, etc., and improve biological safety , Simplify the detection process, the effect of high technical difficulty

Inactive Publication Date: 2012-07-18
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
Comparison scheme
Effect test

Embodiment 1

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

[0037] 2. Extraction of dengue virus RNA: Extract sample RNA according to the QIAamp Viral RNA Mini Kit instruction manual. The specific operation steps are as follows:

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

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

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

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

[0042] ⑤ Pu...

Embodiment 2

[0059] 1. Chikungunya virus RNA extraction: use the QIAamp Viral RNA extraction kit and operate according to the instructions.

[0060] The specific operation steps are as follows:

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

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

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

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

[0065]⑤ Put 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 c...

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PUM

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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 defectsof 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 sensitivemethod 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 flaviviruses and alphaviruses in mosquito samples by real-time fluorescent RT-PCR with double TaqMan probes. Background technique [0002] Arboviruses refer to a group of viruses that cause natural foci diseases and zoonotic diseases caused by blood-sucking arthropods biting sensitive vertebrates. Mainly concentrated in the Flavivirus genus, Togaviridae Alphavirus, Reoviridae and Bunyaviridae, the Flaviviruses are the most harmful to humans. People and livestock are infected by the bites of arthropods carrying viruses, which not only cause human disease and death, but also cause a large number of livestock to become sick or even die, causing huge economic losses and becoming a public health problem in all countries. [0003] At present, the gold standard for arbovirus identification is virus isolation, but it is difficult to be popularized and applied in practice because of its cumbersome ...

Claims

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Application Information

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Patent Type & Authority Applications(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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