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Non-diagnostic method for detecting alpha-virus by use of TaqMan probe through real-time fluorescent everse transcription-polymerase chain reaction (RT-PCR)

A RT-PCR, real-time fluorescence technology, applied in fluorescence/phosphorescence, biochemical equipment and methods, microbial determination/inspection, etc., to achieve the effects of high sensitivity, simple steps and fast speed

Inactive Publication Date: 2012-07-11
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most of the RT-PCR and real-time fluorescent PCR currently used are only for the detection of a certain arbovirus

Method used

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  • Non-diagnostic method for detecting alpha-virus by use of TaqMan probe through real-time fluorescent everse transcription-polymerase chain reaction (RT-PCR)
  • Non-diagnostic method for detecting alpha-virus by use of TaqMan probe through real-time fluorescent everse transcription-polymerase chain reaction (RT-PCR)
  • Non-diagnostic method for detecting alpha-virus by use of TaqMan probe through real-time fluorescent everse transcription-polymerase chain reaction (RT-PCR)

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] 1. The sample to be tested is the supernatant of Geta virus-infected cells, the positive control is Geta virus mRNA transcribed in vitro, and the negative control is RNase-Free Water.

[0028] 2. Extraction of RNA from cell supernatant: extract sample RNA according to the QIAamp Viral RNA Mini Kit instruction manual.

[0029] The specific operation steps are as follows:

[0030] ①According to the number of samples, aliquot the lysate AVL, 560 μl per tube;

[0031] 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;

[0032] ③ Add 560 μl of absolute ethanol (96%-100%) to each tube, shake for 15 seconds to mix well, and centrifuge quickly;

[0033] 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;

[0034] ⑤ P...

Embodiment 2

[0049] 1. The sample to be tested is the serum of chikungunya fever patients, and the extraction of viral RNA: use the QIAamp Viral RNA extraction kit and operate according to the instructions.

[0050] 2. Preparation of reaction system

[0051] Each reaction tube contains the following reagents:

[0052]

[0053] 3. On the machine, the amplification is carried out under the following conditions, the amplification conditions: the standard amplification conditions in this field are adopted.

[0054] 15 minutes at 45°C

[0055] 95°C for 10 minutes

[0056]

[0057] 4. Collect fluorescence signal and detect Ct value

[0058] 5. Result judgment. Such as figure 2 Shown is the chikungunya virus RNA amplification curve, in which: red is the positive control Ct value of 22.6, blue is the Chikungunya virus RNA Ct value of 20.7, and light blue is the negative control Ct value that cannot be detected.

[0059] The above-mentioned virus is extracted from a kit. The detection ...

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Abstract

The invention discloses a non-diagnostic method for detecting alpha-virus by use of a TaqMan probe through a real-time fluorescent everse transcription-polymerase chain reaction (RT-PCR), and the method comprises the following steps of: using a pair of primers and the probe to carry out real-time quantitative detection on a sample to be detected, and using PCR to carry out efficient amplificationon deoxyribonucleic acid (DNA). The defects of the conventional PCR detection can be overcome due to the high specificity of a probe technology and the sensitivity and quantitative characteristic of a light spectrum technology; and the non-diagnostic method has the characteristics of being visual, high in specificity, rapid in detection speed, simple in steps, good in repeatability, high in sensitivity and capable of carrying out multiple quantification. In the actual rapid diagnosis, the non-diagnostic method is excellent in operability.

Description

technical field [0001] The invention relates to a non-diagnostic method for detecting alphaviruses by real-time fluorescent RT-PCR with TaqMan probes. Background technique [0002] Togaviridae alphaviruses, Flaviviruses, Reoviridae, and Bunyaviridae belong to the family of arboviruses, which are vertebrates that are sensitive to the bite of arthropods that suck blood. A zoonotic natural foci disease caused by it. Once humans and animals are bitten and infected by arthropods carrying the virus, it will bring disease and death. When the virus breaks out in a large area, it will bring huge losses to the economy of the outbreak area and cause social panic. It is possible for public health agencies in all countries the problem we are facing. [0003] At present, the main means of identification of alphaviruses is virus isolation, but due to its strict requirements on experimental conditions, complicated operation, high technical difficulty and long detection time accompanied by...

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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