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Non-diagnostic method for detecting flavivirus by TaqMan probe through real-time fluorescent reverse transcription-polymerase chain reaction (RT-PCR)

A RT-PCR, real-time fluorescence technology, applied in the direction of fluorescence/phosphorescence, biochemical equipment and methods, microbial determination/inspection, etc.

Inactive Publication Date: 2012-07-04
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 flavivirus by TaqMan probe through real-time fluorescent reverse transcription-polymerase chain reaction (RT-PCR)
  • Non-diagnostic method for detecting flavivirus by TaqMan probe through real-time fluorescent reverse transcription-polymerase chain reaction (RT-PCR)
  • Non-diagnostic method for detecting flavivirus by TaqMan probe through real-time fluorescent reverse transcription-polymerase chain reaction (RT-PCR)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] 1. The sample to be tested is JE virus vaccine, the positive control is JE virus strain RNA, and the negative control is RNase-Free Water.

[0025] 2. Extraction of JE virus vaccine RNA: extract sample RNA according to the instruction manual of QIAamp Viral RNA Mini Kit.

[0026] The specific operation steps are as follows:

[0027] ① Dispense lysate AVL according to the number of samples, 560 μl per tube.

[0028] ② Take 140 μl virus solution and add it to 560 μl aliquoted AVL, vortex for 15 seconds, mix well, and incubate at room temperature for 10 minutes.

[0029] ③ Add 560 μl of absolute ethanol (96%-100%) to each tube, vortex for 15 seconds to mix well, and centrifuge quickly.

[0030] 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.

[0031] ⑤ Put the filter column into a new 2ml collection tube, suck a...

Embodiment 2

[0047] 1. Extraction of yellow fever virus vaccine RNA: use the QIAamp Viral RNA extraction kit and operate according to the instructions.

[0048] 2. Preparation of reaction system

[0049] Each reaction tube contains the following reagents:

[0050]

[0051]

[0052] 3. On the machine, amplification is carried out under the following conditions, amplification conditions: standard amplification conditions in the field are adopted.

[0053] 15 minutes at 45°C

[0054] 95°C 10 minutes

[0055]

[0056] 4. Collect fluorescence signal and detect Ct value

[0057] 5. Result judgment. Such as figure 2 Shown is the yellow fever virus vaccine RNA amplification curve, in which: red is the positive control Ct value of 19.2, brown is the yellow fever virus vaccine RNA Ct value of 25, and blue is the negative control Ct value that cannot be detected.

[0058] The above-mentioned virus is extracted from a kit. The detection method disclosed in the present invention is not ...

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PUM

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Abstract

The invention discloses a non-diagnostic method for detecting flavivirus by a TaqMan probe through real-time fluorescent reverse transcription-polymerase chain reaction (RT-PCR). The non-diagnostic method comprises one pair of primers and the probe which are used for performing real-time quantitative detection on a sample to be detected. The invention aims to solve the problem that convenience is brought to monitoring and detecting emerging arboviruses because the flavivirus has multiple members and the detection is complicated. The defects existing in traditional and modern methods are overcome by the non-diagnostic method. The primers and the probe have the characteristics that the specificity is high, the repeatability is high, the sensitivity is high and multiple quantitation can be performed.

Description

technical field [0001] The invention relates to a non-diagnostic method for detecting flaviviruses by real-time fluorescent RT-PCR with TaqMan probes. Background technique [0002] Flaviviridae, Flaviviruses, Togaviridae Alphaviruses, Reoviridae, and Bunyaviridae are all arboviruses, which are vertebrates that are sensitive to the bite of blood-sucking arthropods And cause a kind of zoonotic natural foci diseases, wherein the greatest harm to humans with the flavivirus. 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 identification method of flaviviruses is virus isolation, but due to the harsh requirements for experimental conditions, complicated operation, high tec...

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方艳辉杨鹏飞
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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