M-gene based fluorescent RT-PCR (Reverse Transcription Polymerase Chain Reaction) detection method of Nipah virus

A RT-PCR, Nipah virus technology, applied in the field of inspection and quarantine, can solve the problems of no effective treatment and prevention, Nipah disease threat, etc., to achieve the effects of simple operation, enhanced specificity, and high specificity

Inactive Publication Date: 2012-07-11
ANIMAL & PLANT & FOOD INSPECTION CENT OF TIANJIN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

Although Nipah disease has not yet appeared in my country, due to the threat of epidemics from neighboring countries and the ecological environment and animal distribution similar to the original place of NiV in the southeastern coastal areas of my country, Nipah disease has posed an obvious threat to my country
[0006] There is currently no effective method for treatment and prevention of this disease, and related vaccines are still under study

Method used

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  • M-gene based fluorescent RT-PCR (Reverse Transcription Polymerase Chain Reaction) detection method of Nipah virus

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Experimental program
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Effect test

Embodiment

[0043] 1. Sample processing and RNA extraction

[0044] Take 100mg of pig product tissue and grind it for RNA extraction.

[0045] Take 200 μL of the pseudovirus and add it to an EP tube containing 750 μL Trizol reagent without RNase, shake it well for 15 seconds, and let it stand at room temperature for 5 minutes. Add 200 μL of RNase-free chloroform, shake for 15-30 seconds, let stand at room temperature for 2-3 minutes, centrifuge at 12,000 r / min at 4°C for 15 minutes; carefully absorb the upper aqueous phase and transfer it to a new EP tube; add unused RNase-free iso Propanol 500μL, mix well, let stand at room temperature for 10min; centrifuge at 4℃, 12000r / min for 10min; discard the supernatant, add 1mL of 75% ethanol prepared with DEPC water, shake, fully wash the precipitate, 4℃, 12000r / min Centrifuge at 1 / min for 5 min; discard the supernatant, dry in vacuum, add 5-10 μL RNase-free triple distilled water to dissolve the precipitate, and set aside. Take the ground 100m...

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Abstract

The invention discloses an M-gene based fluorescent RT-PCR (Reverse Transcription Polymerase Chain Reaction) detection method of Nipah virus, and a kit thereof. The method adopts a one-step fluorescent RT-PCR method to detect the Nipah virus in porcine samples, is simple, convenient and fast to operate, and has higher specificity and sensitivity. According to the method, false viruses without infectivity are adopted by the positive control sample, the property is stable, false viruses do not need to be cultured, and the laboratory biosafety is good.

Description

technical field [0001] The invention relates to a Nipah virus fluorescent RT-PCR detection method, in particular to a Nipah virus TaqMan-MGB probe fluorescent RT-PCR detection method, which belongs to the technical field of inspection and quarantine. Background technique [0002] Nipah disease first broke out in Malaysia in 1998, causing morbidity and mortality in pigs and humans. Pathogen ecology and epidemiological surveys found that fruit-eating bats of the family Pteropus are the natural host of the pathogen of this disease, Nipah Virus (NiV), and play a decisive role in the process of spreading the disease to animals and humans. NiV is not pathogenic to bats, has certain pathogenicity to pigs, but is highly pathogenic to humans, and the fatality rate of humans after infection with NiV reaches 40% to 70%. Therefore, NiV is listed as the most dangerous biosafety level 4 pathogenic microorganism. [0003] Together with Hendra virus, NiV belongs to the Henipavirus genus o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
Inventor 王建华王玉玲王乃福赵祥平陈本龙吴绍强
Owner ANIMAL & PLANT & FOOD INSPECTION CENT OF TIANJIN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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