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Method for detecting real-time fluorescence quota PCR of epidemic encephalitis B virus and kit

A real-time fluorescence quantitative and Japanese encephalitis virus technology, which is applied in the field of virus molecular biology detection, achieves the effects of high sensitivity, improved detection efficiency, and pollution prevention

Active Publication Date: 2011-01-12
NAT INST FOR THE CONTROL OF PHARMA & BIOLOGICAL PROD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no real-time fluorescent quantitative PCR technology with probe method for the quantitative detection of JE virus in clinical patients and miniature pig samples

Method used

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  • Method for detecting real-time fluorescence quota PCR of epidemic encephalitis B virus and kit
  • Method for detecting real-time fluorescence quota PCR of epidemic encephalitis B virus and kit
  • Method for detecting real-time fluorescence quota PCR of epidemic encephalitis B virus and kit

Examples

Experimental program
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Effect test

Embodiment 1

[0037] Example 1. Design of primers and TaqMan probes for real-time fluorescent quantitative PCR detection of Japanese encephalitis virus (JEV)

[0038] Refer to the full sequence of the Japanese encephalitis virus SA14 genome (GeneBank: U14163) included in GenBank, and compare the sequences of the dengue virus and West Nile virus, which are very similar in structure to the Japanese encephalitis virus genome. Select the E gene conserved region, and use ABI Primer Express 3.0 Real-time fluorescent quantitative PCR primer design software, design and synthesize TaqMan probes and primers. For the fluorescent label of the probe, FAM (5' end) is selected as the reporter luminescent group, and NFQ (3' end) is the quenching group. The sequence is as follows:

[0039] Upstream primer (JEV-1): 5'-GAGAAACAGAGAACTCCTCATGGAA-3' (SEQID NO: 1 in the sequence listing);

[0040] Downstream primer (JEV-2): 5'-CTGTGACCCAAGAGCAACAAC-3' (SEQ ID NO: 2 in the sequence listing).

[0041] TaqMan probe: 5'FA...

Embodiment 2

[0042] Example 2. Real-time fluorescent quantitative PCR detection of Japanese encephalitis virus

[0043] 1. The establishment of standard curve

[0044] 1. Construction of pMD-JEV-E recombinant plasmid

[0045] 1.1 Target gene-clone of Japanese encephalitis virus E gene

[0046] 1.1.1 Extraction of Japanese encephalitis virus RNA

[0047] The following operations must be carried out in the negative pressure biological safety cabinet of the P2 laboratory in strict accordance with the requirements.

[0048] (1) Under aseptic conditions, put 50mg of JE animal brain tissue sample or 500μL of cerebrospinal fluid of JE patients or 300μL of JE virus fluid into a 1.5mL clean centrifuge tube, add 500μL of Trizol Reagents (purchased from Promega, USA), and shake well. Let stand at room temperature for 5 min.

[0049] (2) Add 100 μL of chloroform, shake vigorously for 10 sec, stand at room temperature for 5 min, and centrifuge at 4° C., 12000 r / min for 10 min.

[0050] (3) Carefully transfer the u...

Embodiment 3

[0084] Example 3 Real-time fluorescent quantitative PCR detection kit for Japanese encephalitis virus probe method

[0085] JEV TaqMan assay mix 50μL (TaqMan probe concentration 250nM, upstream and downstream primer concentrations are 900nM each), JEV TaqMan mix 500μL, JEV positive quality control product 50μL, negative control 50μL of the product and 500μL of RNase-free water are packaged together to obtain a real-time fluorescent quantitative PCR detection kit for Japanese encephalitis virus (50 reactions).

[0086]

[0087]

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Abstract

The invention discloses a method for detecting real-time fluorescence quota PCR of epidemic encephalitis B virus and a kit. A primer and a TaqMan probe is designed in accordance with the conserved region of the epidemic encephalitis B virus E gene, which is used for quantitatively detecting nucleic acid copy number of the epidemic encephalitis B virus in samples. Particularly, the forward primer of the primer has a nucleotide sequence of SEQ ID NO: 1 in a sequence table, the reverse primer of the primer has a nucleotide sequence of SEQ ID NO: 2 in the sequence table, and TaqMan probe has a nucleotide sequence in SEQ ID NO: 3 in the sequence table. The invention has larger valuable significance in the field of controlling the quality of biological products related to the human and animal sources and inspecting and quarantining the imported and exported animals, ensures the quality of the related biological products and controls the spread of the epidemic encephalitis B and the medication safety of people. The detection method and the kit of the invention can be used for inspecting the epidemic encephalitis B virus on cerebrospinal fluid samples of the clinical patients, miniature pig brain tissue samples and the biological products, and have wide application prospect.

Description

Technical field [0001] The invention relates to a method for molecular biology detection of viruses in the field of biotechnology, in particular to a real-time fluorescent quantitative PCR detection method and kit for Japanese encephalitis virus. Background technique [0002] Japanese encephalitis virus was first isolated from the brain tissue of patients in Japan (1953), so it was called Japanese encephalitis virus (JEV), and the disease caused by it was called Japanese encephalitis (Japanese encephalitis, JBE). In 1950, my country conducted a large number of etiological and epidemiological studies on the disease. In order to distinguish it from A encephalitis, it was named Japanese encephalitis (Japaneseencephalitis), or Japanese encephalitis for short. According to the relevant regulations of my country's "Infectious Disease Prevention and Control Law", Japanese encephalitis is a Class B infectious disease in my country. Japanese encephalitis is an acute infectious disease o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68G01N21/64C12N15/11
Inventor 高正琴贺争鸣
Owner NAT INST FOR THE CONTROL OF PHARMA & BIOLOGICAL PROD
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