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Identification and detection method for hepatitis E virus by utilizing quadruple fluorescence quantitative PCR (Polymerase Chain Reaction)

A fluorescence quantification, identification and detection technology, applied in the direction of determination/inspection of microorganisms, fluorescence/phosphorescence, biochemical equipment and methods, etc., to achieve the effect of improving detection efficiency and reducing detection cost

Active Publication Date: 2011-02-02
THE INSPECTION & QUARANTINE TECH CENT ZHEJIANG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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Problems solved by technology

At present, there is no report on the method of quickly identifying each genotype of hepatitis E virus simultaneously.

Method used

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  • Identification and detection method for hepatitis E virus by utilizing quadruple fluorescence quantitative PCR (Polymerase Chain Reaction)
  • Identification and detection method for hepatitis E virus by utilizing quadruple fluorescence quantitative PCR (Polymerase Chain Reaction)
  • Identification and detection method for hepatitis E virus by utilizing quadruple fluorescence quantitative PCR (Polymerase Chain Reaction)

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Experimental program
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Embodiment Construction

[0033] 1 Materials and methods

[0034] 1.1 Establishment of HEV multiplex fluorescent quantitative PCR method

[0035] 1.1.1 Design and synthesis of primers and probes

[0036] After analyzing the complete sequences of HEV strains of different genotypes that have been published, a pair of conservative primers were designed for the HEV ORF3 sequence, which can simultaneously amplify different types of HEV strains. At the same time, four type-specific probes were designed for the variable region of this amplified region, respectively labeled with different fluorescent groups, and used for the one-step fluorescent quantitative PCR detection of HEV (Table 1):

[0037] Table 1 Primer and probe sequences

[0038]

[0039] a In the merger primer, D represents the base A, G or T; Y represents the base T or C; W represents the base A or T; R represents the base A or G

[0040] 1.1.2 Optimization of fluorescent quantitative PCR reaction conditions

[0041] Using HEV positive RN...

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Abstract

The traditional pig source hepatitis E has no effective vaccine for prevention, which adopts a control measure that the finding is carried out as soon as possible and the epidemic condition in epidemic areas is monitored at any time to block the spread of the disease. The invention discloses a rapid detection method for identifying each gene type of hepatitis E virus by utilizing multiple fluorescence quantitative PCR, which is characterized in that the a pair of conservative amplification primers and 4 strip-shaped specificity TaqMan probes are designed aiming at an HEVORF 3 sequence; the four probes are respectively designed aiming at the respective ORF 3 metamorphosis region sequence in a type specificity mode; and the identification and the detection of different gene types can be realized. The invention maintains the characteristics of high sensitivity and high accuracy of a PCR method and has the advantages that the quadruple fluorescence quantitative PCR enhances the detection efficiency and reduces the detection cost; the purposes of identification and diagnosis can be simultaneously realized; and the invention lays a foundation for work of infection source survey, spread environment, virus source tracing, etc.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and relates to a rapid detection method for identifying each genotype of hepatitis E virus by multiple fluorescent quantitative PCR in human serum, animal serum and products thereof, food and environment. Background technique [0002] Hepatitis E (Hepatitis E) is caused by Hepatitis E virus (HEV). It is an acute infectious disease that is transmitted through feces and oral cavity and is mainly jaundice. The fatality rate of sick pregnant women can be as high as 21%. . Since the first major outbreak in India in 1955, hepatitis E has become widespread in many countries in Asia, Africa and Latin America. In recent years, the disease has also spread in Japan and other developed countries. my country is a country with a high incidence of hepatitis E, and Xinjiang, Liaoning, Shandong and other provinces all have a large number of epidemics, which greatly endanger human health. There is ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/70G01N21/64
Inventor 张晓峰帅江冰吴姗陈笑梅何永强
Owner THE INSPECTION & QUARANTINE TECH CENT ZHEJIANG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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