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Real-time fluorescent reverse transcription PCR detection primer, probe, detection kit and detection method for hepatitis e viruses

A technology for hepatitis E virus and detection kits, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve the lack of mature technical information on technical parameters and the lack of real-time detection kits. , the difficulty of primer design, etc., to achieve the effects of accurate and reliable detection results, short detection time and high sensitivity

Inactive Publication Date: 2017-10-24
广州白云机场海关综合技术服务中心
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Problems solved by technology

However, due to the complexity of the hepatitis E virus genotype, it is difficult to design primers when using the PCR method, including the sequence design and selection of primer probes, the amount of primers / probes, the amount of magnesium ions, reaction conditions and negative / positive A number of technical parameters such as reference substances still lack mature technical information, so there is no real-time detection kit with a wide range of adaptation, easy operation, accuracy and reliability, which limits the promotion and popularization of this method

Method used

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  • Real-time fluorescent reverse transcription PCR detection primer, probe, detection kit and detection method for hepatitis e viruses
  • Real-time fluorescent reverse transcription PCR detection primer, probe, detection kit and detection method for hepatitis e viruses
  • Real-time fluorescent reverse transcription PCR detection primer, probe, detection kit and detection method for hepatitis e viruses

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Embodiment 1

[0035] 1. Design and selection of detection probes and detection primers

[0036] 1.1 Design of detection primers and detection probes

[0037] 1.1.1 Design principles of detection primers and detection probes

[0038] According to the TaqMan fluorescent probe PCR detection method, the following primer and probe design principles are determined:

[0039] (1) The length of primers and probes: each primer is 18-25 bases in length, and the fluorescent probe is 20-30 bases in length.

[0040] (2) The GC% in primers and probes should be 30% to 70%, and the Tm value of the fluorescent probe should be higher than the Tm value of the primer by more than 5°C.

[0041] (3) Avoid base pairing as much as possible between the primer and the probe itself, and the 3'end between the primer and the probe.

[0042] (4) As far as possible, avoid more than 6 consecutive base pairings between the primer and the probe itself, between the primer and the probe.

[0043] (5) The selected primers and probes are req...

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Abstract

The invention discloses a real-time fluorescent reverse transcription PCR detection primer, a probe, a detection kit and a detection method for hepatitis e viruses. Aiming at hepatitis e viruses, the invention providesa specific primer, a probe and a detection kit containing the primer and the probe, and provides a detection method which utilizes the detection kit for confirming that whether the sample to be detected contains the hepatitis e viruses through real-time fluorescent reverse transcription PCR amplification. The detection kit and the detection method can realize quick detection on all genotypes of the hepatitis e viruses, have the advantages of strong specificity, high sensitivity, wide application range, simple, convenient and quick operation, accurate and reliable detection result and short detection time, can enable the sensitivity to reach 1*10<3>copies / mL, can solve the problems of long time consumption, low sensitivity and the like of the traditional detection method, are especially suitable for quick detection of large-scale samples, and can lay the foundation for infection source survey, propagation environments, virus sources and the like.

Description

Technical field: [0001] The invention belongs to the field of biotechnology, and specifically relates to a hepatitis E virus real-time fluorescent reverse transcription PCR detection primer, probe, detection kit and detection method. Background technique: [0002] Hepatitis E (Hepatitiv E) is an acute infectious disease caused by Hepatitis E virus (HEV) that seriously endangers human health. In recent years, the absolute number and incidence of hepatitis E have shown a continuous and rapid increase. Therefore, it is of great significance to improve the diagnosis and treatment of hepatitis E. Currently, there are many diagnostic methods for hepatitis E, such as virus isolation and identification, enzyme-linked immunosorbent assay, polymerase chain reaction, etc. Among them, the polymerase chain reaction (Polymease Chain Reaction (PCR)) has made breakthroughs in theory and application, and has received great attention in the industry. However, due to the complex type of hepatitis...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/707C12Q2561/113C12Q2563/107C12Q2521/107
Inventor 孙薇陆敏苏艳丽毕英杰
Owner 广州白云机场海关综合技术服务中心
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