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Recombinant polymerase isothermal amplification detection method of hepatitis B viruses in ducks

A hepatitis B virus, isothermal amplification technology, applied in the field of molecular biology, to achieve the effect of good specificity, rapid diagnosis and simple operation

Pending Publication Date: 2020-08-28
POULTRY INST SHANDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

but So far, there has been no report on the detection of duck hepatitis B virus by RPA technology

Method used

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  • Recombinant polymerase isothermal amplification detection method of hepatitis B viruses in ducks
  • Recombinant polymerase isothermal amplification detection method of hepatitis B viruses in ducks
  • Recombinant polymerase isothermal amplification detection method of hepatitis B viruses in ducks

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Design and optimization of primers

[0041] 1. Design of primers:

[0042] In this embodiment, by comparing the C and S gene sequences of 40 strains of duck hepatitis B virus reported in NCBI, a series of RPA primers were designed by selecting the conserved regions of duck hepatitis B virus C and S genes, see for details Table 1.

[0043] Table 1: RPA primers designed for the conserved regions of duck hepatitis B virus C and S genes

[0044]

[0045]

[0046] Utilize the RPA amplification reaction to screen the primers in Table 1, select the standard duck hepatitis B virus serum sample, extract the virus DNA as a template, and carry out RPA detection. The specific steps are as follows:

[0047] Extract the DNA template of the duck hepatitis B virus sample according to the EasyPure@ViralDNA / RNA Kit kit instructions, and perform the RPA reaction system according to the TwistAmp DNA Amplification Kits kit instructions: 1.2 μL each of the upstream and dow...

Embodiment 2

[0050] Embodiment 2: Optimization of RPA reaction conditions

[0051] The usage amount of primers F1 and R1 in primer pair 1 was optimized respectively. The optimization results showed that the concentration of primers F1 and R1 was 20 μmol / μL, and the effect was the best when the usage amount was 1.2 μL. In addition, 6 concentration gradients were set up for the amount of MgAc used. The amount of MgAc added to the RPA system at a concentration of 280 mM was 0.5 μL, 1 μL, 1.5 μL, 2 μL, 2.5 μL and 3 μL. Amplification failed. When 1.5μL, 2μL, 2.5μL and 3μL MgAc were added to the reaction system, amplification products could be obtained, and the amplification effect was the best when the amount of MgAc was 2.5μL.

[0052] The optimized RPA reaction system is: 1.2 μL each of upstream and downstream primers (20 μmol / μL), RehydrationBuffer 29.5 μL, Template 3 μL, ddH 2 O 12.6 μL, 280 mM MgAc 2.5 μL.

[0053] In addition, the RPA reaction time and reaction temperature were optimize...

Embodiment 3

[0055] Embodiment 3: specificity test

[0056] Duck hepatitis B virus, duck hepatitis A virus type 1 (DHAV-1), duck hepatitis A virus type 3 (DHAV-3), duck plague virus (DPV), duck-derived Escherichia coli (E.coli), duck-derived Xincheng The DNA / cDNA template of epidemic virus (NDV), duck origin H9 subtype avian influenza virus (AIV-H9) and Riemerella anatipestifer (RA) carries out RPA by the optimized reaction condition of embodiment 2, by gel Electrophoretic detection. Experimental results such as figure 1 As shown, only the duck hepatitis B virus is positive, and the others are all negative, indicating that the RPA detection system of the present invention has good specificity.

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Abstract

The invention discloses a recombinant polymerase isothermal amplification detection method of hepatitis B viruses in ducks, a special RPA primer pair as shown in SEQ ID No. 1 and SEQ ID No. 2 and a kit. Specific primers are designed according to the gene sequence of the duck hepatitis B viruses, and the recombinant polymerase isothermal amplification method for quickly and accurately detecting theduck hepatitis B viruses is further optimized and established. Through the method, the duck hepatitis B viruses can be detected specifically, the minimum detection template amount is 146 pg through the method, and the sensitivity is equivalent to that of traditional PCR. Since only one temperature is needed during the whole gene amplification, no special instruments and equipment are needed, theoperation is simpler and quicker, and the method and kit are suitable for on-site quick detection of the duck hepatitis B virus during production.

Description

technical field [0001] The invention relates to an isothermal amplification detection method for duck hepatitis B virus recombinant polymerase, belonging to the technical field of molecular biology. Background technique [0002] Hepatitis B virus (HBV) infection is a major public health problem worldwide. Due to the narrow range of HBV hosts, obvious hepatotropism and species specificity, the artificial culture of HBV has not been successful, and the establishment of ideal HBV-infected cell and animal models is also very difficult, which has become a major obstacle limiting the development and evaluation of anti-HBV drugs. Duck hepatitis B virus (DHBV) and HBV belong to the family Hepadnaviridae, and both have many similarities in morphology, nucleic acid composition, biological characteristics, pathogenesis and relative hepatotropism. Susceptible, rich in sources, low in price and easy to raise, it is often used as an ideal animal model for screening anti-HBV drugs and stu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/706C12Q1/6844
Inventor 马秀丽朱蕴暖刘娜黄兵刘存霞胡峰郭效珍李玉峰于可响宋敏训艾武亓丽红
Owner POULTRY INST SHANDONG ACADEMY OF AGRI SCI
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