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One-step dual RT-PCR detection kit of 1-type and 3-type duck hepatitis A virus, primer pairs and method thereof

A technology for duck hepatitis A virus and a detection method is applied in the field of primer sets for detecting type 1 duck hepatitis A virus and type 3 duck hepatitis A virus, which can solve the problem that the detection primers are not applicable and cannot diagnose and analyze type 3 duck hepatitis A virus strains. , There are no problems such as one-step double RT-PCR detection technology of duck hepatitis A virus type 1 and duck hepatitis A virus type 3, which can avoid repeated detection and low cost.

Active Publication Date: 2013-11-06
SICHUAN AGRI UNIV
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0005] At present, there are single RT-PCR molecular detection technology and two-step multiple RT-PCR detection technology for type 1 duck hepatitis A virus and type 3 duck hepatitis A virus, but there is no one-step dual method for type 1 duck hepatitis A virus and type 3 duck hepatitis A virus. Report on RT-PCR Detection Technology
At the same time, because the small RNA virus to which duck hepatitis A virus belongs is very prone to gene mutation and recombination, it is found through BLAST verification that the detection primers provided in the existing detection technology do not have wide applicability, and cannot be used for many types of duck hepatitis A virus and type 1. Accurate Diagnosis and Analysis of Type 3 Duck Hepatitis A Virus Strains

Method used

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  • One-step dual RT-PCR detection kit of 1-type and 3-type duck hepatitis A virus, primer pairs and method thereof
  • One-step dual RT-PCR detection kit of 1-type and 3-type duck hepatitis A virus, primer pairs and method thereof
  • One-step dual RT-PCR detection kit of 1-type and 3-type duck hepatitis A virus, primer pairs and method thereof

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Experimental program
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Effect test

Embodiment 1

[0044] Embodiment 1, design and synthesis of primers

[0045] According to the whole genome sequences of duck hepatitis A virus type 1 and duck hepatitis A virus type 3 in GenBank, the Primer Premier5.0 software was used to design primers in their relatively conserved regions; two pairs of specific primers (Table 1) were synthesized by Shanghai Invitrogen Company.

[0046] Table 1 Identification of DHAV-1 and DHAV - 3 primer sequences

[0047]

Embodiment 2

[0048] Example 2, one-step double RT-PCR detection of type 1 duck hepatitis A virus and type 3 duck hepatitis A virus

[0049] 1. Preparation of samples to be tested

[0050] According to the operation manual of TRNzol total RNA extraction reagent, extract the RNA of duck hepatitis A virus type 1 and duck hepatitis A virus type 3, muscovy duck parvovirus, duck plague virus, infectious laryngotracheitis virus, Marek's virus, duck plague The DNA of Riemerella, Pasteurella, Staphylococcus aureus, Duck Escherichia coli and Duck Salmonella were tested for their nucleic acid concentration and purity using a nucleic acid protein detector (Bio Rad, Smartspec3000), and stored at -70°C for later use.

[0051] 2. Establishment of one-step double RT-PCR reaction

[0052] Use the One Step RT-PCR kit to perform one-step double RT-PCR. By optimizing the amount of primer pair 1 and primer pair 2 used in the reaction solution and the annealing temperature, time, and cycle times of the double ...

Embodiment 3

[0068] Embodiment 3, double PCR detection sample to be tested

[0069] 1. Sample preparation

[0070] Grind the collected 20 copies (numbered 1-20) of suspected duck hepatitis A virus-infected duck disease material (liver) into a suspension, freeze and thaw 3 times and then centrifuge at high speed, then take the supernatant and inoculate it into 11-day-old non-specific Pathogen in the allantoic cavity of healthy duck embryos, 20 pieces / plant, collect the allantoic fluid of dead embryo bodies within 24-120 hours, and obtain 20 samples to be tested.

[0071] 2. Double RT-PCR detection

[0072]1. Preparation of the template: Extract the total RNA in 20 samples to be tested according to step 1 of Example 2. Specifically refer to the instruction manual of TRNzol total RNA extraction reagent, and use a nucleic acid protein detector (Bio Rad, Smartspec3000) to measure its nucleic acid concentration and purity, and store it at -70°C for later use.

[0073] 2. Double RT-PCR detecti...

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Abstract

The invention discloses a one-step dual RT-PCR detection kit of 1-type and 3-type duck hepatitis A virus, primer pairs and a method thereof. The primer 1 is composed of a primer 1 and a primer 2 which are used in detection of 1-type duck hepatitis A virus, and nucleotide sequences of the primer 1 and the primer 2 are respectively single chain DNA as shown in SEQ ID NO:1 and SEQ ID NO:2. The primer pair 2 is composed of a primer 3 and a primer 4 which are used in detection of 3-type duck hepatitis A virus, and nucleotide sequences of the primer 3 and the primer 4 are respectively single chain DNA as shown in SEQ ID NO:3 and SEQ ID NO:4. The detection technology for simultaneously detecting two pathogens of 1-type and 3-type duck hepatitis A virus by one RT-PCT reaction is adopted to avoid repeated conventional PCR detection, and has advantages of low cost, high efficiency and the like. In addition, amplification results are directly determined by the utilization of different lengths of amplified fragments for the design of the primers such that the method is simpler, more visual and practical during result determination.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a primer set, a kit and a detection method for detecting type 1 duck hepatitis A virus and type 3 duck hepatitis A virus. Background technique [0002] Duck virus hepatitis (DVH) is an acute and highly contagious disease caused by ducklings infected by duck hepatitis virus (DHV). The disease mainly affects ducklings less than four weeks old, and has the characteristics of acute onset, rapid spread, short course of disease and high mortality; the main clinical manifestations are convulsions before the duckling dies, and the head is tilted back to the back, showing "opistonus" , The pathological changes are mainly hepatomegaly and inflammation and a large number of hemorrhagic spots seen in necropsy, which is one of the main diseases that endanger the duck industry. At present, the disease exists in major duck-raising areas in the world, and has the charac...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
Inventor 程安春文兴建汪铭书朱德康陈孝跃
Owner SICHUAN AGRI UNIV
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