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Combination of primer and probe for distinguishing duck tembusu virus wild virulent strain and vaccine strains

A technology of duck Tembusu virus and wild strain, applied in the direction of recombinant DNA technology, microbial measurement/inspection, biochemical equipment and methods, etc., can solve the identification and detection method of unretrieved DTMUV wild strain and vaccine strain, etc. problem, achieve the effect of shortening the required time, high accuracy and improving efficiency

Active Publication Date: 2018-07-24
INST OF ANIMAL HEALTH GUANGDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the detection of DTMUV mainly includes virus isolation and identification, nested RT-PCR, RT-LAMP, fluorescent quantitative RT-PCR, double-antibody sandwich ELISA, and no identification detection method for DTMUV wild strains and vaccine strains has been found.

Method used

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  • Combination of primer and probe for distinguishing duck tembusu virus wild virulent strain and vaccine strains
  • Combination of primer and probe for distinguishing duck tembusu virus wild virulent strain and vaccine strains
  • Combination of primer and probe for distinguishing duck tembusu virus wild virulent strain and vaccine strains

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Primers and Probes

[0036] After the inventors of the present application screened a large number of primers and probes designed, they found that the primer F / R and probe P had the best effect on the melting curve analysis method for distinguishing DTMUV wild strains from vaccine strains. The sequence is as follows.

[0037] Primer F: 5'-ATAACAGTCAACCCATACGTGTC-3' (SEQ ID NO: 1);

[0038] Primer R: 5'-CACTTCTATGCCACTGGTACCT-3' (SEQ ID NO: 2);

[0039] Probe P: 5'-CCACTTCCACCATTATCTTGGCACCCG-3' (SEQ ID NO: 3), wherein the 5' end of the probe P is bound to a fluorescent reporter group, and the 3' end of the probe P is bound to a fluorescent quencher group.

Embodiment 2

[0040] The preparation of embodiment 2 standard samples, fluorescent PCR amplification and melting curve analysis

[0041] 1) Extraction of duck Tembusu virus RNA

[0042] Take DTMUV live vaccine WF100 strain and FX2010-180P strain respectively, add 3mL PBS hydrochloric acid buffer solution to dissolve, respectively take 200μL of DTMUV wild strain W strain cell culture solution according to the instructions of MiniBEST Viral RNA / DNAExtraction Kit Ver.4.0 of TAKARA company Perform nucleic acid extraction.

[0043] 2) Preparation of standard samples

[0044] In order to verify the feasibility and reliability of the method of the present invention, construct a standard positive sample (correctly determined by sequence) at the same time, and provide a positive control for subsequent clinical sample detection. In this embodiment, the DTMUV wild strain W and the vaccine strain WF100 strain and Positive standard samples of FX2010-180P strain p-W, p-WF100 and p-FX2010-180P.

[0045...

Embodiment 3

[0061] Embodiment 3 is used for distinguishing the method for duck Tembusu virus wild strain and vaccine strain

[0062] An embodiment of the method for distinguishing duck Tembusu virus wild strain and vaccine strain of the present invention, comprises the steps:

[0063] 1) Extracting viral nucleic acid from the sample: the method is the same as the nucleic acid extraction method in Example 2 above.

[0064] 2) Using the extracted viral nucleic acid as a template, perform fluorescent PCR amplification reaction and melting curve analysis respectively; meanwhile, use the positive standard products p-W, p-WF100 and p-FX2010-180P in Example 2 as positive controls.

[0065] Wherein, the PCR reaction system is as follows:

[0066]

[0067]

[0068] The PCR amplification reaction procedure is as follows:

[0069] Reverse transcription at 50°C for 30min; pre-denaturation at 95°C for 2min; denaturation at 95°C for 20s, annealing at 50°C for 20s, extension at 72°C for 20s; cyc...

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Abstract

The invention discloses a method and corresponding primer and probe for distinguishing duck tembusu virus wild virulent strain and vaccine strains and corresponding primer and probe. According to themethod, a fluorescent PCR (Polymerase Chain Reaction) detection method capable of quickly distinguishing the duck tembusu virus wild virulent strain and vaccine strains (a WF100 strain and an FX2010-180P strain) is established for the first time; the detection method is simple to operate; all the whole operation processes does not exceed 3 hours; the time needed by the identification and detectionof the virus wild virulent strain and vaccine strains is obviously shortened, and moreover, the method is high in accuracy, good in specificity and good in repeatability, can be used for carrying outanalysis in an accurate, quick and high-throughput manner, and is beneficial to the popularization and application in clinical practice.

Description

technical field [0001] The invention relates to the technical field of identifying duck Tembusu virus wild strains and vaccine strains, in particular to a combination of primers and probes, a kit and a method for distinguishing duck Tembusu virus wild strains and vaccine strains. Background technique [0002] Duck tembusu virus disease is a new duck infectious disease caused by duck tembusuvirus (DTMUV), with an infection rate of 100% and a morbidity and mortality rate of 5% to 30%. Clinical symptoms such as slow growth, high fever, loss of appetite, sharp decline or even stop of egg production, and death of sick ducks have caused serious economic losses to the duck industry in my country. DTMUV is an enveloped, single-stranded positive-sense RNA virus belonging to the family Flaviviridae, the genus Flavivirus, and the mosquito-borne virus-like Entayavirus group. DTMUV can infect not only ducks, but also poultry such as chickens, geese, and sparrows. The virus has even been ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/701C12Q2563/107
Inventor 刘志成孙敏华张春红董嘉文李林林沈海燕孙俊颖张建峰
Owner INST OF ANIMAL HEALTH GUANGDONG ACADEMY OF AGRI SCI
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