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Nest type PCR detection method for different variants of ostreid herpes virus

An oyster herpes virus, the technology to be detected, applied in the direction of microbial determination/inspection, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problem of inability to accurately detect the existence of mutant strain infection and the inability of OsHV-1 detection primers Combination and other issues

Inactive Publication Date: 2016-05-04
YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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Problems solved by technology

At the same time, the molecular epidemiological monitoring data and research results of OsHV-1 in recent years show that the virus has undergone strong strain differentiation since 2008; new mutant strains have been produced due to changes in the base composition or sequence of the C region of the genome. As a result, OsHV-1 detection primers designed based on the nucleotide sequence of this region cannot bind to the target site; eventually, various common and nested PCR detection methods based on the C region cannot accurately detect part of OsHV-1 Presence of Variant Infection

Method used

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  • Nest type PCR detection method for different variants of ostreid herpes virus
  • Nest type PCR detection method for different variants of ostreid herpes virus
  • Nest type PCR detection method for different variants of ostreid herpes virus

Examples

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Embodiment 1

[0028] Embodiment 1: PCR primer design

[0029] Using the Primer5.0 primer design software, based on the nucleotide sequence of the key functional domain of the DNA polymerase gene, the nested PCR detection primers were designed: the upstream sequence of the outer primer, Pol-F: 5'-GATTTCAACTCGCAATACC-3', the outer primer The downstream sequence of the primer, Pol-R: 5'-GGCAGACACAGATTCCACA-3', the amplified fragment size is 573bp; the upstream sequence of the internal primer, nPol-F: 5'-GTGTTTCTACATTGCTGG-3', the downstream sequence of the internal primer nPol-R: 5' -CTGTTGGCGTTGACCTTC-3', the size of the amplified fragment is 386bp.

[0030] Optimization of the first-round PCR reaction system and amplification program: 2 μL of DNA from a scallop sample known to be infected with OsHV-1 was used as a positive template, DNA polymerase (5U / μL) was 0.4 μL, 10×PCR buffer was 5.0 μL, dNTPs (2.5 mmol / L) 4.0μL, Mg 2+(25.0mmol / L) 4.0μL, upstream and downstream primers (10.0μmol / L) 2....

Embodiment

[0036] Embodiment: Nested PCR detection method detects different shellfish OsHV-1 infection situation

[0037] (1) DNA extraction: Take about 30 mg of the mantle tissue of different species of shellfish disease materials collected in different years and collected in the laboratory, and use the marine animal tissue genome kit of Tiangen Biochemical Technology (Beijing) Co., Ltd., provided according to the kit instructions The steps are to extract sample DNA and use it as a template for nested PCR detection. The nucleic acid of Chlamys farreri positive for OsHV-1 was used as a positive control, and the nucleic acid of Chlamys scallop negative for OsHV-1 was used as a negative control.

[0038] (2) The first round of PCR reaction: using TaKaRaEx of Treasure Bioengineering (Dalian) Co., Ltd. Add the following reagents to a 50 μL PCR reaction system: DNA polymerase (5U / μL) 0.4 μL, 10×PCR buffer 5.0 μL, dNTPs (2.5 mmol / L) 4.0 μL, Mg 2+ (25.0mmol / L) 4.0μL, upstream and downstream ...

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Abstract

The invention provides a nest type PCR method for detecting different variants of an ostreid herpes virus-1 (OsHV-1). According to the detection method, an ostreid herpes virus DNA polymerase gene functional domain nucleotide sequence is adopted as a template, two pairs of nested primers are designed, and the nest type PCR detection method is designed based on the theme. Due to the fact that DNA polymerase genes are highly conservative on a herpes virus intraspecific category, the two pairs of nested primers can be combined with all currently-known OsHV-1 variant genomes, specific DNA nucleic acid fragments of the variants are qualitatively detected, and high sensitivity and good specificity are achieved. The severe defect that an existing molecular detection method can only detect part of the variants of the OsHV-1 can be overcome through the detection method. The detection method can be used for tracking and detection on the ostreid herpes virus during breeding of shells in all periods, and has high use value. The method is expected to replace prior relevant molecular detection methods for the ostreid herpes virus.

Description

technical field [0001] The invention belongs to the technical field of shellfish pathogen detection, and in particular relates to a method for detecting oyster herpes virus (Ostreidherpesvirus 1, OsHV-1) by nested polymerase chain reaction (Polymerase Chain Reaction, PCR). Background technique [0002] Since the late 1990s, OsHV-1 has caused mass die-offs of marine cultured bivalve molluscs in 11 countries and regions around the world, including my country. Currently, 11 species of host species are known to be affected, including oysters (6 species), scallops (2 species), clams (2 species) and cockles; among them, Chlamys farreri cultivated in my country is the most severely affected (Chlamysfarreri) and the European-raised long oyster (Crassostreagigas). The establishment of accurate, sensitive and rapid detection methods is the premise and basis for early detection of disease pathogens and corresponding prevention and control measures. [0003] Currently, the most commonl...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/6848C12Q1/705C12Q2531/113C12Q2549/119
Inventor 白昌明王崇明高文辉汪清晨史杰张庆利
Owner YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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