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Duplex polymerase chain reaction (PCR) method for rapidly identifying duck circovirus genotype

A duck circovirus and genotyping technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/testing, etc., can solve problems such as inability to distinguish two genotypes of DuCV mixed infection

Inactive Publication Date: 2014-09-24
SHANDONG AGRICULTURAL UNIVERSITY
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  • Claims
  • Application Information

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Problems solved by technology

At present, PCR, fluorescent quantitative PCR, nucleic acid probe hybridization, and ELISA methods for detecting antibodies have been established to detect DuCV pathogens, but these methods cannot distinguish the mixed infection of the two genotypes of DuCV
There is still no report on the typing and detection of the two types of duck circovirus

Method used

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  • Duplex polymerase chain reaction (PCR) method for rapidly identifying duck circovirus genotype
  • Duplex polymerase chain reaction (PCR) method for rapidly identifying duck circovirus genotype
  • Duplex polymerase chain reaction (PCR) method for rapidly identifying duck circovirus genotype

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Embodiment Construction

[0023] 1 Primer design

[0024] By comparing the sequences of DuCV-1 and DuCV-2 published on the reference GenBank, select the conserved regions of DuCV-1 and DuCV-2 to design a pair of general specific primers SEQ1 / SEQ2, 1 for DuCV respectively The type duck circovirus specific primer SEQ3 and the type 2 duck circovirus specific primer SEQ4 were used to amplify the two virus samples by conventional methods.

[0025] SEQ1: 5'-CGG GAA ATG ACG TAG TCG TCA TG-3',

[0026] SEQ2: 5'-GG(A / C)(C / T)T(G / A)AAC ATG AGA TGG GC-3',

[0027] SEQ3: 5'-GTT CAC TCC(G / T)GT TGT GTT GTC(C / T)GG-3',

[0028] SEQ4: 5'-GAT AAT GCG AC(C / T)GGC GAC G-3';

[0029] DuCV's universal primers SEQ1 / SEQ2 amplified a fragment size of 1032bp, DuCV-1 specific primers SEQ2 / SEQ3 amplified a fragment size of 446bp, and DuCV-2 specific primers SEQ1 / SEQ4 amplified a fragment size of 599bp. All primers were sterile ddH 2 O (Rnase free) was prepared at a concentration of 25 pmol / μl for use.

[0030] 2 DNA extractio...

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Abstract

The invention provides a duplex polymerase chain reaction (PCR) method for rapidly identifying duck circovirus genotype. The method comprises the following steps: comparing sequences DuCV-1 and DuCV-2 released on reference GenBank, selectively and respectively designing a pair of general specific primers SEQ1 / SEQ2 aiming at DuCV, a type 1 duck circovirus specific primer SEQ3 and a type 2 duck circovirus specific primer SEQ4 in a conserved region of the DuCV-1 and DuCV-2. According to the method, novel specific primers are designed and screened, various parameters in the reaction process are optimized, the dual PCR sequence-based typing method aiming at the duck circovirus established by utilizing the primers is high in specificity and high in sensitivity, and the sequences DuCV-1 and DuCV-2 can be rapidly and accurately identified.

Description

(1) Technical field [0001] The invention relates to a double PCR method for quickly identifying different genotypes of duck circovirus, belonging to the field of virus molecular biology. (2) Background technology [0002] Duck circovirus (Duck circovirus, DuCV) is a new member of the family Circoviridae. Epidemiological surveys have shown that DuCV infection is common in ducks in my country, and is often associated with Riemerella anatipestifer, duck viral hepatitis virus, Mixed infection of pathogenic microorganisms such as Escherichia coli in ducks occurred. Studies have shown that ducks infected with DuCV will show symptoms such as feather disorder, growth retardation, and weight loss; histopathological analysis of DuCV-infected ducks shows that the virus infection mainly causes duck bursa lymphocyte reduction, atrophy, and histiocytosis; In situ fluorescence detection showed that DuCV infected ducks could form obvious lesions in the spleen, thymus and bursa of Fabricius;...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/70C12Q2531/113C12Q2537/143
Inventor 姜世金李志国王鑫张瑞华
Owner SHANDONG AGRICULTURAL UNIVERSITY
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