Artificial synthesis of VP60 conserved gene fragment of rabbit hemorrhagic disease virus type 2 and preliminary study of its RT-PCR detection method

doi:10.3969/j.issn.1004-1524.2016.10.04

›› 2016, Vol. 28 ›› Issue (10): 1650-1656.DOI: 10.3969/j.issn.1004-1524.2016.10.04

• Animal Science • Previous Articles Next Articles

Artificial synthesis of VP60 conserved gene fragment of rabbit hemorrhagic disease virus type 2 and preliminary study of its RT-PCR detection method

YANG Ze-xiao¹, ZHAO Xi-lun¹, LI Yan², WANG Bo¹, YAO Xue-ping¹, WANG Yin^{1, 3, *}, GENG Yi¹, MENG Zheng-qun¹, BAI Yu¹

1. College of Veterinary Medicine,Sichuan Agricultural University, Wenjiang 611130, China;
2. College of Animal Science and Technology, Sichuan Agricultural University, Wenjiang 611130, China;
3. Key Laboratory of Animal Disease and Human Health of Sichuan Province, Wenjiang 611130, China

Received:2016-01-11 Online:2016-10-15 Published:2016-11-20

Abstract

Abstract: In order to develop a rapid detection method for RHDV2, 4 specific primers and 10 overlapping oligo primers were designed to amplify the conserved RHDV2 VP60 gene specific DNA fragment according to the genome sequences of RHDV and RHDV2 published in GenBank. Based on the synthesis of a conserved part of RHDV2 sequence using overlap extension PCR and the construction of recombinant plasmid, RT-PCR assay was firstly established and evaluated in China after a series of tests, including optimization of reaction conditions, sensitivity and specificity tests, and the application tests of 35 samples. It was shown that a 435 bp specific DNA fragment of the RHDV2 capsid protein (VP60) gene was synthesized in vitro and the recombinant plasmid pMD-19T-RHDV2 was constructed. The sensitivity of the established RT-PCR detection method for RHDV2 could reach about 230 copies of cloned viral genomic fragments of RHDV2, and there was no amplification for RHDV, pGM-T-EBHSV, Pasteurella multocida, Escherichia coli and Salmonella from rabbits by this method. Besides, the application results showed that there were not RHDV2 in the experimental infection RHDV samples and clinical samples. The present research set basis for RHDV2 control study and supplied a back-up method for the rapid detection and prevention of RHDV2.

Key words: rabbit hemorrhagic disease, RHDV2, RT-PCR, overlap extension PCR

CLC Number:

S855.3

YANG Ze-xiao, ZHAO Xi-lun, LI Yan, WANG Bo, YAO Xue-ping, WANG Yin, GENG Yi, MENG Zheng-qun, BAI Yu. Artificial synthesis of VP60 conserved gene fragment of rabbit hemorrhagic disease virus type 2 and preliminary study of its RT-PCR detection method[J]. , 2016, 28(10): 1650-1656.

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Artificial synthesis of VP60 conserved gene fragment of rabbit hemorrhagic disease virus type 2 and preliminary study of its RT-PCR detection method

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