Preliminary Study on The SYBR Green I RT- qPCR for Rabbit Hemorrhagic Disease Virus 2 (RHDV2) Detection

DOI

10.2991/bep-16.2017.9How to use a DOI?

Keywords

rabbit hemorrhagic disease; RHDV2; RT-qPCR; overlapping PCR; detection

Abstract

To develop a rapid method for the detection RHDV2, 4 specific primers and 12 overlapping PCR primers were designed to amplify the conserved RHDV2 specific DNA fragment according to the genome sequences of RHDV and RHDV2 published in GenBank. Based on the synthesis of a conserved part of the RHDV2 sequence using overlap extension PCR, a SYBR Green I RT-qPCR assay was first preliminary developed and evaluated in China after a series of tests, including, reaction conditions optimization, establishment of standard curve, the sensitivity and specificity tests, intra-assay , and the application tests of 30 samples. The results showed that a 435bp specific DNA fragment of the RHDV2 capsid protein(VP60)gene was synthesized in vitro and the recombinant plasmid pMD-19T-RHDV2 was constructed, and the RT-qPCR method for RHDV2 rapid detection had good specificity, sensitivity and repeatability, which performance was linear ranging from 128 copies/µL to 1.28x108 copies/µL for the cloned viral genomic fragments of RHDV2.The sensitivity of the RT-qPCR could reach about 128 copies/µL of target fragments, and there was no amplification for RHDV, pGM-T-EBHSV, Pasteurella multocida, E.coli and Salmonella from rabbits detection by this method. And the application tests results showed that there were not RHDV2 in the experimentally infected samples and clinical samples.

Copyright

© 2017, the Authors. Published by Atlantis Press.

Open Access

This is an open access article distributed under the CC BY-NC license (http://creativecommons.org/licenses/by-nc/4.0/).

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TY  - CONF
AU  - Zexiao YANG
AU  - Yadong LIU
AU  - Zhengqun MENG
AU  - Bo WANG
AU  - Xueping YAO
AU  - Yin WANG
AU  - Yi GENG
AU  - Yan LI
PY  - 2016/12
DA  - 2016/12
TI  - Preliminary Study on The SYBR Green I RT- qPCR for Rabbit Hemorrhagic Disease Virus 2 (RHDV2) Detection
BT  - Proceedings of the 2016 International Conference on Biological Engineering and Pharmacy (BEP 2016)
PB  - Atlantis Press
SP  - 39
EP  - 44
SN  - 2468-5747
UR  - https://doi.org/10.2991/bep-16.2017.9
DO  - 10.2991/bep-16.2017.9
ID  - YANG2016/12
ER  -