鸭肠炎病毒疫苗株EvaGreen实时荧光定量PCR方法的建立

doi:10.11843/j.issn.0366-6964.2018.07.027

Abstract

Abstract:

A live attenuated duck enteritis virus(DEV) vaccine has been used routinely to control lethal DEV in ducks since the 1960s, which is safety, stable, efficacious, and cost effective to produce. Recently, DEV vaccine strain has been developed as a vector for expressing foreign antigens for vaccine purposes, which offered the advantage of efficiently generating both humoral and cellular immune responses and overcomed pre-existing antibodies. To the best of our knowledge, no real time fluorescent quantitative PCR(qPCR) method was reported for only detection attenuated duck enteritis virus(DEV) vaccine strain. In this study, nucleotide comparison analysis showed the UL2 had significant characteristics with 528 bp continuous gene deletion between the virulent DEVs and attenuated vaccine strains. Based on the UL2 gene characterization, an EvaGreen real time qPCR method was developed with conditional optimization. The results demonstrated that the established qPCR had good linear correlation when UL2 gene content was 5.25×10¹-5.25×10⁶ copies·μL^-1 with a linear correlation(R²) of 0.999 and efficiency of 100% between the cycle threshold value and the logarithm of the plasmids copy number. The axial intercept of standard curve equation was 34.95 and the slope was -3.218. The lowest limit of detection concentration was 52.5 copies·μL^-1. The melting curve analysis showed one specific peaked with a melting temperature(Tm) was(90.62±0.28)℃,with no primer-dimers peak represent. No cross amplification was detected from other common duck pathogens(such as wild duck enter virus, Muscovy duck parvovirus, duck circovirus, Muscovy duck origin goose parvovirus, novel recombinant duck parvovirus, duck adenovirus A, goose hemorrhagic polyomavirus, Escherichia coli, Rimerella anatipstifer and Pasteurella multocida). Reproducibility test showed that the intra-and inter-assay were ranged from 0.55%-1.72% and 0.92%-2.49%, respectively. In conclusion, this study provided an EvaGreen real time qPCR method for DEV vaccine strain, which lays good foundation for mechanism research with live attenuated DEV vector based genetic engineering vaccines.

Key words: duck enteritis virus, vaccine strain, UL2 gene, EvaGreen, real-time fluorescent quantitative PCR method

CLC Number:

S852.659.1

WAN Chun-he, LIU Rong-chang, CHENG Long-fei, FU Guang-hua, SHI Shao-hua, CHEN Hong-mei, FU Qiu-ling, HUANG Yu. Establishment of a EvaGreen Real-Time Fluorescent Quantitative PCR Method for Vaccine Strain of Duck Enteritis Virus[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(7): 1558-1566.

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Establishment of a EvaGreen Real-Time Fluorescent Quantitative PCR Method for Vaccine Strain of Duck Enteritis Virus

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[1]	WAN Chun-he, LIU Rong-chang, CHENG Long-fei, FU Guang-hua, SHI Shao-hua, CHEN Hong-mei, FU Qiu-ling, HUANG Yu. Establishment of a EvaGreen Real-Time Fluorescent Quantitative PCR Method for Vaccine Strain of Duck Enteritis Virus [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(7): 1558-1566.
[2]	WU Yu-zi, XING Xian-ping, WEI Yan-na, XIONG Qi-yan, FENG Zhi-xin, SHAO Guo-qing. Comparison of Nucleotide and Deduced Amino Acid Sequences of P46 and P97 for the Different Live Vaccine Strains of Mycoplasmal Pneumonia of Swine [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(12): 2365-2373.
[3]	WU Yu-zi, XING Xian-ping, WEI Yan-na, XIONG Qi-yan, FENG Zhi-xin, SHAO Guo-qing. Comparison of Nucleotide and Deduced Amino Acid Sequences of P46 and P97 for the Different Live Vaccine Strains of Mycoplasmal Pneumonia of Swine [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(12): 2365-2373.
[4]	MA Yun-chao, CHENG An-chun, WANG Ming-shu, WU Ying. Progress of Recombinant Duck Enteritis Virus-vectored Vaccines [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(11): 2015-2022.
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