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

摘要/Abstract

摘要：

鸭肠炎病毒（duck enteritis virus，DEV）疫苗株自1960年以来一直被用于防控DEV感染，该疫苗安全稳定、生产技术成熟、可快速诱导机体体液免疫和细胞免疫。DEV的基因组庞大，复制非必需区多，可容纳多个外源基因的插入，被广泛用来作为基因工程载体来开发疫苗。当前，尚未见仅针对DEV疫苗株的实时荧光定量PCR检测方法相关研究报道。本研究通过分析DEV疫苗株和强毒株UL2基因特点，明确DEV疫苗株在UL2基因上存在528 bp连续核苷酸缺失，设计针对该差异的实时荧光定量PCR引物，建立了EvaGreen实时荧光定量PCR检测DEV疫苗株的方法。结果表明，建立的检测DEV疫苗株实时荧光定量检测方法，当UL2基因含量为5.25×10¹~5.25×10⁶拷贝·μL^-1时有良好的线性扩增，其标准曲线方程Y轴截距为34.95，斜率为-3.218，扩增相关系数为0.999，扩增效率为100%；敏感性强，最低检测限为52.5拷贝·μL^-1；特异性好，对DEV疫苗株扩增产物的熔解曲线分析，仅出现1个单特异峰值[Tm=（90.62±0.28）℃]，无引物二聚体，对其他鸭源传染病病原（如鸭肠炎病毒强毒、番鸭细小病毒、鸭圆环病毒、番鸭源鹅细小病毒、新型基因重组型鸭细小病毒、鸭腺病毒A型、鸭源鹅多瘤病毒、鸭源大肠杆菌、鸭疫里默菌和鸭源禽多杀性巴氏杆菌）检测均为阴性；重复性好，组内变异系数和组间变异系数分别为0.55%~1.72%和0.92%~2.49%。本研究建立了DEV疫苗株实时荧光定量检测方法，为评价DEV活载体基因工程疫苗免疫防控机制提供参考。

关键词: 鸭肠炎病毒, 疫苗株, UL2基因, EvaGreen, 实时荧光定量PCR方法

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

中图分类号:

S852.659.1

万春和, 刘荣昌, 程龙飞, 傅光华, 施少华, 陈红梅, 傅秋玲, 黄瑜. 鸭肠炎病毒疫苗株EvaGreen实时荧光定量PCR方法的建立[J]. 畜牧兽医学报, 2018, 49(7): 1558-1566.

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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