Duplex fluorescence melting curve analysis as a new tool for rapid detection and differentiation of genotype I, II and Bartha-K61 vaccine strains of pseudorabies virus

文献类型： 外文期刊

第一作者： Liu, Zhicheng

作者： Liu, Zhicheng;Zhang, Chunhong;Shen, Haiyan;Sun, Junying;Zhang, Jianfeng

作者机构：

关键词： PRV; Duplex FMCA; Genotyping

期刊名称：BMC VETERINARY RESEARCH （ 影响因子：2.741； 五年影响因子：2.955 ）

ISSN： 1746-6148

年卷期： 2018 年 14 卷

页码：

收录情况： SCI

摘要： Background: Recently, pseudorabies (PR) outbreaks have been reported in a large number of swine herds vaccinated with the Bartha-K61 vaccine in China, the current pseudorabies virus (PRV) belonging to Genotype II is differential genetically from Bartha-K61 vaccine belonging to Genotype I. Furthermore, it has been proved that the Bartha-K61 vaccine cannot provide sufficient protection against the current PRVs in China. Therefore, the accurate and rapid identification of PRVs is essential. The objective of this study is to develop a duplex fluorescence melting curve analysis (FMCA) capable of rapid, simple, high-throughput differentiation of Chinese, European/American and Bartha-K61 vaccine strains of PRV. Results: Primers 6F/6R and probes P1/P2, combined with three recombinant plasmids p-B (Bartha-K61), p-N (Genotype I), and p-H (Genotype II), were used to establish the Bicolor FMCA. FAM Tm values (probe P1) and HEX (probe P2) channels of p-B were used as reference values. Tm differences (Delta Tm) between detected samples and reference plasmid p-B were calculated in each channel. Bartha-K61 vaccine samples had Delta Tm values of 1 degrees C in both FAM and HEX channels, Genotype I samples had Delta Tm values of +/- 1 degrees C in the FAM channel and 4.38 +/- 1 degrees C in the HEX channel, and Genotype II samples had Delta Tm values of 6.52 +/- 1 degrees C in the FAM channel and 4.38 +/- 1 degrees C in the HEX channel. The minimum detection limit of the duplex FMCA was approximately 1x10(0) copies per reaction for p-B, p-N, and p-H. The duplex FMCA technique was used to detect and different 198 suspected clinical samples, of which 18 (9%) were positive for Genotype II strains and eight (4%) were positive for Bartha-K61 vaccine strains, and the results were compared with sequencing and phylogenetic analyses, which confirmed that the Bicolor FMCA worked correctly for all samples. Conclusions: In this study, we developed a duplex FMCA of dual-labeled, self-quenched probes that was performed for rapid detection and differentiation of Genotype I, II and Bartha-K61 vaccine strains of PRV. The duplex FMCA was rapid, simple, and high-throughput, and will likely prove useful for molecular epidemiological investigations and pathogen surveillance of PRV.

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