Liu Zhicheng, Zhang Chunhong, Shen Haiyan, Sun Junying, Zhang Jianfeng
Institute of Animal Health, Guangdong Academy of Agricultural Sciences, Key Laboratory of Livestock Disease Prevention of Guangdong Province, Scientific Observation and Experiment Station of Veterinary Drugs and Diagnostic Techniques of Guangdong Province,Ministry of Agriculture, P.R.China, Guangzhou, 510640, Guangdong, China.
BMC Vet Res. 2018 Nov 28;14(1):372. doi: 10.1186/s12917-018-1697-4.
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.
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 (ΔTm) between detected samples and reference plasmid p-B were calculated in each channel. Bartha-K61 vaccine samples had ΔTm values of ±1 °C in both FAM and HEX channels, Genotype I samples had ΔTm values of ±1 °C in the FAM channel and 4.38 ± 1 °C in the HEX channel, and Genotype II samples had ΔTm values of 6.52 ± 1 °C in the FAM channel and 4.38 ± 1 °C in the HEX channel. The minimum detection limit of the duplex FMCA was approximately 1 × 10 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.
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.
最近，中国大量接种了Bartha-K61疫苗的猪群中报告了伪狂犬病（PR）疫情，当前属于基因II型的伪狂犬病病毒（PRV）在基因上与属于基因I型的Bartha-K61疫苗不同。此外，已证明Bartha-K61疫苗不能为中国目前的PRV提供足够的保护。因此，准确快速地鉴定PRV至关重要。本研究的目的是开发一种双链荧光熔解曲线分析（FMCA）方法，能够快速、简单、高通量地区分中国、欧美和Bartha-K61疫苗株的PRV。
使用引物6F/6R和探针P1/P2，结合三个重组质粒p-B（Bartha-K61）、p-N（基因I型）和p-H（基因II型），建立双色FMCA。以p-B的FAM熔解温度（Tm）值（探针P1）和HEX（探针P2）通道作为参考值。计算每个通道中检测样品与参考质粒p-B之间的Tm差异（ΔTm）。Bartha-K61疫苗样品在FAM和HEX通道中的ΔTm值均为±1°C，基因I型样品在FAM通道中的ΔTm值为±1°C，在HEX通道中的ΔTm值为4.38±1°C，基因II型样品在FAM通道中的ΔTm值为6.52±1°C，在HEX通道中的ΔTm值为4.38±1°C。双链FMCA的最低检测限约为每个反应1×10个拷贝的p-B、p-N和p-H。使用双链FMCA技术检测并区分了198份疑似临床样品，其中18份（9%）为基因II型毒株阳性，8份（4%）为Bartha-K61疫苗株阳性，并将结果与测序和系统发育分析进行了比较，证实双色FMCA对所有样品均能正确工作。
在本研究中，我们开发了一种双链FMCA方法，该方法采用双标记、自淬灭探针，用于快速检测和区分PRV的基因I型、II型和Bartha-K61疫苗株。双链FMCA快速、简单且高通量，可能对PRV的分子流行病学调查和病原体监测有用。
