Dataset Information

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.

ABSTRACT: 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 (?Tm) between detected samples and reference plasmid p-B were calculated in each channel. Bartha-K61 vaccine samples had ?Tm values of ±1?°C in both FAM and HEX channels, Genotype I samples had ?Tm values of ±1?°C in the FAM channel and 4.38?±?1?°C in the HEX channel, and Genotype II samples had ?Tm 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?×?100 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.

SUBMITTER: Liu Z

PROVIDER: S-EPMC6264625 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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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.

Liu Zhicheng Z Zhang Chunhong C Shen Haiyan H Sun Junying J Zhang Jianfeng J

BMC veterinary research 20181128 1

<h4>Background</h4>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. ...[more]

PMID: 30486818

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Dataset Information

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.

Publications

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.

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