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 (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 × 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.

Keywords: Duplex FMCA; Genotyping; PRV.

Fig. 1

Schematic illustration of the duplex FMCA method. (a) Relative binding positions of primers and probes along the gC gene of PRV. Melting peak calculation by derivative plotting -dF/dT versus temperature in the FAM channel (b) and the HEX channel (c). Red, blue, and green lines represent Bartha-K61 vaccine, European/American (Genotype I), and Chinese (Genotype II) strains, respectively

Fig. 2

Specificity of the duplex FMCA method. Melting curves obtained from duplex FMCA in the FAM channel (a) and the HEX channel (b) with 12 samples comprising 3 PRV positive samples, and five other viruses known to cause similar clinical symptoms in pig. Reference recombinant plasmids p-B (Bartha-K61 vaccine), p-N (Genotype I), and p-H (Genotype II) served as positive controls corresponding to red, blue, and green lines, and a No Template Control (NTC) served as the negative control (grey line)

Fig. 3

Simultaneous detection of PRV Bartha-K61vaccine and Genotype II strains in mixed infections. Two co-existing genotypes were detected in the FAM channel (a) and the HEX channel (b). Melting curves of artificial plasmid templates containing p-B and p-H at various ratios (10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, 0:10) were tested. The overall template concentration was 10⁸ copies per reaction. The NTC negative control is represented by a grey line