The updated one-step multiplex RT-qPCR method for PRRSV classical strains, highly pathogenic strains and NADC30-like...

PRRSV-2 prevalent strains mainly include C-PRRSV, HP-PRRSV, and NADC30-like, with the latter being the current dominant lineage. Due to the high recombination and genetic variation of PRRSV, existing RT-qPCR assays face an increasing risk of false negatives. Therefore, based on the current prevalent strain sequences, it is of great significance to update and establish a one-step multiplex RT-qPCR method that can simultaneously detect C-PRRSV, HP-PRRSV and NADC30-like.

By downloading the latest prevalent strain full genome sequences from NCBI and isolating them in our laboratory, the conserved and type-specific target regions of the three strains were screened in the high-variable region of the nsp2 gene. TaqMan MGB probes and primers were designed. After optimizing the reaction conditions, the standard curve, amplification efficiency, sensitivity, specificity, repeatability and clinical application effect of this method were evaluated.

The established standard curve showed a good linear relationship within the range of 1 × 108 to 1 × 10³ copies/μL, with a correlation coefficient R² of 0.998 for all. The amplification efficiency ranged from 97.58 to 103.53%. The minimum detection limits for C-PRRSV, HP-PRRSV and NADC30-like were 10.128 copies, 8.998 copies and 8.458 copies, respectively. This method showed no cross-reaction with common porcine pathogens such as PRRSV-1, PCV2 and CSFV, and the intra-batch and inter-batch coefficient of variation was less than 2%. The positive rate of PRRSV in 588 samples was 15.5% (91/588), which was higher than 13.94% (82/588) of the reported methods. The consistency Kappa of the two methods was 0.87.

This study successfully established a one-step multiplex RT-qPCR method based on current prevalent strain sequences, which offers high sensitivity, strong specificity, and good repeatability, and can be used for rapid differential diagnosis of the three PRRSV subtypes in clinical samples, thereby supporting precise diagnosis, epidemiological monitoring, and prevention and control of PRRSV in China.