Development of a novel duplex crystal digital PCR for the detection of PRRSV–1 and PRRSV–2

TL;DR

A new digital PCR method was developed to accurately detect two types of PRRSV in pigs, offering high sensitivity and specificity.

Contribution

The study introduces a novel duplex crystal digital PCR assay for differential detection of PRRSV–1 and PRRSV–2 with improved sensitivity.

Findings

The developed assay showed a strong linear relationship (R2 of 0.998) between template concentration and Ct values.

The method detected PRRSV–1 and PRRSV–2 with LODs of 4.507 and 5.607 copies/reaction, respectively, 30 times more sensitive than qPCR.

The assay demonstrated high repeatability with intra- and inter-assay CVs of 0.74%–0.93% and 0.63%–1.62%, respectively.

Abstract

Porcine reproductive and respiratory syndrome (PRRS) is a widely prevalent disease of reproductive failure of pregnant pigs and respiratory syndromes in pigs of different ages, especially in piglets. The etiological agents include PRRS virus (PRRSV) genotypes 1 (PRRSV–1) and PRRSV–2, whereas their clinical symptoms are similar and hard to differentiate. It is necessary to establish accurate and reliable methods for differential detection of PRRSV-1 and PRRSV-2.

Two pairs of specific primers and probes were designed basing on the PRRSV–1 and PRRSV–2 ORF6 gene. The reaction conditions and procedures of the duplex crystal digital PCR (cdPCR) were optimized. The specificity, sensitivity, and repeatability of the developed assay were evaluated. The application of the developed assay was assessed by testing 2,185 clinical tissue samples.

The results indicated that the concentration of the templates and their Ct values had good linear relationship with R2 of 0.998. This method could specifically detect PRRSV–1 and PRRSV–2, without cross–reaction with other swine viruses. The limits of detection (LODs) of the assay were 4.507 copies/reaction and 5.607 copies/reaction for PRRSV–1 and PRRSV–2, respectively, which was approximately 30 times more sensitive than that of the duplex real-time quantitative PCR (qPCR). The repeatability test showed that the intra– and inter–assay coefficients of variation (CVs) were 0.74%–0.93% and 0.63%–1.62%, respectively. This method was validated by testing 2,185 clinical samples from Guangxi Province in South China, and the positivity rates of PRRSV–1 and PRRSV–2 were 2.20% (48/2,185) and 23.43% (512/2,185), respectively. The coincidence rates of the developed assay with the qPCR assay recommended by the World Organisation of Animal Health (WOAH) were 99.73% and 99.73%, respectively, while with the duplex qPCR developed in this study were 99.82% and 99.77%, respectively.

These results indicated that a rapid and accurate duplex cdPCR method with high sensitivity and excellent specificity had been successfully developed for the differential detection of PRRSV–1 and PRRSV–2.

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