International Journal of Molecular Veterinary Research, 2025, Vol.15, No.1, 32-42 http://animalscipublisher.com/index.php/ijmvr 37 Figure 2 RNA-seq data quality and detection of viral transcripts in PRRSV-infected and bystander PAMs (Adopted from Chaudhari et al., 2021) 5.2 Nucleic acid detection techniques Molecular techniques have become the norm for diagnosing PRRSV. RT-PCR and qPCR are both sensitive and specific and enable the early detection and enumeration of viral RNA in various sample types (serum, swabs, tissue). RT-LAMP and RT-RPA are isothermal amplification approaches that enable rapid equipment-limited alternatives suitable for application in the field. Digital PCR (dPCR) and crystal digital PCR (cdPCR) are still more sensitive and allow absolute quantification, which is more sensitive than conventional qPCR in detecting low viral loads (Tian et al., 2022; Chae et al., 2023; Long et al., 2023; Yan et al., 2024). 5.3 Application of high-throughput sequencing and digital PCR in PRRSV detection Next-generation sequencing (NGS), high-throughput, permits whole genome characterization of PRRSV from clinical samples and assists in the identification of mixed infections, new variants, and recombinants. NGS is valuable in genotyping and epidemiological monitoring but is not yet routine in diagnostic laboratories due to its complexity and expense. Digital PCR, including droplet and crystal digital PCR, provides ultra-sensitive, specific, and quantitative detection with multiplexing and simultaneous identification capabilities of PRRSV and co-infecting pathogens (Kojabad et al., 2021; Long et al., 2023). 5.4 Rapid on-site diagnostic technologies (POCT and microarray-based systems) POCT assays such as lateral flow assays, microfluidic, and CRISPR/Cas-based assays enable rapid, sensitive, and simple detection of PRRSV at the farm level. RT-RPA combined with lateral flow dipsticks and CRISPR/Cas12a-based fluorescence assays have the ability to yield results within 30 minutes with high specificity and minimal instrumentation needs. On the horizon are microarray technology and AI-enhanced POCT instruments, with multiplex detection and rapid turnaround being provided for field testing (Liu et al., 2021; Tian et al., 2022; Lee et al., 2024; Augustine et al., 2025). 5.5 Multiplex detection and genotyping strategies Multiplex RT-qPCR and digital PCR assays allow simultaneous detection and differentiation of PRRSV genotypes (PRRSV-1, PRRSV-2) and prevailing lineages, and co-infecting respiratory pathogens. The assays are highly sensitive, specific, and effective for bulk surveillance and outbreak investigation. High-density multiplexing with sequencing or Sanger validation facilitates vigorous genotyping and epidemiological surveillance of circulating strains (Long et al., 2023; Yan et al., 2024). 6 Emerging Research Directions: Molecular Evolution and Viral Tracing of PRRSV 6.1 Global molecular evolution characteristics and transmission dynamics of PRRSV PRRSV is an extremely diverse RNA virus with rapid evolution driven by mutation and recombination and marked by the ever-emergent and replacing lineages in the world. In China, recent monitoring (2021-2023) indicated PRRSV-2 dominates, and sub-lineages 1.8 (NADC30-like), 1.5 (NADC34-like), and 8 (HP-PRRSV-like)
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