Reverse transcriptase droplet digital PCR to identify the emerging vesicular virus Senecavirus A in biological...

Area of Science:

• Veterinary Virology
• Molecular Diagnostics
• Picornaviridae Research

Background:

• Senecavirus A (SVA) emerged as a significant pathogen causing vesicular disease in swine, presenting clinical signs indistinguishable from Foot-and-mouth disease.
• SVA outbreaks in Brazil since 2015 have caused substantial economic losses, necessitating improved diagnostic capabilities.
• Existing diagnostic methods require enhancement for accurate and efficient detection of SVA.

Purpose of the Study:

• To standardize a diagnostic tool for Senecavirus A (SVA) using RNA reverse transcriptase droplet digital PCR (RT-ddPCR).
• To compare the analytical sensitivity and specificity of RT-ddPCR with real-time PCR (RT-qPCR).
• To evaluate the utility of RT-ddPCR for absolute viral RNA quantification in biological samples.

Main Methods:

• Standardization of one-step and two-step RT-ddPCR assays for SVA detection.
• Parallel analytical sensitivity and specificity testing against one-step and two-step RT-qPCR.
• Optimization of RT-ddPCR using double-quenched probes and temperature gradients.
• Validation using biological samples from Brazilian SVA outbreaks and swine serum pools.

Main Results:

• One-step RT-ddPCR and RT-qPCR demonstrated superior analytical sensitivity and specificity compared to two-step methods.
• High concordance (94.2%) was observed between one-step RT-ddPCR and RT-qPCR in biological samples.
• RT-ddPCR exhibited enhanced performance over RT-PCR when analyzing swine serum pools.
• Key optimization factors for RT-ddPCR included probe design and temperature gradient optimization.

Conclusions:

• The one-step RT-ddPCR assay is a highly sensitive and specific tool for Senecavirus A diagnosis.
• RT-ddPCR enables absolute quantification of viral RNA, crucial for disease monitoring and control.
• Both one-step RT-ddPCR and RT-qPCR are recommended as auxiliary diagnostic tools for SVA.
• These advanced molecular diagnostic tools contribute to effective vesicular disease control programs in swine.