Veterinary Science & Technology

Rapid Detection of Type 2 Porcine Reproductive and Respiratory Syndrome Virus by a Duplex Reverse Transcription Insulated Isothermal PCR on a Field-Deployable System

Abstract

Hung-Chih Kuo, Dan-Yuan Lo, Chiu-Lin Chen, Chien-Hsien Lee, Yu-Han Shen, Yung-Long Tsai, Pei-Yu Alison Lee and Hsiao-Feng Grace Chang

Porcine reproductive and respiratory syndrome virus (PRRSV) is an important porcine pathogen globally. Reverse transcription-polymerase chain reaction (RT-PCR) for PRRSV detection is an important tool for disease management and control. Clinical sensitivity of RT-PCR for PRRSV detection is compromised to a certain degree by the high genetic diversity in the PRRSV genome. A duplex RT-insulated isothermal PCR (RT-iiPCR) for the North America lineage of PRRSV (PRRSV-NA) has been developed by targeting both ORF6 and ORF7 to increase test inclusivity. In this study, its limit of detection 95% was determined to be about 5 genome equivalents per reaction by testing a serial dilution of in-vitro transcribed RNA. The PRRSV-NA duplex RT-iiPCR was compared with an ORF7 real-time RT-PCR (rRT-PCR) published previously for the evaluation of analytical and clinical performance. Both tests did not react with seven common swine pathogens. The two methods had similar detection endpoints for viral RNA of two PRRSV-NA isolates. Further tests with 187 swine samples showed that 14 of the 90 rRT-PCR-negative and 2 of the 97 rRT-PCR-positive samples were positive and negative by the duplex RT-iiPCR, respectively. The two methods had 91.44% agreement (95% confidential interval: 87.26 - 95.62%, ?=0.83). Repeat testing could not resolve 13 of the discrepant samples (all negative by rRT-PCR and positive by RT-iiPCR). Further RT-nested PCR analysis and DNA sequencing analysis of the ORF7 region supported that the target RNA was present in these samples. Therefore, the PRRSV-NA duplex RT-iiPCR appeared to have higher clinical sensitivity than the reference rRT-PCR. Working on a field-deployable device, the PRRSV-NA duplex RT-iiPCR has potential to serve as a fast and sensitive tool for PRRSV detection at points of need.