Reverse transcription recombinase polymerase amplification assay for the rapid detection of type 2 porcine reproductive and respiratory syndrome virus

Jan 26, 2017Journal of virological methods

Fast detection test for type 2 pig reproductive and respiratory syndrome virus using reverse transcription recombinase polymerase amplification

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Abstract

The fluorescence reverse transcription RPA assay detects type 2 PRRSV at a sensitivity of 690 copies.

The assay can identify both classical and highly pathogenic strains of PRRSV without cross-reacting to other pathogens.
Testing of 60 field samples showed a detection rate of 86.6% for the RT-RPA assay compared to 83.3% for real-time RT-PCR.
The reaction occurs at 40°C and takes only 20 minutes to complete.
This method may provide a rapid and reliable means of detecting PRRSV in point-of-care settings.

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Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important pathogens in pigs, and has tremendous negative economic impact on the swine industry worldwide. PRRSV is classified into the two distinct genotypes: type 1 and type 2, and most of the described PRRSV isolates in China are type 2. Rapid and sensitive detection of PRRSV is of great importance for the disease control and regional eradication programs. Recombinase polymerase amplification (RPA) has emerged as a novel isothermal amplification technology for the molecular diagnosis of infectious diseases. In this study, a fluorescence reverse transcription RPA (RT-RPA) assay was developed to detect the type 2 PRRSV using primers and exo probe specific for the viral nucleocapsid gene. The reaction was performed at 40°C within 20min. The RT-RPA assay could detect both the classical (C-PRRSV) and highly pathogenic PRRSV (HP-PRRSV), but there was no cross-reaction to other pathogens. Using the in vitro transcribed PRRSV RNA as template, the analytical sensitivity of RT-RPA was 690 copies. The assay performance was evaluated by testing 60 field samples and compared to real-time RT-PCR. The detection rate of RT-RPA was 86.6% (52/60), while the detection rate of real-time RT-PCR was 83.3% (50/60). This simple, rapid and reliable method could be potentially applied for rapid detection of PRRSV in point-of-care and rural areas.

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Reverse transcription recombinase polymerase amplification assay for the rapid detection of type 2 porcine reproductive and respiratory syndrome virus

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