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A rapid and specific real-time reverse-transcription recombinase polymerase amplification assay (RT-RPA) was developed to detect the transmissible gastroenteritis virus (TGEV) in this study. The primers and exo probe were designed to be specific for a portion of spike (S) gene conserved in TGEV, but absent in the closely related porcine respiratory coronavirus (PRCV). The amplification was performed at 40 °C for 20 min. The assay could only detect the TGEV, and there was no cross-reaction with other pathogens tested. Using the in vitro transcribed TGEV RNA as template, the limit of detection of the developed RT-RPA was 100 copies per reaction. The assay performance was evaluated by testing 76 clinical samples by RT-RPA and a real-time RT-PCR. Fourteen samples were TGEV RNA positive in RT-RPA (18.4%, 14/76), which were also positive in the real-time RT-PCR. The diagnostic agreement between the two assays was 100% (76/76). The R2 value of RT-RPA and real-time RT-PCR was 0.959 by linear regression analysis. The developed RT-RPA assay provides a useful alternative tool for rapid, simple and reliable detection of TGEV in resource-limited diagnostic laboratories and on-site facilities.

Porcine epidemic diarrhea (PED), which is caused by porcine epidemic diarrhea virus (PEDV), is an acute, highly contagious enteric disease characterized by severe watery diarrhea, vomiting, dehydration, and high mortality in suckling piglets. A real-time reverse-transcription recombinase polymerase amplification assay (RT-RPA) was developed based on the nucleocapsid gene of PEDV. RT-RPA assay was performed at 40 °C for 20 min. The assay could detect both the classical and variant PEDV strains, and there was no cross-reaction with other pathogens tested. Using the in vitro transcribed PEDV RNA as template, the analytical sensitivity was 23 copies per reaction. The assay performance was evaluated by testing 76 clinical samples. PEDV RNA positive rate was 55.3% (42/76) by RT-RPA and 59.2% (45/76) by real-time RT-PCR. The diagnostic agreement between the two assays was 96.1% (73/76), and the R2 value of the two assays was 0.903 by linear regression analysis. The developed RT-RPA assay provides a useful alternative tool for simple, rapid and reliable detection of PEDV in resource-limited diagnostic laboratories and on-site facilities.

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.

Recombinase polymerase amplification (RPA), an isothermal amplification technology, has been developed as an alternative to PCR in pathogen detection. A real-time RPA assay (rt-RPA) was developed to detect the porcine parvovirus (PPV) using primers and exo probe specific for the VP2 gene. The amplification was performed at 39°C for 20min. There was no cross-reaction with other pathogens tested. Using the recombinant plasmid pPPV-VP2 as template, the analytical sensitivity was 103 copies. The assay performance was evaluated by testing 115 field samples by rt-RPA and a real-time PCR assay. The diagnostic agreement between assays was 100%, and PPV DNA was detected in 94 samples. The R2 value of rt-RPA and real-time PCR was 0.909 by linear regression analysis. The developed rt-RPA assay provides a useful alternative tool for rapid, simple and reliable detection of PPV in diagnostic laboratories and at point-of-care, especially in remote and rural areas.

Highlights • Visual, rapid molecular assay for detection of CDV was developed. • Assay is based on RT-RPA and use of a lateral flow strip to visualize product. • Assay was performed in closed fists using body heat for 15 min. • Assay analytical sensitivity and specificity was similar to a real-time RT-PCR. • Assay was demonstrated to be simple, convenient, rapid and reliable for detection of CDV., Canine distemper, caused by Canine distemper virus (CDV), is a highly contagious and fatal systemic disease in free-living and captive carnivores worldwide. Accurate, rapid and simple detection of CDV is critical to improve disease management and prevent outbreaks. In this study, a visible and incubation instrument-free reverse-transcription recombinase polymerase amplification assay combined with lateral flow strip (LFS RT-RPA) was developed to detect CDV using primers and lateral flow (LF) probe specific for the nucleocapsid (N) protein gene. The CDV LFS RT-RPA assay was performed in a closed fist using body heat for 15 min, and the products were visible to the naked eyes on the LFS within 5 min. The assay could detect CDV, and there was no cross-reaction with the other viruses tested. Using the in vitro transcribed CDV RNA as template, the analytical sensitivity was 9.4 × 101 copies per reaction, which was the same result as that of a real-time RT-PCR. The assay performance was further evaluated by testing 32 nasal/oropharyngeal swab samples, and CDV RNA positive rate was 62.0% (20/32) by LFS RT-RPA, which was the same result as that of the real-time RT-PCR assay. The performance of the LFS RT-RPA was comparable to real-time RT-PCR, while the LFS RT-RPA assay was much faster and easier to perform. The novel CDV LFS RT-RPA assay provides an attractive and promising tool for rapid and reliable detection of CDV in the underequipped laboratory and point-of-need facility, which is of great significance in CD control in low resource settings.

Peste des petits ruminants (PPR) is a severe infectious disease of small ruminants caused by PPR virus (PPRV). Rapid and sensitive detection of PPRV is critical for controlling PPR. This report describes the development and evaluation of a conventional reverse transcription recombinase polymerase amplification (RT-RPA) assay and a real-time RT-RPA assay, targeting the PPRV N gene. Sensitivity analysis revealed that the conventional RT-RPA assay could detect 852 copies of standard PPRV RNA per reaction at 95% probability within 20 min at 41 °C, and the real-time RT-RPA assay could detect 103 copies of RNA molecules per reaction at 95% probability. Specificity analysis showed that both assays have no cross-reactivity with nucleic acid templates prepared from other selected viruses or common pathogens. Clinical evaluation using 162 ovine and hircine serum and nasal swab samples showed that the performance of both the real-time RT-RPA assay and the conventional RT-RPA assay were comparable to that of real-time RT-PCR. The overall agreements between real-time RT-PCR and real-time RT-RPA, and conventional RT-RPA were 99.4% (161/162) and 98.8% (160/162), respectively. The R2 value of real-time RT-RPA and real-time RT-PCR was 0.900 by linear regression analysis. Our results suggest that both RT-RPA assays have a potential application in the rapid, sensitive and specific detection of PPRV.