Evaluation of an incubation instrument-free reverse transcription recombinase polymerase amplification assay for rapid and point-of-need detection of canine distemper virus

• Published in: Journal of virological methods Vol. 260; pp. 56 - 61
• Main Authors: Wang, Jianchang, Wang, Jinfeng, Li, Ruiwen, Shi, Ruihan, Liu, Libing, Yuan, Wanzhe
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01-10-2018
• Subjects: Animals; CDV; Distemper - diagnosis; Distemper - virology; Distemper Virus, Canine - isolation & purification; Dogs; LF probe; LFS; N gene; Nucleic Acid Amplification Techniques; Nucleocapsid Proteins - genetics; Recombinases - genetics; Reproducibility of Results; Reverse Transcription; RNA, Viral - genetics; RT-RPA; Sensitivity and Specificity; LFS; LF probe; N gene; RT-RPA; CDV
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2018.07.007

•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.