An on-site detection assay for screening of sendai virus by using palm-sized handheld system based on the reverse transcription multienzyme isothermal rapid amplification

Abstract

Sendai virus (SV) is the required inspection item for specific-pathogen-free grade rabbits and clean grade mice in the China National standard (GB14922.2–2011). Health monitoring should be performed regularly to avoid microbiological interference and obtain reliable experimental results. Reverse transcription multienzyme isothermal rapid amplification (RT-MIRA) technology allows for a high degree of specificity and sensitivity in pathogen detection. The aim of this study was to develop an on-site RT-MIRA assay with a handheld digitally thermostatic device to permit rapid visualization SV detection with naked eyes or displaying on phones connected via Bluetooth. SV-specific primers were targeted at the well-conserved L gene. The assay obtained results under isothermal conditions at 39 °C for 20 min. And the 95 % limit of detection was 33.5 copies/μL of SV RNA standards. Absence of non-specific amplification was confirmed by other common murine viruses. A nucleic acid releasing agent was used to lyse and release SV RNA within 5 min. Diagnostic performance of RT-MIRA on RNA extracted from 80 mice biological samples was compared with the reference method RT-PCR. SV was detected in 29 of 30 artificial positive samples and 0 of 50 clinical samples. The obtained results statistical analysis demonstrated that a good concordance was found between on-site RT-MIRA and the reference method RT-PCR (Kappa = 0.949). The on-site RT-MIRA assay exhibited high sensitivity and specificity, and the total time from sample collection to final result was approximately 30 min. We introduced a novel amplification technology and described an easy and efficient RT-MIRA assay for SV detection. This assay could be applied for simple on-site visual detection of SV.