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.

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.