Application of portable real-time recombinase-aided amplification (rt-RAA) assay in the clinical diagnosis of ASFV and prospective DIVA diagnosis.

African swine fever, a serious infectious disease, has been found in many countries around the world over the last nearly 100 years, and causes untold damage to the economy wherever it occurs. Diagnosis is currently performed by real-time PCR, which is highly sensitive but can only be carried out in a diagnostic laboratory environment with sophisticated equipment and expertise. A sensitive, rapid diagnostic method that can be implemented in agricultural settings is thus urgently needed for the detection and control of African swine fever virus (ASFV) infection. In this study, we developed an isothermal amplification technology to achieve molecular diagnosis of ASFV in clinical samples, using recombinase-aided amplification (RAA) assay combined with a portable instrument. This assay method avoids the limitations of traditional real-time PCR and offers detection times within 20 min, enabling detection of as few as 10 copies of ASFV DNA molecules per reaction without cross-reaction with other common swine viruses. We evaluated clinical performance using 200 clinical blood samples. The coincidence rate of the detection results between rt-RAA and RT-qPCR was 96.94% positive, 100% negative, and 97.50% total. We have also developed an rt-RAA system for the detection of ASFV targeting the EP402R gene, with detection of as few as 10 copies of DNA per reaction; this offers the possibility of DIVA (differentiating infected from vaccinated animals) diagnosis, because CD2V gene-deleted ASFV could soon be approved to be the leading candidate for live attenuated vaccine in China. The rt-RAA assay is a reliable, rapid, highly sensitive method, and it offers a reasonable alternative to RT-qPCR for point-of-care detection of ASFV. KEY POINTS: • The RT-RAA assay can detect as few as 10 copies of ASFV genome per reaction within 20 min. • The rt-RAA assay system targeting different genes can achieve differentiating infected from vaccinated diagnosis.

Establishment of reverse transcription loop-mediated isothermal amplification for rapid detection and differentiation of canine distemper virus infected and vaccinated animals.

Although widespread vaccination against canine distemper virus (CDV) has been conducted for many decades, several canine distemper outbreaks in vaccinated animals have been reported frequently. In order to detect and differentiate the wild-type and vaccine strains of the CDV from the vaccinated animals, a novel reverse transcription loop-mediated isothermal amplification (RT-LAMP) method was developed. A set of four primers-two internal and two external-were designed to target the H gene for the specific detection of wild-type CDV variants. The CDV-H RT-LAMP assay rapidly amplified the target gene, within 60 min, using a water bath held at a constant temperature of 65°C. The assay was 100-fold more sensitive than conventional RT-PCR, with a detection limit of 10(-1)TCID50ml(-1). The system showed a preference for wild-type CDV, and exhibited less sensitivity to canine parvovirus, canine adenovirus type 1 and type 2, canine coronavirus, and canine parainfluenza virus. The assay was validated using 102 clinical samples obtained from vaccinated dog farms, and the results were comparable to a multiplex nested RT-PCR assay. The specific CDV-H RT-LAMP assay provides a simple, rapid, and sensitive tool for the detection of canines infected with wild-type CDV from canines vaccinated with attenuated vaccine.