Rapid and Visual Detection of Type 2 Porcine Reproductive and Respiratory Syndrome Virus by Real-Time Fluorescence-Based Reverse Transcription Recombinase-Aided Amplification

Abstract

Porcine reproductive and respiratory syndrome (PRRS) is one of the most important diseases that has brought significant economic losses to the swine industry worldwide. Rapid and accurate PRRS virus (PRRSV) detection is one of the key factors for PRRS prevention and control. This study developed a real-time fluorescence-based reverse transcription recombinase-aided amplification (RF-RT-RAA) method for type 2 PRRSV (PRRSV-2) detection. The RF-RT-RAA assay could be performed at 42 °C for 20 min with the optimal primers and a probe. RF-RT-RAA results could be monitored using real-time fluorescence read-out or visually observed with the naked eye using a portable blue light transilluminator. The method had a strong specificity; no cross-reaction was identified with the detected common swine viruses. Moreover, the technique yielded high sensitivity with the lowest detection limit of 101 copies/μL and exhibited good repeatability and reproductively with the coefficients of variation (CV) less than 10%. Eighty-seven clinical samples were tested using RF-RT-RAA and a commercial PRRSV-2 RT-qPCR detection kit. The coincidence rate was 100% between RF-RT-RAA (real-time fluorescence read-out) and RT-qPCR, and 97.7% between RF-RT-RAA (visually observed) and RT-qPCR. The RF-RT-RAA assay provides a new method for rapid and visual detection of PRRSV-2.

Establishment and Application Prospect of Reverse Transcriptase Recombinase-Aided Amplification Assay for Subgroup C Avian Metapneumovirus

“…The primer then searches for complementary bases on the template DNA using DNA polymerase to form a new DNA duplex, and this process is repeated several times to complete the amplification [ 44 ]. RT-RAA can directly amplify RNA templates, saving time and costs [ 45 ]. After the amplification, special probes are used to detect the target DNA via real-time fluorescence.…”

Rapid Visual Detection of Glaesserella parasuis with a Real-Time Recombinase-Aided Amplification Assay

“…As a new emerging technology with good application prospects, RAA has recently undergone steady development in the field of clinical diagnostics for animals and humans and has a broad application spectrum [26][27][28]. Compared with the qPCR, the real-time RAA for G. parasuis showed several advantages.…”

Advances in the application of recombinase-aided amplification combined with CRISPR-Cas technology in quick detection of pathogenic microbes

“…In addition, when designing RAA primers, it is better not to have a palindrome sequence, a continuous single-base repeat sequence, and an internal secondary structure region in the sequence, and the melting temperature (Tm value) is not an important factor to be considered. Because the amplification efficiency of different primers is different, it is usually necessary to design a series of candidate primers for an amplified gene, and finally select the best primer pair through experimental optimization (Xia et al, 2022).…”