Establishment of a Real-Time Reverse Transcription Recombinase-Aided Isothermal Amplification (qRT-RAA) Assay for the Rapid Detection of Bovine Respiratory Syncytial Virus

Abstract

Background: Bovine respiratory syncytial virus (BRSV) is a significant cause of bovine respiratory disease, resulting in significant losses to the cattle industry. For rapid detection of BRSV, a real-time recombinase-aided isothermal amplification assay (qRT-RAA) based on the F gene of BRSV was developed in this study.

Results: The developed qRT-RAA assay showed good exponential amplification of the target fragment in 20 min at a constant temperature of 39 °C. And this assay displayed a high specificity for BRSV, without cross-reactions with Infectious Bovine Rhinotracheitis Virus (IBRV), Bovine Parainfluenza Virus Type 3 (BPIV3), Bovine Viral Diarrhea Virus (BVDV), and Bovine Coronavirus (BCoV). With the standard RNA of BRSV serving as a template, the limit of detection for qRT-RAA was 102 copies/μL. We examined ninety-seven clinical samples from cattle with respiratory disease using this method and determined a positive rate of 7.2% (7/97), consistent with results using the classical PCR method reported previously.

Conclusions: A qRT-RAA assay for BRSV detection was established in this study. The method is specific and sensitive and can be completed within 20 min at 39 °C. These works demonstrate that the generated qRT-RAA assay is an effective diagnostic tool for rapidly detecting BRSV in resource-limited settings, which may be applied for the clinical detection of BRSV.

Keywords: bovine respiratory syncytial virus; detection assay; qRT-RAA; sensitivity; specificity.

Figure 1

Determination of optimal primers and reaction conditions for BRSV RT-RAA methods. (A) Lane M: DL2000 DNA Marker (Takara). Determination of optimal primer pairs for BRSV RT-RAA that target the F gene (numbers 1–3 correspond to BRSVF1/R1, BRSVF2/R2, and BRSVF3/R3). A 5 μL template containing 10⁴ copies/μL of F gene RNA was added to each reaction system and carried out at 39 °C for 30 min. The amplification product was analyzed by electrophoretic in 1% (w/v) agarose gel. (B) Initial specificity assays. The primer pairs BRSV F1/R1 and 5 μL template were added (numbers 1–5 correspond to BRSV, BVDV, BPIV, IBRV, and BCoV). The reactions were performed at 39 °C for 30 min. (C) Results of optimal temperature screening for BRSV RT-RAA. Primer pair BRSV F1/R1 was selected for amplification using 5 μL template harboring 10⁴ copies/μL of F gene RNA with an incubation time of 30 min and different incubation temperatures (numbers 1–4 correspond to 37 °C, 38 °C, 39 °C, and 40 °C). (D) Results of optimal time screening for BRSV real-time fluorescent RT-RAA. Primer pair BRSV F1/R1 was selected for basal-type amplification using F gene RNA as a template. The incubation temperature was 39 °C, and the incubation time was different (numbers 1–4 correspond to 10 min, 20 min, 30 min, and 40 min). All the amplification products were electrophoresed on 1% agarose gel.

Figure 2

Evaluation of the BRSV RT-RAA detection method. RFU, Relative Fluorescence Unit. Each cycle denotes a 30 s reaction time. (A) The RT-RAA was performed with an RNA standard of 10¹⁰ copies under 39 °C for 20 min. (B) Specificity analysis of RT-RAA assays. Nucleic acids of BRSV, BVDV, BPIV, IBRV, BCoV, and ddH₂O were extracted and used as templates for RT-RAA assay under optimal conditions. (C) Sensitivity evaluation. RNA standards were serially 10-fold diluted to a range of 10⁸ to 10⁰ copies/μL and subjected to RT-RAA assays. (D) M: DL2000 DNA Marker (Takara). The former diluted RNA standards were taken as templates for typical PCR, as reported previously.

Figure 3

Repeatability tests. RNA standards with concentrations of 10⁸, 10⁶, and 10⁴ copies/μL were assayed using the RT-RAA system at 39 °C for 20 min. Each cycle denotes a 30 s reaction time. RFU is Relative Fluorescence Unit. (A) Inter-batch repeat test; (B) intra-batch repeat test.