Bovine viral diarrhea virus (BVDV) is an important pathogen in cattle that can cause bovine viral diarrhea (BVD) and is also an agent of bovine respiratory disease, which is distributed globally and can cause significant economic losses[26] [27].Based on phylogenetic analysis of partial 5'UTR sequences into two distinct genetic species, BVDV-1 (1a-1t) and BVDV-29 (2a-17c)[28].The BVDV subtypes currently prevalent in China are diversified and include 1 subtype: 1a, 1b, 1c, 1d, 1m, 1o, 1p and 1q [29].RPA is an emerging isothermal nucleic acid amplification technique that has been applied to detect pathogens in a variety of samples, such as blood[30], food [31], and feces [32].In this study, we established BVDV RPA, RPAS assays based on the BVDV 5'UTR gene and validated them in bovine nasal swabs, anal swabs and blood samples.
The BVDV RPA reaction established in this study can be assayed in only 25 minutes at 37°C, without expensive or large instruments, with simple incubation conditions, even at body temperature[33].In contrast, conventional PCR reactions require agarose gel electrophoresis and fluorescent quantitative PCR methods require a thermal cycler. In terms of reaction time, the RPA method showed faster time to results (< 45 min) than the PCR method (> 20.1 h)[34].Higher cost of the RPA platform (~$60.25) compared to the cost of conventional PCR (~$3.70) and real-time fluorescent quantitative PCR (~$2)[35] [36].However, RPA is resistant to a variety of known PCR inhibitors, including hemoglobin, heparin, undiluted serum, and ethanol[37].
In this study, we added SYBR Green I to the RPA assay for endpoint monitoring. The endpoint assay uses less instrumentation than real-time monitoring, thus reducing the overall cost of the test and making it suitable for resource-limited areas. SYBR Green I is a phorbol ester dye that has been used as a nucleic acid stain for the assay platform in several studies. It binds preferentially to double-stranded DNA, producing a DNA dye complex that emits a green light and is detectable by the naked eye[38] [39, 40].Focused detection of RPA can also be achieved using the LF strip assay, which is relatively costly compared to the SYBR Green I assay [41].In addition to rapidity, specificity and cost, the sensitivity of the RPA assays was also tested. We compared the sensitivity of the RPA-AGE, RPA-AGE, and RPAS methods. the lowest detection threshold for BVDV RPA-AGE was 1×10− 1 copies/µL, and the lowest detection threshold for PCR-AGE was 1×104 copies/µL. under sunlight, the lowest detection threshold for RPAS was 1×103 copies/µL; under UV, BVDV The detection threshold of RPAS was 1×101 copies/µL. The detection limit of RPAS was 100-fold higher than that of PCR-AGE under sunlight and 1,000-fold higher than that under UV.
RPA technology is expected to challenge the dominant position of PCR molecular detection with its advantages of rapid amplification and no need to rely on large-scale instrumentation, which can make the experiments more convenient and faster. However, the RPA method still has some limitations. False positives may exist in the RPA assay. The reaction tubes should be opened and closed carefully and the gloves should be changed frequently when performing PRA; SYBR Green I should be added to the tube cap in advance when visualizing the assay. Meanwhile, the technology is more costly and less popular, but the premixing of the RPA reaction system can be completed in advance, and the total volume can be reduced to 12.5 µL (1/4 of the original volume), thus reducing the cost of the assay for each sample. In addition, low-cost commercial nucleic acid extraction methods for field samples based on magnetic bead technology and the heated NaOH method can be used to further reduce the cost of the RPA detection [42]. Furthermore, a mini UV flashlight was equipped to increase the sensitivity of sample detection and also circumvent the subjectivity of visual judgment.
In summary, the developed BVDV RPA RPAS assay with high specificity and sensitivity is simple, rapid and reliable for BVD detection. It makes it suitable for rapid testing in under-equipped diagnostic laboratories as well as for BVDV diagnosis in quarantine stations and cattle farms, which is of great significance for controlling bovine viral diarrhea.