A lateral flow dipstick combined with reverse transcription recombinase polymerase amplification for rapid and visual detection of the bovine respirovirus 3

Abstract

Bovine respirovirus 3 also known as Bovine parainfluenza virus type 3 (BPIV3) is one of the most important viral respiratory agents of both young and adult cattle. Rapid diagnosis could contribute greatly in containing epidemics and thus avoid economic losses. However, the lack of robust isothermal visual method poses difficulty. In this study, a novel isothermal assay for detecting BPIV3 was established. The method includes a lateral flow dipstick (LFD) assay combined with reverse transcription recombinase polymerase amplification (RT-RPA). First, the analytical sensitivity and specificity of BPIV3 LFD RT-RPA were tested. The LFD RT-RPA assay has a detection limit of up to 100 copies per reaction in 30 min at 38 °C. Then the performance of LFD RT-RPA was evaluated using 95 clinical samples. Compared to qPCR, the LFD RT-RPA assay showed a clinical sensitivity of 94.74%, a clinical specificity of 96.05% and 0.8734 kappa coefficient. These results have demonstrated the efficiency and effectiveness of the method to be developed into a point of care protocol for the diagnosis of BPIV3.

Keywords: Bovine respirovirus 3; Lateral flow dipstick; Molecular diagnosis; Recombinase polymerase amplification.

Fig. 1

Optimization of BPIV3 LFD RT-RPA assay. (A) Agarose gel electrophoresis of RPA products generated using designed primers/probes. Lane M: molecular weight standard (DNA Marker 1000). Lane PC: positive control (supplied by Twist Amp nfo kit); Lane NC: negative control (DNase-free water); Lane 1 to 6 was designed primer and probe sets: F1/LF/R1, F1/LF/R2, F2/LF/R1, F2/LF/R2, F3/LF/R1, and F3/LF/R2, respectively. Specifically, Lane 6 was primers/probe set F3/LF/R2, and the expected size of the product was 311/210 bp. (B) Lateral-flow strip end-point analysis of RPA products generated by using six set of primers and probe combination. Lane PC: positive control (supplied by Twist Amp nfo kit); Lane NC: negative control (DNase-free water); Lane 1 to 6: BPIV3 amplicons performed with RPA primers and probe combination, the order was the same as Lane 1 to 6 of (A). (C) Optimization of incubation temperature for BPIV3 LFD RT-RPA assay. The amplification performance of LFD RT-RPA was effectively within the range of 30 °C–45 °C. Samples were tested in triplicate with one reaction displayed in figure for each triplicate. (D) Optimization of incubation reaction time for BPIV3 LFD RT-RPA assay. After 10 min of amplification reaction, the test line was clearly visible on the strip. Samples were tested in triplicate with one reaction displayed in figure for each triplicate.

Fig. 2

Testing the BPIV3 LFD RT-RPA assay for analytical sensitivity. (A) Sensitivity of the LFD-RPA assay. Molecular sensitivity test results of LFD RT-RPA using 10-fold serially diluted template of RNA molecular standard. BPIV3 templates of lane 1 to 7 in these reactions ranged from 10⁷ to 10¹ copies per reaction, respectively. Samples were tested in triplicate with one reaction displayed in figure for each triplicate. (B) BPIV3 RPA reaction products were detected on a 2% agarose gel. BPIV3 templates of Lane 1 to Lane 7 in these reactions ranged from 10⁷ to 10¹ copies per reaction, respectively.

Fig. 3

Testing the BPIV3 LFD RT-RPA assay for analytical specificity. (A) Specificity of the LFD RT-RPA assay. RPA products detected using LFD assay yield visually positive results only when tested using cDNA synthesis from BPIV3; results are visually negative for all other bovine viral pathogens with similar clinical symptoms. Samples were tested in triplicate with one reaction displayed in figure for each triplicate. (B) The results of amplification products of the LFD RT-RPA on 2% agarose gel. Lanes 1 to 6: BEFV, VSV, BcoV, BRSV, BVDV, and IBRV, respectively; Lane 7: positive control of BPIV3. (C) Quality detection of RNA/DNA of BEFV, VSV, BcoV, BRSV, BVDV, IBRV and BPIV3. The RNA/DNA of different virus was prepared for specificity detection by PCR reaction with viral specific primers. The positive amplification results were shown in Lane 2, Lane 4, Lane 6, Lane 8, Lane 10, Lane 12, Lane 14, respectively. Lane 1, Lane 3, Lane 5, Lane 7, Lane 9, Lane 11, Lane 13 were negative controls with DNase-free water as template.

Fig. S1

Details (location and sequence) for each primer and LF probe set used in BPIV3 LFD RT-RPA assay. The target region spanned nucleotides HN gene of BPIV3 (nucleotides 6818 to 8536 of the Genbank accession number: LC000638.1).