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. 2022 Feb;167(2):493-499.
doi: 10.1007/s00705-021-05349-8. Epub 2022 Jan 7.

Rapid visual detection of porcine reproductive and respiratory syndrome virus via recombinase polymerase amplification combined with a lateral flow dipstick

Xiao-Xiao Tian  1 , Tao Wang  1 , Xing-Yang Cui  1 , Xin-Yi Huang  1 , Yue Sun  1 , Da-Song Xia  1 , Yong-Bo Yang  1 , Xue-Hui Cai  1 , Tong-Qing An  2
Affiliations

Rapid visual detection of porcine reproductive and respiratory syndrome virus via recombinase polymerase amplification combined with a lateral flow dipstick

Xiao-Xiao Tian et al. Arch Virol. 2022 Feb.

Abstract

Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically devastating infectious diseases in the global swine industry. A rapid and sensitive on-site detection method for PRRS virus (PRRSV) is critically important for diagnosing PRRS. In this study, we established a method that combines reverse transcription recombinase polymerase amplification (RT-RPA) with a lateral flow dipstick (LFD) for detecting North American PRRSV (PRRSV-2). The primers and probe were designed based on the conserved region of all complete PRRSV-2 genomic sequences available in China (n = 512) from 1996 to 2020. The detection limit of the assay was 5.6 × 10-1 median tissue culture infection dose (TCID50) per reaction within 30 min at 42 °C, which was more sensitive than that of reverse transcription polymerase chain reaction (RT-PCR) (5.6 TCID50 per reaction). The assay was highly specific for the epidemic lineages of PRRSV-2 in China and did not cross-react with pseudorabies virus, porcine circovirus 2, classical swine fever virus, or porcine epidemic diarrhea virus. The assay performance was evaluated by testing 179 samples and comparing the results with those of quantitative RT-PCR (RT-qPCR). The results showed that the detection coincidence rate of RT-RPA and RT-qPCR was 100% when the cycle threshold values of RT-qPCR were < 32. The assay provides a new alternative for simple and reliable detection of PRRSV-2 and has great potential for application in the field.

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Figures

Fig. 1
Fig. 1
Optimization of the reaction temperature and time of the PRRSV-2 RT-RPA assay. A The assay functions in the temperature range of 30–45 ℃, with 42 ℃ or 45 ℃ being the optimal reaction temperature. B A positive signal is seen in 5 min, and the brightness of the band gradually increases with increasing amplification time. No difference was observed between 30 min and 35 min. M DNA marker
Fig. 2
Fig. 2
Sensitivity of the PRRSV-2 RT-RPA assay. The amplification products of the PRRSV-2 RT-RPA were analyzed by LFD and agarose gel electrophoresis. A In the LFD, the sensitivity was 5.6 × 10-1 TCID50 per reaction. The top line on the LFD is the control line, and the bottom line is the test line. B In agarose gel electrophoresis, the sensitivity was 5.6 TCID50 per reaction. M DNA marker, NC negative control, LFD lateral flow dipstick
Fig. 3
Fig. 3
Specificity of the PRRSV-2 RT-RPA assay. Four main lineages of PRRSV-2 in China (PRRSV-L1, PRRSV-L3, PRRSV-L5, and PRRSV-L8) and four other swine pathogens—CSFV, PCV2, PRV, and PEDV—were used as templates. NC negative control
Fig. 4
Fig. 4
Correlation between RT-RPA and RT-qPCR for detecting PRRSV-2. Each solid dot represents a sample. Red solid dots indicate that the sample tested positive by RT-qPCR, and green indicates a negative result.
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