A rapid visual detection method for porcine teschovirus through reverse transcription recombinase-aided amplification coupled with lateral flow dipstick

Abstract

Background: Porcine teschovirus (PTV) is an important enteropathogen, associated with symptoms of polioencephalomyelitis, pneumonia, pericarditis, myocarditis, diarrhea, and reproductive disorders in pigs. Rapid and precise diagnostic methods are essential for managing PTV infections. The study introduced a simple, quick, and visual approach for detecting PTV through the use of RT-RAA coupled with LFD.

Results: The procedures of RT-RAA-LFD for PTV could be carried out with 1.0 μmol/L primer concentration and 2.0 μmol/L probe concentration at 37 °C for 20 min, and the amplification result could be visualized within 5 min through LFD detection. Meanwhile, the assay established in this study showed no interaction with other associated diarrhea viruses, and has high specificity to PTV, with a minimal detection limit of 10 copies/μL and good repeatability. 128 clinical samples suspected of having a PTV infection were tested by RT-PCR and RT-RAA-LFD, respectively. The total diagnostic coincidence rate was 98.44% (126/128) with a Kappa value of 0.96(K ≥ 0.75), demonstrating a high degree of agreement to detect PTV for the two methods.

Conclusions: The RT-RAA-LFD assay created in this research displayed quick response, specificity, and sensitivity, capable of successfully detecting PTV in less than 25 min, providing an easy-to-use diagnostic instrument for rapid and visual PTV detection, especially suitable for labs and low-resource environments.

Keywords: Lateral flow dipstick; Porcine teschovirus; Recombinase-aided amplification; Visual detection method.

Fig.1

The screening results of RT-RAA primers. A The lateral flow dipstick detected results of amplification products by RT-RAA assessed with different candidate primer. “T”: test line, “C”: control line. “ + ”: the PUC57-PTV (10⁵ copies/μL) was used as amplification template in RT-RAA reaction, “-”: the ddH₂O was used as amplification template in RT-RAA-LFD reaction as a negative control group. B The Image J software was employed to analyze the intensity of the test line for primer combinations F1/R1 and F1/R2

Fig.2

The optimized results of RT-RAA-LFD reaction conditions for PTV detection. A Primer concentration optimization. The primer concentrations range for RT-RAA reaction was 0.1, 0.5, 1.0, 2.0, and 5.0 μmol/L. C Probe concentration optimization. RT-RAA reactions were assessed in the range of prober concentrations at 0.1, 0.5, 1.0, 2.0, and 3.0 μmol/L. E Reaction temperature optimization. The RT-RAA reaction was assessed at seven temperatures: 10, 25, 30, 35, 37, 41, and 43 °C. G Optimization of reaction time. Conducted at 37 °C, the RT-RAA reactions were timed for 5, 10, 15, 20, and 25 min each. The produced LFD was used to visualize the amplification results. Densitometry of the LFD's test lines' pixel intensity by Image J (B, D, F, and H). Distinct symbols signify noteworthy variations among every group ((P < 0.05)

Fig. 3

Specificity analysis of RT-RAA-LFD for PTV. The assay specificity was assessed using the RNA of PTV and other swine-associated viruses, including PDCoV, TGEV, PoRV, and PEDV

Fig. 4

Sensitivity and repeatability analysis of RT-RAA-LFD. A Sensitivity test, tenfold serial concentration ranged from 10⁰ to 10⁴ copies/μL were detected according to the optimal conditions. B Repeatability test. The plasmid pUC57-PTV at of 10¹, 10², and 10³ copies/μL were detected in three rounds