A reverse transcription recombinase-aided amplification assay for the rapid detection of goose astrovirus

Abstract

China's burgeoning animal husbandry sector has witnessed a notable expansion in goose farming. Among the various health challenges, a novel goose astrovirus (GoAstV) has emerged as a significant threat to the industry, necessitating prompt detection strategies to mitigate its economic impact. This research introduces a novel detection approach using real-time fluorescence-based reverse transcription recombinase-aided amplification (RT-RAA), offering a rapid and reliable method for GoAstV identification. We meticulously designed specific primers and probes, and optimized the RT-RAA reaction conditions. The assay's specificity, sensitivity, repeatability, and clinical efficacy were rigorously assessed. Our method achieves detection within a swift 26-min window at a constant temperature of 39.00 ˚C, boasting a detection threshold as low as 1.19 × 10² copies per μL. Notably, the assay exhibited no cross-reactivity with closely related viruses, including Newcastle disease virus, avian influenza virus H9 subtype, goose circovirus, goose parvovirus, duck Tembusu virus, and avian adenovirus type 4. Validation through testing of 40 clinical samples confirmed a 100% agreement with pre-existing data. The study's outcomes underscore the high specificity, sensitivity, and operational simplicity of the developed RT-RAA assay, positioning it as an ideal candidate for the rapid and on-site detection of GoAstV.

Keywords: Clinical diagnosis; Constant temperature detection; Goose astrovirus; Reverse transcription recombinase-aided amplification.

Fig. 1

Screening of primers for goose astrovirus detection by reverse transcription recombinase-aided amplification. RFU: relative fluorescence unit.

Fig. 2

Screening of the concentration of probe and primer for goose astrovirus detection by reverse transcription recombinase-aided amplification. RFU: relative fluorescence unit. A) Screening of the concentration of probe: 1: 10.00 μM; 2: 6.70 μM; 3: 3.30 μM; 4: Negative control; B) Screening of the volumes of primer: 1: 6.00 μM; 2: 10.00 μM; 3: 8.00 μM; 4: 4.00 μM; 5: Negative control.

Fig. 3

Screening of reaction temperature for goose astrovirus detection by reverse transcription recombinase-aided amplification. RFU: relative fluorescence unit. The amplification time is set to 20 min. A) 37.00 ˚C; B) 38.00 ˚C; C) 39.00 ˚C; D) 40.00 ˚C; E) 41.00 ˚C; F) 42.00 ˚C. According to the speed of the peak time of the amplification curve and strength of the fluorescence signal, the reaction temperature is set to 39.00 ˚C.

Fig. 4

Screening of reaction time for goose astrovirus detection by reverse transcription recombinase-aided amplification. RFU: relative fluorescence unit. Reaction temperature is set to 39.00 ˚C. A) 7 min; B) 10 min; C) 13 min; D) 17 min; E) 20 min; F) 23 min. According to the speed of the peak time of the amplification curve and strength of the fluorescence signal, the reaction time is set to 20 min.

Fig. 5

Specificity analysis of real-time fluorescence reverse transcription recombinase-aided amplification detection method. RFU: relative fluorescence unit. 1-7: Negative controls for Newcastle disease virus, avian influenza virus H9 subtype, goose circovirus, goose parvovirus, duck Tembusu virus, and avian adenovirus type 4, respectively; 8: Goose astrovirus (GoAstV). Apart from GoAstV, no amplification curves were observed for other viral nucleic acids and the negative control.

Fig. 6

Sensitivity test of real-time fluorescence reverse transcription recombinase-aided amplification detection method. RFU: relative fluorescence unit. The results showed that the lowest detection line was 1.19 × 10² copies per μL. 1: Negative control; 2: 1.19 copies per μL; 3: 1.19 × 10¹ copies per μL; 4: 1.19 × 10² copies per μL; 5: 1.19 × 10³ copies per μL; 6: 1.19 × 10⁴ copies per μL; 7: 1.19 × 10⁵ copies per μL.