Sensitivity of RNA viral nucleic acid-based detection of avian influenza virus, Newcastle disease virus, and African horse sickness virus on flinders technology associates card using conventional reverse-transcription polymerase chain reaction

Abstract

Background and aim: The flinders technology associates (FTA) card is a cotton-based cellulose membrane impregnated with a chaotropic agent that inactivates infectious microorganisms, lyses cellular material, and fixes DNA and/or RNA within the fiber matrix. However, little is known about the effectiveness of these cards for detecting RNA viruses in animals. This study aimed to evaluate the sensitivity of RNA virus detection using conventional reverse-transcription polymerase chain reaction (RT-PCR) on FTA cards.

Materials and methods: A highly virulent Newcastle disease virus (NDV) and an avian influenza virus (AIV) with low pathogenicity were propagated using chicken embryonic eggs. Three days after inoculation, the allantoic fluid was harvested, stored at -80°C, and the stock virus was tested for virus titration. African horse sickness virus (AHSV) was obtained from a live attenuated vaccine that was dissolved and stored at -80°C. For sample preparation, each stock virus was 10-fold serially diluted and each dilution was inoculated onto an FTA card, followed by drying in a Class II safety cabinet. Both the stock virus and infected FTA card were genomically isolated using an extraction kit, FTA purification kit, and extraction kit with Tris-EDTA (TE) buffer. The target genome was then detected by one-step RT-PCR for NDV and AIV, and two-step RT-PCR for African horse sickness, including gel electrophoresis for the detection of specific nucleic acids.

Results: The detection limit of stock AIV was compared on FTA cards, using the FTA purification kit, and with TE buffer with an extraction kit. The corresponding results were 1.47, 1.17, and 2.18 log₁₀ EID₅₀, respectively, while for NDV the results were 4.13, 4.83, and 4.84 log₁₀ ELD₅₀. Finally, detection limit of stock AHSV and AHSV on the FTA card extracted using TE buffer with an extraction kit were 4.30 and 4.01 log₁₀ plaque-forming units, respectively.

Conclusion: This study demonstrated that the detection limit or sensitivity of all tested RNA viruses on FTA cards did not differ when compared with those of the stock virus and in both methods for RNA isolation on FTA cards. These cards are suitable for collecting and transporting samples infected with RNA viruses, particularly AIV, NDV, and AHSV. Flinders technology associates cards also provide hazard-free samples, a reliable source of RNA for molecular characterization, and sufficient quantity for diagnostic applications based on nucleic acid-based detection.

Keywords: African horse sickness virus; Newcastle disease virus; avian influenza virus; flinders technology associates card; sensitivity.

Figure-1

Illustration of the FTA card and biopsy dermal puncher: (a) Virus inoculation area (diameter 25 mm) = 491.07 mm² (b) sampling area for 2.5 mm puncture = 4.91 mm², ratio B: A = 1:100 (c) sampling area for 8.0 mm puncture = 50.28 mm², ratio C: A = 1:9.76. FTA=Flinders technology associates.

Figure-2

Gel electrophoresis of avian influenza virus: (a) stock virus, (b) Flinders technology associates (FTA) card using the isolation kit, (c) FTA card using TE buffer with extraction kit. M: ladder; N: negative control; 0: undiluted; −1: diluted at 1 log₁₀; −2: diluted at 2 log₁₀; −3: diluted at 3 log₁₀; −4: diluted at 4 log₁₀; −5: diluted at 5 log₁₀; −6: diluted at 6 log₁₀; −7: diluted at 7 log₁₀; −8: diluted at 8 log₁₀.

Figure-3

Gel electrophoresis of Newcastle disease virus: (a) stock virus (b) Flinders technology associates (FTA) card using isolation kit (c) FTA card using TE buffer with extraction kit. M: ladder; N: negative control; 0: undiluted; −1: diluted at 1log₁₀; −2: diluted at 2 log₁₀; −3: diluted at 3 log₁₀; −4: diluted at 4 log₁₀; −5: diluted at 5 log₁₀; −6: diluted at 6 log₁₀; −7: diluted at 7 log_10.

Figure-4

Gel electrophoresis of African horse sickness virus: (a) stock virus (b) Flinders technology associates card using TE buffer with extraction kit. M: ladder; N: negative control; 0: undiluted; 1: diluted at 1 log₁₀; −2: diluted at 2 log₁₀; −3: diluted at 3 log₁₀; −4: diluted at 4 log₁₀; −5: diluted at 5 log₁₀.