An affordable detection system based on RT-LAMP and DNA-nanoprobes for avian metapneumovirus

Abstract

Airborne animal viral pathogens can rapidly spread and become a global threat, resulting in substantial socioeconomic and health consequences. To prevent and control potential epidemic outbreaks, accurate, fast, and affordable point-of-care (POC) tests are essential. As a proof-of-concept, we have developed a molecular system based on the loop-mediated isothermal amplification (LAMP) technique for avian metapneumovirus (aMPV) detection, an airborne communicable agent mainly infecting turkeys and chickens. For this purpose, a colorimetric system was obtained by coupling the LAMP technique with specific DNA-functionalized AuNPs (gold nanoparticles). The system was validated using 50 different samples (pharyngeal swabs and tracheal tissue) collected from aMPV-infected and non-infected chickens and turkeys. Viral detection can be achieved in about 60 min with the naked eye, with 100% specificity and 87.88% sensitivity for aMPV. In summary, this novel molecular detection system allows suitable virus testing in the field, with accuracy and limit of detection (LOD) values highly close to qRT-PCR-based diagnosis. Furthermore, this system can be easily scalable to a platform for the detection of other viruses, addressing the current gap in the availability of POC tests for viral detection in poultry farming. KEY POINTS: •aMPV diagnosis using RT-LAMP is achieved with high sensitivity and specificity. •Fifty field samples have been visualized using DNA-nanoprobe validation. •The developed system is a reliable, fast, and cost-effective option for POCT.

Keywords: Avian metapneumovirus (aMPV); Molecular detection; Nanoprobes; Point of Care (POC) test; Reverse transcription loop mediated isothermal amplification (RT-LAMP).

Fig. 1

Representative DNA electrophoresis of end-point RT-LAMP products after 1-h reaction at temperatures of 60 °C, 63 °C, or 65 °C using the aMPV-F RT-LAMP. SHS, nucleic acids purified from aMPV vaccine in duplicate; N1, nucleic acids from ILTV vaccine (negative control); NTC, non-template control

Fig. 2

DNA electrophoresis of end-point RT-LAMP products at 65 °C for 30 min reaction using the aMPV-F RT-LAMP primer set without (left panel) or with UDG (right panel). M1 and M2, two positive aMPV samples; N1, ILTV vaccine (negative control); NTC, non-template control

Fig. 3

Representative RT-qLAMP amplification plot using aMPV-F LAMP primers and purified nucleic acids from several aMPV-positive samples (M1 to M5). To discard any potential cross-reactivity of the assay nucleic acids purified from IBV- and ILTV-positive samples, a live-attenuated vaccine for NDV was used (M14, M15, and N2 samples, respectively). Purified aMPV RNA from the SHS vaccine was used as a positive control

Fig. 4

Validation of aMPV-F DNA-nanoprobes for visualization of RT-LAMP reactions targeting the aMPV-F gene. A Schematic representation of DNA-nanoprobe behavior depending on the presence of specific or unspecific RT-LAMP products. B RT-qLAMP amplification plot for 45 min of purified nucleic acids from representative aMPV samples (M6 to M11); purified RNA from NDV was used as a negative control (N2). C Digital pictures of reactions taken after 0 min or 30 min incubation at 37 °C of M6 to M11 RT-LAMP products with aMPV-F DNA-nanoprobes. Histograms represent their absorbance intensity at λ = 540 nm at 30 min of incubation at 37 °C

Fig. 5

Representative results for the validation of the specificity of DNA-nanoprobes. A DNA electrophoresis of end-point RT-LAMP products at 65 °C for 30 min reaction using the indicated RT-LAMP primer sets for aMPV G-gene (lanes 2 and 3), aMP-F gene (lanes 4 and 5), ILTV-TK gene (lanes 6 and 7), or IBV-5′UTR (lanes 8 and 9) using their corresponding purified viral genome, or non-template controls ( −). B Binding results after incubation at 37 °C for 0 min or 30 min of respective RT-LAMP products with different DNA-nanoprobes. Tube 1, aMPV-F DNA-nanoprobe with an aMPV-F RT-LAMP product. Tube 2, competition assay of aMPV-F DNA-nanoprobe with an aMPV-F RT-LAMP product in the presence of 100 nM of a free aMPV-F oligonucleotide probe. Tube 3, non-functionalized AuNPs with an aMPV-F RT-LAMP product. Tube 4, aMPV-F DNA-nanoprobes with an aMPV-F RT-LAMP product of a negative sample. Tube 5, ILTV-TK DNA-nanoprobes with an ILTV-TK RT-LAMP product. Tube 6, ILTV-TK DNA-nanoprobes with an aMPV-F RT-LAMP product. Tube 7, aMPV-F DNA-nanoprobes with an ILTV-TK RT-LAMP product. Tube 8, aMPV-F DNA-nanoprobes with an IBV-5′UTR RT-LAMP product. Tube 9, aMPV-F DNA-nanoprobes with an aMPV-G RT-LAMP product

Fig. 6

Results for the LOD determination assay using aMPV-F DNA-nanoprobes. A aMPV-F RT-qLAMP amplification plot after 50 min of incubation using fivefold dilutions of transcripted RNA from the TOPO-aMPV-F gene construct. B Binding results after 0 min or 30 min incubation at 37 °C of respective RT-qLAMP products with aMPV-F DNA-nanoprobes