RESUMEN
Infectious hematopoietic necrosis virus (IHNV) severely and lethally infects salmonid fish, including Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) worldwide. Rapid and accurate viral detection is crucial for preventing pathogen spread and minimizing damage. Although several IHNV detection assays have been developed, their analytical and diagnostic performances have not been evaluated and field usability assessments have not been completely validated. Here, we developed a reverse-transcription cross-priming amplification-based lateral flow assay (RT-CPA-LFA) and validated its diagnostic performance. To detect the IHNV, primers were designed based on the consensus sequence of the nucleocapsid (N) gene. Notably, when combined with a lateral flow dipstick, it could visualize the IHNV amplification products within 5â¯min and the detection limit of the developed RT-CPA-LFA was 3.28×105 copies/µL. The diagnostic sensitivity and specificity in fish samples (n=140) were 98.88â¯% and 96.08â¯%, respectively. Moreover, the IHNV detection rate by RT-CPA-LFA in dead rainbow trout artificially injected with the virus was 100â¯%, consistent with to the results obtained from second conventional and real-time PCR, indicating its applicability for rapid IHNV detection and presumptive IHN diagnosis during the endemic period.
Asunto(s)
Cartilla de ADN , Enfermedades de los Peces , Virus de la Necrosis Hematopoyética Infecciosa , Oncorhynchus mykiss , Infecciones por Rhabdoviridae , Sensibilidad y Especificidad , Virus de la Necrosis Hematopoyética Infecciosa/genética , Virus de la Necrosis Hematopoyética Infecciosa/aislamiento & purificación , Animales , Infecciones por Rhabdoviridae/veterinaria , Infecciones por Rhabdoviridae/diagnóstico , Infecciones por Rhabdoviridae/virología , Enfermedades de los Peces/diagnóstico , Enfermedades de los Peces/virología , Oncorhynchus mykiss/virología , Cartilla de ADN/genética , Salmo salar/virología , Técnicas de Amplificación de Ácido Nucleico/métodos , Técnicas de Amplificación de Ácido Nucleico/veterinaria , Transcripción Reversa , Técnicas de Diagnóstico Molecular/métodosRESUMEN
Epizootics of Koi herpesvirus (KHV) cause mass mortality in koi carp (Cyprinus rubrofuscus) and common carp (Cyprinus carpio) worldwide. Rapid and accurate virus detection technology is crucial for preventing pathogen spread and minimizing damage. Although several diagnostic assays have been developed for KHV, the analytical and diagnostic performance of the detection methods has not been evaluated. In this study, we developed and validated the diagnostic performance of two molecular diagnostic assays, cross-priming amplification-based lateral flow assay (CPA-LFA) and TaqMan probe-based real-time polymerase chain reaction (PCR). To detect KHV, primers and probe were designed based on the thymidine kinase (TK) genes. The detection limits of developed CPA-LFA and real-time PCR assays were determined to be 675.69 copies/µL and 8.384 copies/µL, respectively. The diagnostic sensitivity and specificity of the developed assay were determined using fish samples (n = 179). CPA-LFA was found to be 93.67% and 100%, respectively, and real-time PCR was found to be 100% and 100%, respectively. Therefore, the newly developed CPA-LFA and real-time PCR assays accurately and rapidly detect KHV. CPA-LFA is particularly suitable for point-of-care diagnosis because of its simple diagnostic process, and real-time PCR analysis is most suitable for precise diagnosis because it can detect low viral loads.
