Diagnostic performance of cross-priming amplification-based lateral flow assay (CPA-LFA) and real-time PCR for koi herpesvirus (KHV) detection

• Published in: Journal of virological methods 2024-04, Vol.325, p.114890-114890, Article 114890
• Main Authors: Kim, Guk Hyun, Jeong, Ye Jin, Jeon, Yu Gyeong, Yang, Yun Jung, Min, Joon Gyu, Kim, Do-Hyung, Il Kim, Kwang
• Format: Article
• Language: English
• Subjects: Cross-priming amplification; Diagnostic performance; Koi herpesvirus; Lateral flow assay; TaqMan probe-based real-time polymerase chain reaction
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2024.114890

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. •Developed and validated CPA-LFA and TaqMan probe real-time PCR for KHV detection.•Primers and probe designed from consensus sequence of enlarged TK genes in KHV Asian and European types.•Evaluation of the diagnostic sensitivity and specificity of the developed method obtained values higher than 95%.•The CPA-LFA and TaqMan probe real-time PCR facilitate the rapid and accurate detection of KHV.