Se-Hee An , Na-Yeong Kim , Gyeong-Beom Heo, Yong-Myung Kang, Youn-Jeong Lee, Kwang-Nyeong Lee
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引用次数: 0
Abstract
H5, H7 and H9 are the major subtypes of avian influenza virus (AIV) that cause economic losses in the poultry industry and sporadic zoonotic infection. Early detection of AIV is essential for preventing disease spread. Therefore, molecular diagnosis and subtyping of AIV via real-time RT-PCR (rRT-PCR) is preferred over other classical diagnostic methods, such as egg inoculation, RT-PCR and HI test, due to its high sensitivity, specificity and convenience. The singleplex rRT-PCRs for the Matrix, H5 and H7 gene used for the national surveillance program in Korea have been developed in 2017; however, these methods were not designed for multiplexing, and does not reflect the sequences of currently circulating strains completely. In this study, the multiplex H5/7/9 rRT-PCR assay was developed with sets of primers and probe updated or newly designed to simultaneously detect the H5, H7 and H9 genes. Multiplex H5/7/9 rRT-PCR showed 100% specificity without cross-reactivity with other subtypes of AIVs and avian disease-causing viruses or bacteria, and the limit of detection was 1–10 EID50/0.1 ml (50% egg infectious dose). Artificial mixed infections with the three different subtypes could be detected accurately with high analytical sensitivity even under highly biased relative molecular ratios by balancing the reactivities of each subtype by modifying the concentration of the primers and probes. The multiplex H5/7/9 rRT-PCR assay developed in this study could be a useful tool for large-scale surveillance programs for viral detection as well as subtyping due to its high specificity, sensitivity and robustness in discriminating viruses in mixed infections, and this approach would greatly decrease the time, cost, effort and chance of cross-contamination compared to the conventional method of testing three subtypes by different singleplex rRT-PCR methods in parallel or in series.
期刊介绍:
The Journal of Virological Methods focuses on original, high quality research papers that describe novel and comprehensively tested methods which enhance human, animal, plant, bacterial or environmental virology and prions research and discovery.
The methods may include, but not limited to, the study of:
Viral components and morphology-
Virus isolation, propagation and development of viral vectors-
Viral pathogenesis, oncogenesis, vaccines and antivirals-
Virus replication, host-pathogen interactions and responses-
Virus transmission, prevention, control and treatment-
Viral metagenomics and virome-
Virus ecology, adaption and evolution-
Applied virology such as nanotechnology-
Viral diagnosis with novelty and comprehensive evaluation.
We seek articles, systematic reviews, meta-analyses and laboratory protocols that include comprehensive technical details with statistical confirmations that provide validations against current best practice, international standards or quality assurance programs and which advance knowledge in virology leading to improved medical, veterinary or agricultural practices and management.