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Development of cross-priming amplification coupled with vertical flow visualization for rapid detection of infectious spleen and kidney necrosis virus (ISKNV) in mandarin fish, Siniperca chuatsi

生物 病毒学 病毒 爆发 脾脏 免疫学
作者
Wenzhi Liu,Yong Zhou,Yuding Fan,Nan Jiang,Kenneth D. Cain,Lingbing Zeng
出处
期刊:Journal of Virological Methods [Elsevier BV]
卷期号:253: 38-42 被引量:24
标识
DOI:10.1016/j.jviromet.2017.12.008
摘要

Infectious spleen and kidney necrosis virus (ISKNV) has been recognized as the causative agent of the most serious disease in cultured mandarin fish, Siniperca chuatsi, in China. Disease outbreaks have resulted in substantial losses to the aquaculture industry. Currently, reliable laboratory detection and identification methods are available for this virus. However, rapid detection methods applicable for on-site diagnosis of this infectious agent are unavailable. To address this need, a nearly instrument-free, cost-effective and simple detection method was developed and optimized and incorporates cross priming amplification coupled with vertical flow visualization for rapid identification of ISKNV (ISKNV-CPA-VF). Results show that cross circulation amplification targeting the conserved region of the major capsid protein (MCP) regiment of the ISKNV genome had a sensitivity 10 times greater than traditional PCR at 64 °C within 60 min. The optimized concentration of dNTPs and the concentration for Mg2+ were 1.0 mmol/L and 10 mmol/L, respectively. No cross-reactions with other viruses or bacteria were observed. When combined with the nucleic acid strip detection technology, visual detection of ISKNV amplified products was realized within 3-5 min following amplification. The simplicity and nearly instrument-free method for this ISKNV-CPA-VF assay shows great potential for on-site diagnostics of ISKNV infection in Siniperca chuatsi.
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