Comparison of Three Terminal Detection Methods Based on Loop Mediated Isothermal Amplification (LAMP) Assay for Spring Viremia of Carp Virus (SVCV)
2019
Liu, Lu | Xu, Yuyan | Zhong, Weifang | Li, Lina | Li, Weizhe | Xiao, Qin
Spring viremia of carp virus (SVCV) is a significant cyprinid-pathogenic virus. SVCV detection is usually performed in a laboratory with apparatus. But the virus outbreaks are generally in fishery banks. The study was developed a loop-mediated isothermal amplification (LAMP) assay, and compared to three different terminal detection methods in order to achieve SVCV field-based detection. A set of six specific primers for LAMP were designed based on SVCV glycoprotein (G) gene. The reaction parameters of LAMP were optimized, and three terminal detection methods (SYBR Green I staining, lateral flow dipstick (LFD), and agarose gel electrophoresis) were applied respectively to the detection of LAMP products. The results showed that 8 mM Mg2+, 320 U/mL Bst DNA polymerase, 1.4 mM dNTP, and 1 M Betaine were optimum at 63 ℃ for 40 min. Among the three terminal ways, LFD was preferred for detection of LAMP products by comparing their sensitivity and specificity. The detection limit of LAMP combined LFD (LAMP-LFD) was 860 fg, and no cross-reaction with other aquaculture viruses. Thus, the presented LAMP-LFD was suitable for field-based detection of SVCV with its advantages of speed, simplicity, and disposability. Meanwhile, the study also provides a valuable alternative to immunoassays and PCR-based tests for other virus or bacteria.
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