Visualized Detection of Ectromelia Virus by Loop-Mediated Isothermal Amplification Assay

doi:10.3969/j.issn.1674-5817.2018.05.004

Abstract

Abstract: Objective To established a loop-mediated isothermal amplification (LAMP) assay for rapid visual detection of ectromelia virus (ECTV). Methods According to the published GenBank sequences (NC_004105.1), 9 pairs of primers were designed targeting the conserved region of ECTV. The amplification was detected with LAMP Real Time Turbidimeter LA-302, and the visudlized detection method of ECTV was established. Meanwhile, the amplified products were colored by fluorescence detection reagent after completion of the reaction, so that the amplification could be detected with naked eyes. Then, methodological evaluation of the LAMP was tested, and the samples were tested for artificial infection. Results The method of LAMP shows a highly efficient amplification for ECTV viral target gene which performed at 63 ℃ for 60 min by the LAMP Real Time Turbidimeter LA-302. The detection limit was 530 fg/µL, was 10³ times higher than that of PCR, and no cross-reaction with other RNA and DNA of viruses from mice. The results of ECTV LAMP reaction visualized the tube directly with naked eyes by the addition of fluorescence detection reagent are consistent with the results detected by Tubidimeter real-time. Conclusion The established LAMP detection method for ECTV is rapid, specificity, high sensitivity, and be easy of operation under simple conditions, which may be suitable for rapid detection of ECTV.

Key words: Ectromelia virus (ECTV), Loop-mediated isothermal amplification (LAMP), Real time, Rapid detection

CLC Number:

Q95-33

ZHOU Jie, TAO Ling-yun, ZHAO Li-juan, HU Jian-hua, GAO Cheng. Visualized Detection of Ectromelia Virus by Loop-Mediated Isothermal Amplification Assay[J]. Laboratory Animal and Comparative Medicine, 2018, 38(5): 343-349.

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