Validation of Loop-Mediated Isothermal Amplification method for Specific Detection of Camelpox Virus Genome

Abstract

Camelpox (CP) is a highly contagious viral disease affecting mostly young animals and characterized by papular eruption on the skin and mucus membrane. The causative agent is an orthopox virus of the family poxviridae. The disease causes high mortality and morbidity in its singlehost species. Camelpox is wide spread in the arid zone of Africa, the Middle East and central Asia. Loop-mediated isothermal amplification (LAMP) was optimized to amplify the hemagglutninin (HA) gene of camelpox virus (CMPV). LAMP is a novel method of gene amplification that amplifies nucleic acid with high specificity, efficiency, and rapidity under isothermal conditions with a set of six specially designed primers that recognize eight distinct sequences of the target. Primers used for gene amplification were designed from the camelpox Hemagglutinin gene. The whole procedure is very simple and rapid, and amplification could be obtained in less than 1 h by incubating the reaction mix in a single tube at 63°C and visualizing the products by agarose gel electrophoresis. Naked-eye visualization for amplification of LAMP method was also possible through observation of a color change following addition of 1 µl (1:1,000) of SYBR Green I dye to the tube. In positive amplification, the original orange colour of the dye changes to green, which can be seen under natural light as well as under UV light (302 nm). The sensitivity and specificity of the LAMP were evaluated with PCR using six extracted DNA samples of different poxvirus-species (camelpox, sheeppox, vaccinia and parapox virus). Both LAMP and PCR showed the same specificity as both detected camelpox without false positive result with other pox virus species. The specificity of the LAMP was observed by the absence of any cross-reaction

iv with other, closely related, members of the pox viruses species. When the sensitivity of the developed LAMP assay was compared to that of PCR, it was found that the LAMP demonstrated 16-fold higher sensitivity compared to PCR with a detection limit of LAMP in serially diluted quantified camelpox DNA of 0.075 ng/µl, compared to that of PCR (1.25 ng/µl). These results demonstrate that the LAMP-based assay is a useful tool for the rapid and sensitive diagnosis of camelpox. It is cost-effective, highly sensitive, and specific. The developed assay is expected to improve laboratory methods for diagnosis of this important disease in the Sudan and in the other countries where camels are raised and subsequently assist in its control.