Development Of A Loop-mediated Isothermal Amplification Method For Rapid Detection Of Capripoxvirus

Capripoxvirus can cause the extensive nodules and edema disease in the skin and organ surface of goats, sheep and cattle, which drastically reduce milkproduction and fur quality, and result in huge economic losses. According to the differences in the virulence between different strains, the fatality rate of susceptible animals is up to 10% to 58% or 75% to 100%.Besides, the goat, sheep, cattle and the product will be restricted in international trade, which infection from Capripoxvirus. It is composed of Goatpox virus (GTPV), sheeppox virus (SPPV), bovine lump skin disease virus (LSDV), which have strictly host specificity and will not occur cross-infection under natural conditions. SPPV and GTPV is worldwide distributed, and widely distributed in Africa, the Middle East, Northern Europe, Mediterranean countries now. In recent years, It happended at many areas in China, e.g. Guangxi, Guizhou, Heilongjiang. Lumpy skin disease was first found in Zambia and then spreay to South Africa, Tanzania, Israel and Egypt. Now LSD is Main distributed in central and southern Africa, and has the trend to spread to Europe and Asia.Although the traditional virological tests and serological tests are lower-cost and simple, there are defects such as spending a long time, lower specificity and sensitivity. With the development of molecular techniques, PCR and real-time PCR are widely Application, however, because of the need of expensive PCR instruments and complex operating procedures, these methods are not conducive to the locale and fast diagnosis. Until now there are no report about the prevalence of the virus of LSDV and the detection research of this disease in China. Therefore, it is practically important to establish a rapid and sensitive method to prevent the introduction of LSDV and strengthen the monitoring of CaPV in China.Loop-mediated isothermal amplification method (LAMP) is a new kind of isothermal nucleic acid amplification technology invented by Notomi et al (2000).This technology uses two pairs of special internal and external primers specifically to identification the six separate areas of target sequences, and Bst DNA polymerase to start the cycle strand displacement reaction. This method can amplify the target DNA fragmentsl09～1010 times within one hour. The method is simple, rapid amplification with high specificity, sensitivity and intuitive identification results, without expensive equipment, and very suitable for the grass-roots level and on-site quarantine. It is now widely used in the detection of viruses, bacteria and parasites such as avian influenza virus, Salmonella and Plasmodium falciparum.In this study, we choose Inverted terminal repeat as Target genes, use Primer Explorer 4.0 (http://primerexplorer.jp/elamp4.0.0/index.html) to design primers by analyzing the conserved genes of sheep pox virus in the Gen Bank sequence. The study use LAMP Real Time Turbidimeter LA-320c instrument for real-time monitoring of turbidity in the reaction process, to analysis the parameters such as sets amplified the starting time, the maximum amplification rate, the time to arrive maximum amplification rate and the time required to enter the platform from different primers, and than screen out LAMP primers which have high effective and specific amplification. The study optimizes significant parameter to the efficiency of the reaction for LAMP, deign optimal reaction system, verify the specificity and lower limit of detection of primers. The study verify the repeatability of CaPV-LAMP by select DNA from same batch and different batch, and detection different infection group, make Preliminary Exploration to develop Detection kit of LAMP poxvirus.The main study results are as follows:1. According above optimize test, the optimal reaction system:60 pmol FIP 1μL,60 pmol BIP lμL,5 pmol F31μL,5 pmol B3 1μL,8 u Bst DNA Polymerase 1μL,2xReaction Mix 12.5μL, Simple DNA 2μL, ddH2O 5.5μL, total volume25μL. Optimize reaction condition:Constant temperature reaction in 62℃60min,80℃,5 min Reaction was terminated.2. Sensitivity test show that Sensitivity of LAMP in this study is far more high than the PCR which OIE recommended, the lower detection limited was 10-1.51TCID50 in LAMP. However lower detection limited in PCR was 101.49TCID50.They isn’t Non-specific amplification in specific reaction, it is show that the method has Good specificity.3. The result can be observe by naked eye when added Calcein before reaction. It avoided the complex operate that use agarose gel electrophoresis detection LAMP LAMP product, and greatly reducing the error for turbidity judgment.4. We detection the DNA from the same batch and different batch, coefficient of variatio (CV%) was 1.65% and 8.66%, the agreement was 98.35% and 91.33%. It is show that the method has Good stability.The CaPV-LAMP detection method established in this study could complete the collection of sample,, the viral nucleic acid extraction and LAMP testing the entire process within 1.5 h。The method could detection in water bath, with advantages as fast, efficient, easy to operate, low cost, low equipment requirements. The detection do not require expensive thermal cycler, can complete the detection in a constant temperature water bath within 60 min, and it was simple and intuitive to determine results, can be able to meet the needs of large quantities of clinical samples for rapid detection. The CaPV-LAMP detection method established in this study provided a new way for the rapid diagnosis of CaPV in early stage and sensitive techniques for on-site quarantine and epidemiological surveillance of CaPV.