Development And Preliminary Application Of LAMP Testing For Deformed Wing Virus

Loop-mediated Isothermal Amplification is a new kind of nucleic acid sequence-based amplification, relying on the automatic cycle chain substitution reaction under the condition of constant temperature, and it can amplify target sequence specifically using the four specific primers with advantage of strong specificity and high sensitivity. Deformed wing virus belongs to Iflavirus, Iflaviridae, Picorna-like viruses, and it can cause wing deformities in honeybees, a bloated, shortened abdomen and discolouration. DWV, often in unapparent infection, can cause serious impact to the swarm. Therefore, establishing a quick and efficient detection on DWV is particularly important. According to the DWV-JL1(Jilin apiary), the thesis cloned the capsid protein coding region, and then gain the capsid, named pMD-18T-DWV-JL1. We also using DNAStar software for analyzing the nucleotide sequences which encoded the capsid protein, and the homology is between 96%-97%, show that the sequence is highly conserved. Then we designed 5 sets of RT-LAMP primers to screen DWV. The author used RT-LAMP detection method for specificity, sensitivity, repeatability and stability analysis. After the reaction, three kinds of methods was used to determine the results. The results were SYBR Green I, by both naked-eye obversation and uv light imaging, agarose gel electrophoresis and the LA320 C instrument connected with the computer software for real-time detection. at 63 ℃ constant temperature for 32 minutes to DWV specific amplification of samples; The sensitivity test showed DWV minimum detectable amount of 3.7 × 101 copies / μL, which is about 10 times of ordinary PCR; After repeatability and stability test, the maximum amplification rate coefficient of variation was less than 10%, indicating that the method had good reproducibility and stability. By using the establish and optimize reaction system of RT-LAMP, ordinary PCR detection methods, real-time fluorescent PCR detection method and the RT-LAMP test method were used to test the 132 samples. The results showed that RT-LAMP was much higher than ordinary PCR detection method, and it had the similarity with real-time fluorescent PCR detection method. This method had advantages of simple, short reaction time, high accuracy and strong specificity, so it can be applied to detection of clinical samples.