Acquisition Of Tetranychus Sinensis Resistance Gene D6 And Establishment Of Real-time PCR Assay

Ticks are a kind of surface parasites with tenacious vitality and adaptabi.lity to the environment.Is the world's second largest biological media,able to spread various virus disease,infected animals and humans.And is widespread in the world,on the animal breeding industry,human health,the natural environment of serious harm,causing huge economic losses each year.At present,the main methods for the elimination of ticks include biological control,chemical control,plant source control,artificial control and so on,the main method of prevention and cure is the most common chemical prevention and control.Chemical drug use for a long time,so that the tick gene mutation with drug resistance,and the residual chemical drugs on the natural environment and the harm of humans and animals are also increasing.In order to be able to quickly,accurately and efficiently detect the resistance of ticks,to avoid the abuse of chemical drugs and to reduce the economic losses caused by chemical drug resistance caused by ticks.The main contents and results are as follows:1?Through to the small cattle ticks preserved in our laboratory,positive genome cloning,construction of positive standard plasmid for common PCR method.By sequencing and uploaded on NCBI sequence to design the ordinary PCR detection method and real-time fluorescent quantitative PCR primers and probe detection method need.300 samples from Hunan,Gansu,China,Zhijiang,China,Qinghai,China,Gansu,China,Lintan,china.Screening specific primers and probes,and general PCR detection methods for comparison sensitivity.2 ? Construction of real time fluorescent quantitative PCR positive plasmid,successfully established ticks sodium channel resistance genes of real-time fluorescent quantitative PCR Taq Man probe testing methods.For quantitative PCR reaction conditions and optimizing the system,get better amplification efficiency and linear correlation.And with ticks of infection pear-shaped insect genome specificity test,the specificity of this method is better.Compared with ordinary PCR detection methods positive test results,display sensitivity is 1000 times the normal PCR detection method,The sensitivity of this method is better.And then use the standard positive plasmid with different concentration gradient to carry out the in group repeatability test and the group repeated test,the results showed that the repeatability coefficient of the group was less than that of Ct 5%,the coefficient of variation between groups was less than 10%,Indicating that the reproducibility of this methodis better.3?A total of 300 genomes of ticks from Hunan Zhijiang bloodstained ticks,Gansu Linxan Qinghai ticks and Gansu Yongqing were detected.Using the real-time quantitative PCR method established in this experiment,According to the standard of Taq Man probe method,263 samples were detected in the 300 samples with sodium channel resistance gene,Positive rate is 87.67%.The specific distribution of specific ticks were that,Hunan zhijiang small cattle ticks of the 79,69 positive cases were detected and the positive rate was 87.34%.Hunan zhijiang Bloodstained ticks of the72,63 positive cases were detected and the positive rate was 87.50%.In the gansu qinghai qinghaiensis ticks of the 65,60 positive cases were detected and the positive rate was 92.31%.Gansu town.the long-horned qinghaiensis ticks of the 84,71 positive cases were detected and the positive rate was 84.52%.The positive rate detected by common PCR was 47.00%.Using the real-time quantitative PCR method established in this experiment,the positive rate was 87.67%.Among them,The positive samples detected by normal PCR were also positive for real-time fluorescence quantitative PCR.110 of the negative samples detected by PCR were positive for quantitative PCR.It is proved that the method of Taq Man probe real-time fluorescence quantitative PCR is more reliable and sensitive.It is the first time to use real time fluorescent quantitative PCR in detection of resistance genes in ticks.It is a great progress for the determination of the resistance gene of ticks by real time quantitative PCR,it also has important significance for the establishment of detection methods of similar genes in ticks.