Establishment And Preliminary Application Of Nucleic Acid Detection Method Of Clonorchis Sinensis

Clonorchis sinensis disease,also known as liver fluke disease,affects approximately 35 million people worldwide,of which more than 12 million are infected in China.Humans or other mammals are infected with the disease by eating fish or shrimp infected raw or half-life with cysticercosis.The cysticercosis of Clonorchis sinensis can develop into an adult in the host after one month.The adult parasites in the human bile duct and gallbladder can cause liver damage,which can lead to hepatobiliary diseases and mixed bacterial infections.After being infected with Clonorchis sinensis,children and adolescents have severe clinical manifestations.In addition to hepatobiliary symptoms,they are often accompanied by malnutrition,anemia,hypoproteinemia,edema,and developmental disorders.It is a serious harm to zoonotic parasites.disease.At present,for the detection of Clonorchis sinensis,the current Kato thick smear method has defects such as missed detection and false detection.The commonly used molecular biology detection methods in the laboratory include traditional PCR,nested PCR,and fluorescent quantitative PCR.The immunological detection method is generally ELISA.However,these traditional molecular biology and immunological detection methods are expensive,cumbersome,and time-consuming,and thus cannot satisfy the rapid detection at the grassroots level.Therefore,the development of low-cost,easy-to-operate,and short-term detection methods of Clonorchis sinensis can greatly improve the detection efficiency of Clonorchis sinensis,and provide new methods and technical support for the prevention and control of Clonorchis sinensis.In this study,four primers were designed for 6 regions of Clonorchis sinensis ITS-2 gene sequence,and the sensitivity and specificity of the system were verified.A loop-mediated isothermal amplification of Clonorchis sinensis(LAMP)was initially established.Detection method.In addition,two long-chain primers were designed for Clonorchis sinensis COX-1 gene sequence,and the sensitivity and specificity of thesystem were verified.The detection method of recombinant enzyme polymerase amplification(RPA)of Clonorchis sinensis was preliminarily established.A set of common PCR detection methods for Clonorchis sinensis was established using the two short-chain primers in the LAMP detection method established above,and the minimum DNA concentration of Clonorchis sinensis detected by the common PCR system was verified.The artificial infection experiment of Beagle dogs was completed by collecting fish samples from each tributary of the Songhua River and collecting the cysticercosis of Clonorchis sinensis by artificial digestion of gastric juice.Then we use known positive samples and negative samples to verify the effectiveness and accuracy of LAMP and RPA technology in the detection of Clonorchis sinensis,and compare and analyze the detection steps and results of the two methods to further evaluate the two methods.It is used to detect the feasibility of Clonorchis sinensis infection.The results show that the LAMP detection method established in this study can detect the minimum DNA concentration of Clonorchis sinensis 90 pg/?L,and there is no cross-amplification reaction with Trichinella spiralis,Clonorchis orientalis,Clonorchis sinensis,Cryptosporidium,etc.RPA detection method can detect the lowest DNA concentration of Clonorchis sinensis is 2 ng/?L,no cross-amplification reaction with Trichinella spiralis,Clonorchis orientalis,Clonorchis sinensis,Cryptosporidium,etc.Ordinary PCR established with LAMP short chain primers can detect Clonorchis sinensis minimum DNA concentration of 11 ng/?L.In this study,the sensitivity of LAMP detection is about 120 times that of ordinary PCR detection,and the sensitivity of RPA detection is about 5 times that of ordinary PCR detection.In the detection of actual samples,by adding the nucleic acid dyes Sybr Green I,LAMP and RPA can quickly detect Clonorchis sinensis and worm eggs under the condition of leaving the PCR instrument and the ultraviolet gel system.In summary,the LAMP detection method and RPA detection method of Clonorchis sinensis established in this study have good specificity and sensitivity.Both systems can quickly detect the infection of Clonorchis sinensis and have good application prospects.