Establishment And Application Of Three Kinds Of Shrimp Pathogens Detection Methods Based On Loop-mediated Isothermal Amplification

With the increasing demand for shrimp and the large-scale expansion of domestic shrimp aquaculture,the existing problem of shrimp disease has become emergent.It is difficult to discern diseased shrimp based on early clinical symptoms,but early detection and early treatment are crucial to the shrimp aquaculture industry.The conventional detection method is not suitable for field detection due to its own defects,however,Loop-mediated isothermal amplification(Lamp)can achieve high sensitivity and high specificity amplification with a shorter time and a simpler instrument rather than a cyclic change of temperature.So our research have developed lamp methods for rapid detection of Enterocytozoon hepatopenae(EHP),White spot syndrome virus(WSSV)and Vibrio parahaemolyticus(VP),and we combined it with a portable field detection box to form a field disease detection technology for shrimp disease.We selected 18 S rRNA,ORF21,and tlh as targets gene to establish lamp detection methods for Enterocytozoon hepatopenae,White spot syndrome virus,and Vibrio parahaemolyticus,based on the optimal experimental system for EHP.And a series of validation experiments were carried out including specificity,sensitivity and fleid samples detection.The test results indicated that these detection methods have good stability and specific detection of corresponding diseases,and the sensitivity detections of plasmid DNA are at least 10 times higher than the PCR method.The sensitivity for EHP and WSSV were 0.1 fg/?L,while the lowest detectable limit for VP was 1 fg/?L,and the detection results of the field samples by the Lamp methods were consistent with PCR method.Also,we combined three kinds of disease detection methods with the portable field detection box,and the results of field test were consistent with the verification results,and the detection time and accuracy were all in line with the requirements.The high sensitivity and high specific Lamp detection technology of three shrimp diseases established in this study,combined with the field detection box can achieve efficient and simple field detection.This technology is suitable for the promotion of shrimp disease detection in grass roots and farms.