Evaluation Of On-site Rapid And High Sensitive Detection Kits For Three Types Of Shrimp Pathogens

In recent years,the major and new diseases in shrimp have been raging,which has caused great economic losses to the shrimp breeding industry in China.At present,breeding disease has become one of the main factors restricting the development of green and high quality shrimp culture in China.Early detection of pathogenic microorganisms of shrimp blight and taking preventive measures to prevent the large-scale occurrence of disease are still effective means to reduce the risk of pathogen epidemic and reduce the serious economic loss caused by the large-scale epidemic of disease in aquaculture.Therefore,the development of shrimp blight pathogen field available,simple and easy to detect technology,and the development of the corresponding detection kit has become an urgent problem in the prevention and control of shrimp disease.To better evaluate the field rapid detection kit for shrimp pathogens developed in the laboratory,to find out its performance parameters and to clarify its specific test efficacy,in this study,the field rapid and highly sensitive detection kits of shrimp hepatopancreas（Enterocytozoon hepatopenaei disease,EHP）,Vibrio parahaemolyticus causing Acute hepatopancreatic necrosis disease(Vp_AHPND)and shrimp blood cell iridovirus（Shrimp hemocyte iridescent virus disease,SHIV）were used as the research objects,an experiment was designed in accordance with the testing requirements of test reagents in the World Animal Health Organization（OIE）Manual for the Diagnosis of Aquatic Animals,Ministry of Agriculture Proclamation No.683and quality Inspection Industry Standards.The kit was randomly selected to test and evaluate its analytical specificity,analytical sensitivity,diagnostic specificity,diagnostic sensitivity,repeatability and stability more comprehensively and scientifically.The following results were obtained.（1）The ASe of the on-site rapid and high sensitive detection kit for shrimp hepatocutaria was 10¹copies/μL,Asp test results show that the kit has no cross-reaction with five kinds of shrimp pathogens and shrimp tissue nucleic acid such as WSSV、CMNV、IHHNV、Vp_AHPNDand SHIV;By testing 298 clinical samples,The DSe of the kit compared to the reported EHP Taq Man-qPCR detection method is 91.7%,DSp is99.2%;The test repetition rate of the kit for negative and strongly positive samples is100%,The detection repetition rate for weakly positive samples was 95.8%;The kit can be stored for at least 7 months under-20℃,at least 12 months under-40℃conditions.On the whole,the EHP field rapid and highly sensitive detection kit is basically the same as the reported EHP Taq Man-qPCR detection method.（2）The ASe of the on-site rapid and high sensitive detection kit for Vp_AHPNDwas10²copies/μL,Asp test results showed that the kit had no cross-reaction with five kinds of shrimp pathogens and shrimp tissue nucleic acid such as WSSV、CMNV、IHHNV、SHIV、EHP;By testing 322 clinical samples,The DSe of the kit compared to the reported Vp_AHPNDTaq Man-qPCR detection method is 62.1%,DSp is 100%;The test repetition rate of the kit for negative and strongly positive samples is 100%,Test repetition rate for weakly positive samples was 83.3%;The kit can be stored for 7 days in 4℃,for at least 6 months in-20℃and for more than 8 months in-40℃.Generally speaking,the Vp_AHPNDfield rapid and highly sensitive detection kit compared with the reported Vp_AHPNDTaq Man-qPCR detection method,the Dsp/Dse is slightly lower,and the other detection performance is similar.（3）The ASe of the on-site rapid and sensitive detection kit for Shrimp hemocyte iridescent virus was 10¹copies/μL,Asp test results showed that the kit had no cross-reaction with five kinds of shrimp pathogens and shrimp tissue nucleic acid such as WSSV,CMNV,IHHNV Vp_AHPNDand EHP;By testing 298 clinical samples,The DSe of the kit compared to the reported Vp_AHPNDTaq Man-qPCR detection method is 96.6%,DSp is 98.5%;The test repetition rate of the kit for negative and strongly positive samples is 100%,The detection repetition rate for weakly positive samples was 91.6%;kit can be stored for 4 days in 4℃,for more than 6 months in-20℃,for more than 8months at-40℃.Overall,the SHIV field rapid and highly sensitive detection kit is basically the same as the reported SHIV Taq Man-qPCR detection method.In the process of detection,the rapid and sensitive detection kits for EHP,Vp_AHPND,and SHIV were used to carry out the clinical sample detection process,,from the sample nucleic acid extraction to the end of the detection time is only 1.5 h,Combined with the results of the above performance test,it can be found that the kits has the advantages of fast,high analytical sensitivity,strong analytical specificity,good repeatability and strong stability.It is suitable for rapid detection of EHP,Vp_AHPND,and SHIV in shrimp production or quarantine practice,and auxiliary diagnosis of shrimp diseases.