In order to establish an efficient,rapid,and simple method for detecting decapod iridovirus 1(DIV1),the DIV1 ATPase gene was used as the detection target in this study.Specific primers and probes were designed,and the reaction time and temperature were optimized by the square array method.The results showed that the optimal reaction temperature was 37℃,the reaction time was 15 minutes,the result observation time was 5 minutes,and the total duration of the entire detection process was 20 minutes.The recombinase polymerase amplification technology combined with the lateral flow test strip method(RPA-LFD)of DIV1 was preliminarily established.Seven other common shrimp pathogen genes and the DIV1 gene were extracted and detected using this method to analyze their specificity;a recombinant plasmid standard pUC57-DIV1 was constructed and detected after 10-fold dilution to analyze its sensitivity;recombinant plasmid standards prepared at different times were used as templates for inter-batch and intra-batch repeatability tests.The specificity test revealed that the DIV1 could be specifically detected,and had no cross reaction with other common susceptible pathogens of shrimp.The sensitivity test indicated that the detection limit for DIV1 was 1×101 copies/μL.Moreover,the repeatability test showed that the detection results within and between three batches of plasmid standards with three concentrations were consistent,indicating good reproducibility of this method.We applied this method and the published fluorescence quantitative PCR method to simultaneously detect 15 samples of Macrobrachium rosenbergii infected with DIV1 and 40 clinical samples.The result showed that the positive detection rate of this method was 69.09%(38/55),and the negative rate was 30.91%(17/55),which is consistent with the results of fluorescence quantitative PCR detection.The positive,negative,and total coincidence rates of the two methods were 100%.In summary,the recombinase polymerase amplification technology established in this study combined with the lateral flow test strip method for detecting decapod iridovirus 1 is rapid,simple,high sensitive,and highly specific,and does not require precise and expensive equipment,providing a feasible technical method for grassroots laboratories and on-site detection.
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