Abstract:Two populations of Litopenaeus vannamei from Haiyang and Weifang in Shandong Province were sampled. TaqMan qPCR was used to measure Enterocytozoon hepatopenaei (EHP) in the shrimp hepatopancreas one by one, and then the extracted DNA samples were pooled by 5-pool (5∶1), 25-pool (25∶1), 50-pool (50∶1), 100-pool (100∶1), and 150-pool (150∶1) to test the EHP in the pooled samples. The amplification used a special threshold cycle value (Ct) of samples lower/higher than an assumed critical cycle value (Ca), differentiated as an analyzed positive/negative ratio in the single sample test and pooled sample test. The relationship between different pooling modes and the analyzed positive rate, diagnostic sensitivity, diagnostic specificity, and quantitative accuracy were compared. The results showed that the detection for high pooling rate samples could be reduced in very low analytic sensitivity. When the positive rate of individuals in the pooled samples was above 30%, the detection results of the high pooling rate were consistent with that of the single sample detection. If the positive rate of individuals with heavy infections in the pooled sample were not less than 6.7%, satisfying results could be obtained in the sample with a pooling rate below 50∶1; while the positive rate of individuals with light infections in the pooled sample were not less than 16%, the satisfying result could be obtained in the sample with a pooling rate below 25∶1. The pooled samples with a positive rate below 1.3% of individuals with heavy infection, or a positive rate below 8% of individuals with light infection, might lead to false negative results in all pooling rates. All of the pooling modes have good diagnostic specificity. The sample with a 50∶1 pooling rate had a similar diagnostic sensitivity to the sample with a 5∶1 pooling rate, which is the highest pooling rate recommended by the OIE standard. It had a very significant correlation between the mean of the detected EHP load of pooled sample, and the mean of the known EHP load calculated according to the single sample, with a ratio of 0.27~2.83. The quantitative test results of the pooled samples could roughly reflect the average EHP load of the single sample at the order of magnitude. This study provides a reference for sample detection in aquatic animal disease diagnosis, and epidemiological investigations.
