Development of Real-Time PCR Assay for Detecting Microsporidian Enterocytozoon hepatopenaei and the Application in Shrimp Samples with Different Growth Rates

Abstract:

In this study we developed fluorescence SYBR Green I real-time quantitative PCR (qPCR) to detect shrimp microsporidian Enterocytozoon hepatopenaei (EHP). A pair of specific primers was designed according to the SSU rDNA sequences of shrimp EHP published in GenBank and the optimized annealing temperature was determined to be 60℃. The melting curve of amplified products exhibited only one specific peak. Between 8.3×101–8.3×108 copies/µl the test results were linearly correlated with the titers of EHP. The cycles of amplification threshold (Ct) and the logarithmic of the initial template quantity [log(Sq)] conformed to Ct = –3.369 log(Sq) + 39.364, of which the coefficient of association R2 was 0.992 and the amplification efficiency was 98.1%. This method had high sensitivity (8.3×101 copies/µl), and generated duplicable results both within a group and between different groups. The test of 31 samples of farmed L. vannamei suggested that the qPCR method was 4 times more sensitive than the previously reported nested PCR method. To further verify this method, we tested hepatopancreatic DNA (HpDNA) samples extracted from 94 samples of L. vannamei that were collected from farms in Jiangsu, Hainan, and Shandong provinces. The results showed that the EHP loads in the hepatopancreas were negatively correlated with the shrimp growth rates. EHP load above 103 copies/(ng HpDNA) indicated high risk. In conclusion, we developed a highly specific, sensitive, rapid and quantitative method, which could be a useful tool in the prevention and control of shrimp microsporidian E. hepatopenaei.