Abstract
Enterocytozoon hepatopenaei (EHP) is an obligate, intracellular, spore-forming parasite, which mainly infects the gastrointestinal tract of shrimp. It significantly hinders the growth of shrimp, which causes substantial economic losses in farming. In this study, we established and optimized a SYBR Green I fluorescent quantitative PCR (qPCR) assay based on the polar tube protein 2 (PTP2) gene for the quantitative analysis of EHP-infected shrimp. The result showed that the optimum annealing temperature was 60 °C for the corresponding relation between the amplification quantitative (Cq) and the logarithmic of the initial template quantity (x), conformed to Cq = −3.2751x + 31.269 with a correlation coefficient R2 = 0.993. The amplification efficiency was 102%. This qPCR method also showed high sensitivity, specificity, and repeatability. Moreover, a microscopy method was developed to observe and count EHP spores in hepatopancreas tissue of EHP-infected shrimp using Fluorescent Brightener 28 staining. By comparing the PTP2-qPCR and microscopy method, the microscopic examination was easier to operate whereas PTP2-qPCR was more sensitive for analysis. And we found that there was a correspondence between the results of these two methods. In summary, the PTP2-qPCR method integrated microscopy could serve for EHP detection during the whole period of shrimp farming and satisfy different requirements for detecting EHP in shrimp farming.
Highlights
Microsporidia are obligate, intracellular, spore-forming parasites, and diverse species infect almost all invertebrates and vertebrates, as well as some protists, with different species exhibiting various degrees of host specificity [1,2]
Enterocytozoon hepatopenaei (EHP) detection methods have been reported via microscopy and molecular diagnosis
The higher sensitivity and specificity of molecular diagnoses have been widely reported for EHP-infected shrimp detection, such as PCR, quantitative PCR (qPCR), nested PCR, loop-mediated isothermal amplification (LAMP), and so on [19,20,21]
Summary
Microsporidia are obligate, intracellular, spore-forming parasites, and diverse species infect almost all invertebrates and vertebrates, as well as some protists, with different species exhibiting various degrees of host specificity [1,2] It is currently considered as a kind of fungi, and approximately 200 genera and 1400 species have been identified [3,4,5]. EHP detection methods have been reported via microscopy and molecular diagnosis. The higher sensitivity and specificity of molecular diagnoses have been widely reported for EHP-infected shrimp detection, such as PCR, qPCR, nested PCR, loop-mediated isothermal amplification (LAMP), and so on [19,20,21]. Instead of SSU rRNA, a more specific diagnosis target needs to be chosen
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