Jaroenlak Pattana, Sanguanrut Piyachat, Williams Bryony A P, Stentiford Grant D, Flegel Timothy W, Sritunyalucksana Kallaya, Itsathitphaisarn Ornchuma
Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok, Thailand.
Center of Excellence for Shrimp Molecular Biology and Biotechnology (Centex Shrimp), Faculty of Science, Mahidol University, Bangkok, Thailand.
PLoS One. 2016 Nov 10;11(11):e0166320. doi: 10.1371/journal.pone.0166320. eCollection 2016.
Hepatopancreatic microsporidiosis (HPM) caused by Enterocytozoon hepatopenaei (EHP) is an important disease of cultivated shrimp. Heavy infections may lead to retarded growth and unprofitable harvests. Existing PCR detection methods target the EHP small subunit ribosomal RNA (SSU rRNA) gene (SSU-PCR). However, we discovered that they can give false positive test results due to cross reactivity of the SSU-PCR primers with DNA from closely related microsporidia that infect other aquatic organisms. This is problematic for investigating and monitoring EHP infection pathways. To overcome this problem, a sensitive and specific nested PCR method was developed for detection of the spore wall protein (SWP) gene of EHP (SWP-PCR). The new SWP-PCR method did not produce false positive results from closely related microsporidia. The first PCR step of the SWP-PCR method was 100 times (104 plasmid copies per reaction vial) more sensitive than that of the existing SSU-PCR method (106 copies) but sensitivity was equal for both in the nested step (10 copies). Since the hepatopancreas of cultivated shrimp is not currently known to be infected with microsporidia other than EHP, the SSU-PCR methods are still valid for analyzing hepatopancreatic samples despite the lower sensitivity than the SWP-PCR method. However, due to its greater specificity and sensitivity, we recommend that the SWP-PCR method be used to screen for EHP in feces, feed and environmental samples for potential EHP carriers.
由肝肠胞虫（EHP）引起的肝胰腺微孢子虫病（HPM）是养殖虾类的一种重要疾病。严重感染可能导致生长迟缓以及收获无利可图。现有的PCR检测方法以EHP小亚基核糖体RNA（SSU rRNA）基因（SSU-PCR）为靶点。然而，我们发现由于SSU-PCR引物与感染其他水生生物的密切相关微孢子虫的DNA发生交叉反应，它们可能会给出假阳性检测结果。这对于调查和监测EHP感染途径来说是个问题。为克服这一问题，开发了一种灵敏且特异的巢式PCR方法用于检测EHP的孢子壁蛋白（SWP）基因（SWP-PCR）。新的SWP-PCR方法不会因密切相关的微孢子虫产生假阳性结果。SWP-PCR方法的第一步PCR比现有的SSU-PCR方法（每反应管106个拷贝）灵敏100倍（每反应管104个质粒拷贝），但在巢式步骤中两者的灵敏度相同（10个拷贝）。由于目前已知养殖虾的肝胰腺除了EHP外未感染其他微孢子虫，尽管SSU-PCR方法的灵敏度低于SWP-PCR方法，但对于分析肝胰腺样本仍然有效。然而，由于其更高的特异性和灵敏度，我们建议使用SWP-PCR方法在粪便、饲料和环境样本中筛查潜在的EHP携带者。
