A nested PCR assay to avoid false positive detection of the microsporidian enterocytozoon hepatopenaei (EHP) in environmental samples in shrimp farms

PublishedJournal Article© 2016 Jaroenlak et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.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.OI acknowledges support from Agricultural Research Development Agency under project CRP5905020530 and Mahidol University. KS received funding from National Research Council Thailand, Division of Plan Administration and Research Budget/2557-79. PJ is supported by the Science Achievement Scholarship of Thailand (SAST). GDS acknowledges support of DG SANCO of the European Commission, and the UK Department of Environment, Food and Rural Affairs under project FB002. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Small subunit rRNA sequences used for multiple sequence alignment analysis.

<p>Small subunit rRNA sequences used for multiple sequence alignment analysis.</p

Spore wall protein sequences used for multiple sequence alignment analysis.

<p>Spore wall protein sequences used for multiple sequence alignment analysis.</p

Alignments of the SSU-PCR primer sequences and confirmation of cross reactions with closely related microsporidia.

<p>(A) Alignments of the SSU primer sequences (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166320#pone.0166320.t003" target="_blank">Table 3</a>) with homologous SSU regions of other microsporidia (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166320#pone.0166320.t001" target="_blank">Table 1</a>). Black highlights indicate matches with the primer sequences, while asterisks under the sequences indicate regions of 100% identity for all of the aligned sequences. (B) Agarose gel of SSU-PCR amplicons from EHP and other microsporidia. In addition to the pGEM-SSU plasmid (+ve) and water (-ve), total DNA obtained from EHP-infected shrimp (I) and naïve shrimp (U) were used as controls. PCR amplicons and false positive test results are marked with arrowheads and asterisks, respectively. The band at 226 bp show amplicons of residual primers ENR779 from the first PCR step and primers ENF176 from the second nested PCR step.</p

Alignments of the SWP-PCR primer sequences and lack of cross reactions with closely related microsporidia.

<p>(A) Alignments of the SWP primer sequences (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166320#pone.0166320.t003" target="_blank">Table 3</a>) with homologous regions of spore wall protein genes of other microsporidia available in databases (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166320#pone.0166320.t002" target="_blank">Table 2</a>). Black highlights indicate matches with the primer sequences, and asterisks indicate regions of 100% identity for all of the aligned sequences. (B) Agarose gel of SWP-PCR amplicons from EHP and other microsporidia. In addition to the pGEM-SWP plasmid (+ve) and water (-ve), total DNA obtained from EHP-infected shrimp (I) and naïve shrimp (U) were used as controls. PCR amplicons are marked with arrowheads. The 180 bp band is PCR products from residual primers SWP1_R from the first PCR step and primers SWP_2F from the second nested PCR step.</p

Higher sensitivity of first step SWP-PCR compared to first step SSU-PCR.

<p>(A) and (C) show agarose gels of amplicons from the first step PCR reactions, while (B) and (D) show agarose gels of amplicons from the nested step PCR reactions carried out using serial dilutions of the plasmid templates pGEM-SWP and pGEM-SSU, respectively.</p

PCR primers used in this study.

<p>PCR primers used in this study.</p