Integrated qPCR and Staining Methods for Detection and Quantification of Enterocytozoon hepatopenaei in Shrimp Litopenaeus vannamei

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.

Development of a Quantitative PCR Method for Specific and Quantitative Detection of Enterocytospora artemiae, a Microsporidian Parasite of Chinese Grass Shrimp (Palaemonetes sinensis)

“…Notably, qPCR is often used in the detection of microsporidia and is considered to be more sensitive than conventional PCR (Naung et al, 2020). Wang et al (2020) used qPCR to detect the polar tube protein 2 gene when quantitatively analyzing EHP in infected shrimp and reported that sensitivity was increased by at least two orders of magnitude compared with that of conventional PCR. Phelps and Goodwin (2007) used the same technology to detect Notemigonus crysoleucas and reported that the minimum gene copy number that could be detected in each reaction was as low as 10.…”

“…Researchers have attempted to use qPCR to detect different microsporidia genes, such as those encoding the SSU rRNA, the polar tube protein, tubulin, and the spore wall protein (Jaroenlak et al, 2016;Han et al, 2018;Piamsomboon et al, 2019;Wang et al, 2020). Of these, the SSU rRNA gene carries the most commonly used detection target sequence.…”

A genome-wide association study identifies a novel association between SDC3 and apparent treatment-resistant hypertension

“…Analysis of SDC3 mRNA expression was performed using the primers as follows: 5′-TGG CGC AGT GAG AAC TTC G-3′ (forward) and 5′-CCC CGA GTA GAG GTC ATC CAG-3′ (reverse). Specific PCRs (20 μL) were set up as described previously [17]. The quantitative measures were obtained using the 2 ΔΔCT method.…”

Pyrococcus furiosus Argonaute Combined with Recombinase Polymerase Amplification for Rapid and Sensitive Detection of Enterocytozoon hepatopenaei

“…From 1 g of the sample homogenate prepared by a handheld third-gen TGrinder, the DNA was extracted with a TIANamp Genomic DNA Kit (Tiangen Biotech Co. Ltd., Beijing, China) and quantified with a NanoDrop Lite Spectrophotometer (Thermo Fisher Scientific Inc., Wilmington, DE). The presence of the respective pathogen in the extracted DNA was confirmed by qPCR, as previously reported. − DNA of clinical shrimp samples were extracted using the same procedure as mentioned above.…”