4.7 Article

Rapid Detection of Enterocytozoon hepatopenaei Infection in Shrimp With a Real-Time Isothermal Recombinase Polymerase Amplification Assay

出版社

FRONTIERS MEDIA SA
DOI: 10.3389/fcimb.2021.631960

关键词

Enterocytozoon hepatopenaei; recombinase polymerase amplification; recombination-dependent replication; spore wall protein gene; molecular detection

资金

  1. National Natural Science Foundation of China [31470275]
  2. Key Natural Science Research Project of the Jiangsu Higher Education Institutions of China [20KJA416002]
  3. Fishery Science and Technology Innovation Program of China [Y2018-14]
  4. Nantong Municipal Science and Technology Plan of China [GJZ17077]
  5. Lianyungang Science and Technology Project of China [SF2003]
  6. Science and Technology Project of Lianyungang High-tech Zone of China [HZ201901]
  7. Openend Funds of Jiangsu Key Laboratory of Marine Pharmaceutical Compound Screening [HY202004, HY201805]
  8. Postgraduate Research and Practice Innovation Program of Jiangsu Province [KYCX19_2296]
  9. Priority Academic Program Development of Jiangsu Higher Education Institutions of China

向作者/读者索取更多资源

The study developed a real-time RPA assay for EHP detection, which showed rapid detection speed, good specificity, sensitivity comparable to PCR and LAMP methods, and significant improvement over the RPA assay analyzed by gel electrophoresis.
Enterocytozoon hepatopenaei (EHP) infection has become a significant threat in shrimp farming industry in recent years, causing major economic losses in Asian countries. As there are a lack of effective therapeutics, prevention of the infection with rapid and reliable pathogen detection methods is fundamental. Molecular detection methods based on polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) have been developed, but improvements on detection speed and convenience are still in demand. The isothermal recombinase polymerase amplification (RPA) assay derived from the recombination-dependent DNA replication (RDR) mechanism of bacteriophage T4 is promising, but the previously developed RPA assay for EHP detection read the signal by gel electrophoresis, which restricted this application to laboratory conditions and hampered the sensitivity. The present study combined fluorescence analysis with the RPA system and developed a real-time RPA assay for the detection of EHP. The detection procedure was completed in 3-7 min at 39 degrees C and showed good specificity. The sensitivity of 13 gene copies per reaction was comparable to the current PCR- and LAMP-based methods, and was much improved than the RPA assay analyzed by gel electrophoresis. For real clinical samples, detection results of the real-time RPA assay were 100% consistent with the industrial standard nested PCR assay. Because of the rapid detection speed and the simple procedure, the real-time RPA assay developed in this study can be easily assembled as an efficient and reliable on-site detection tool to help control EHP infection in shrimp farms.

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