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Visual loop-mediated isothermal amplification (LAMP) for the rapid diagnosis of Enterocytozoon hepatopenaei (EHP) infection

Abstract

The emerging microsporidian parasite Enterocytozoon hepatopenaei (EHP), the causative agent of hepatopancreatic microsporidiosis, has been widely reported in shrimp-farming countries. EHP infection can be detected by light microscopy observation of spores (1.7 × 1 μm) in stained hepatopancreas (HP) tissue smears, HP tissue sections, and fecal samples. EHP can also be detected by polymerase chain reaction (PCR) targeting the small subunit (SSU) ribosomal RNA (rRNA) gene or the spore wall protein gene (SWP). In this study, a rapid, sensitive, specific, and closed tube visual loop-mediated isothermal amplification (LAMP) protocol combined with FTA cards was developed for the diagnosis of EHP. LAMP primers were designed based on the SSU rRNA gene of EHP. The target sequence of EHP was amplified at constant temperature of 65 °C for 45 min and amplified LAMP products were visually detected in a closed tube system by using SYBR™ green I dye. Detection limit of this LAMP protocol was ten copies. Field and clinical applicability of this assay was evaluated using 162 field samples including 106 HP tissue samples and 56 fecal samples collected from shrimp farms. Out of 162 samples, EHP could be detected in 62 samples (47 HP samples and 15 fecal samples). When compared with SWP-PCR as the gold standard, this EHP LAMP assay had 95.31% sensitivity, 98.98% specificity, and a kappa value of 0.948. This simple, closed tube, clinically evaluated visual LAMP assay has great potential for diagnosing EHP at the farm level, particularly under low-resource circumstances.

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Abbreviations

EHP:

Enterocytozoon hepatopeanei

SSU:

Small subunit

SWP:

Spore wall protein

WFS:

White feces syndrome

References

  • Angamuthu R, Baskaran S, Gopal DR, Devarajan J, Kathaperumal K (2012) Rapid detection of the Marek’s disease viral genome in chicken feathers by loop-mediated isothermal amplification. J Clin Microbiol 50(3):961–965

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Hong M, Zha L, Fu W, Zou M, Li W, Xu D (2012) A modified visual loop-mediated isothermal amplification method for diagnosis and differentiation of main pathogens from Mycobacterium tuberculosis complex. World J Microbiol Biotechnol 28(2):523–531

    CAS  Article  PubMed  Google Scholar 

  • Jaroenlak P, Sanguanrut P, Williams BAP, Stentiford GD, Flegel TW, Sritunyalucksana K, Itsathitphaisarn O (2016) A nested PCR assay to avoid false positive detection of the microsporidian Enterocytozoon hepatopenaei (EHP) in environmental samples in shrimp farms. PLoS One 11(11):e0166320. https://doi.org/10.1371/journal.pone.0166320

    Article  PubMed  PubMed Central  Google Scholar 

  • Karthik K, Rathore R, Thomas P, Arun TR, Viswas KN, Dhama K, Agarwal RK (2014) New closed tube loop mediated isothermal amplification assay for prevention of product cross-contamination. Methods X 1:137–143

    CAS  Google Scholar 

  • Karthikeyan K, Sharma A, Mekata T, Itami T, Sudhakaran R (2017) Rapid and sensitive real-time loop-meditated isothermal amplification for the detection of Enterocytozoon hepatopenaei of shrimp. Aquaculture 481:119–123. https://doi.org/10.1016/j.aquaculture.2017.08.036

    CAS  Article  Google Scholar 

  • LAMP restriction digest fragment analysis http://creisle.github.io/creisle.lamprflp/

  • Lau YL, Meganathan P, Sonaimuthu P, Thiruvengadam G, Nissapatorn V, Chen Y (2010) Specific, sensitive, and rapid diagnosis of active toxoplasmosis by a loop-mediated isothermal amplification method using blood samples from patients. J Clin Microbiol 48(10):3698–3702

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Liang C, Cheng S, Chu Y, Wu H, Zou B, Huang H, Xi T, Zhou G (2013) A closed-tube detection of loop-mediated isothermal amplification (LAMP) products using a wax-sealed fluorescent intercalator. J Nanosci Nanotechnol 13(6):3999–4005

    CAS  Article  PubMed  Google Scholar 

  • Liu Z, Zhang QL, Wan XY., Huang J (2015). Development of real-time PCR assay for detection of microsporidian Enterocytozoon hepatopenaei and detection in shrimp samples under different growth rates. Fish. Sci. (in Press Chinese, English Abstr)

  • Nasarudin SNSA, Zainudin NS, Bernadus M, Nawi AM, Hanafiah A, Osman E (2015) Loop-mediated isothermal amplification for rapid molecular detection of Enterocytozoon bieneusi in faecal specimens. J Med Microbiol 64(11):1329–1334

    CAS  Article  PubMed  Google Scholar 

  • Ndzi ES, Asonganyi T, Nkinin MB, Xiao L, Didier ES, Bowers LC, Nkinin SW, Kaneshiro ES (2016) Fast technology analysis enables identification of species and genotypes of latent microsporidia infections in healthy native Cameroonians. J Eukaryot Microbiol 63(2):146–152

    Article  PubMed  Google Scholar 

  • Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T (2000) Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 28(12):E63

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Rajendran KV, Shivam S, Ezhil Praveena P, Joseph Sahaya Rajan J, Sathish Kumar T, Avunje S, Jagadeesan V, Prasad Babu SVANV, Pande A, Navaneeth Krishnan A, Alavandi SV, Vijayan KK (2016) Emergence of Enterocytozoon hepatopenaei (EHP) in farmed Penaeus (Litopenaeus) vannamei in India. Aquaculture 454:272–280. https://doi.org/10.1016/j.aquaculture.2015.12.034

    Article  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989). Molecular cloning: a laboratory manual (No. Ed. 2). Cold spring harbor laboratory press

  • SISA online statistical software www.quantitativeskills.com/sisa/

  • Subrungruang I, Mungthin M, Chavalitshewinkoon-Petmitr P, Rangsin R, Naaglor T, Leelayoova S (2004) Evaluation of DNA extraction and PCR methods for detection of Enterocytozoon bienuesi in stool specimens. J Clin Microbiol 42(8):3490–3494

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Suebsing R, Prombun P, Srisala J, Kiatpathomchai W (2013) Loop-mediated isothermal amplification combined with colorimetric nanogold for detection of the microsporidian Enterocytozoon hepatopenaei in penaeid shrimp. J Appl Microbiol 114(5):1254–1263. https://doi.org/10.1111/jam.12160

    CAS  Article  PubMed  Google Scholar 

  • Tang KFJ, Pantoja CR, Redman RM, Han JE, Tran LH, Lightner DV (2015) Development of in situ hybridization and PCR assays for the detection of Enterocytozoon hepatopenaei (EHP), a microsporidian parasite infecting penaeid shrimp. J Inverteb Pathol 130:37–41. https://doi.org/10.1016/j.jip.2015.06.009

    CAS  Article  Google Scholar 

  • Tang KFJ, Han JE, Aranguren LF, White-Noble B, Schmidt MM, Piamsomboon P, Risdiana E, Hanggono B (2016) Dense populations of the microsporidian Enterocytozoon hepatopenaei (EHP) in feces of Penaeus vannamei exhibiting white feces syndrome and pathways of their transmission to healthy shrimp. J Invertebr Pathol 140:1–7. https://doi.org/10.1016/j.jip.2016.08.004

    Article  PubMed  Google Scholar 

  • Tangprasittipap A, Srisala J, Chouwdee S, Somboon M, Chuchird N, Limsuwan C, Srisuvan T, Flegel TW, Sritunyalucksana K (2013) The microsporidian Enterocytozoon hepatopenaei is not the cause of white feces syndrome in whiteleg shrimp Penaeus (Litopenaeus) vannamei. BMC Vet Res 9(1):139. https://doi.org/10.1186/1746-6148-9-139

    Article  PubMed  PubMed Central  Google Scholar 

  • Tanner NA, Zhang Y, Evans TC Jr (2015) Visual detection of isothermal nucleic acid amplification using pH-sensitive dyes. BioTechniques 58:59–68

    CAS  Article  PubMed  Google Scholar 

  • Tomita N, Mori Y, Kanda H, Notomi T (2008) Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products. Nat Protoc 3(5):877–882

    CAS  Article  PubMed  Google Scholar 

  • Viera AJ, Garrett JM (2005) Understanding interobserver agreement: the kappa statistic. Fam Med 37(5):360–363

    PubMed  Google Scholar 

  • Yan W, Shen Z, Tang X, Xu L, Li Q, Yue Y, Xiao S, Fu X (2014) Detection of Nosema bombycis by FTA cards and loop-mediated isothermal amplification (LAMP). Curr Microbiol 69(4):532–540

    CAS  Article  PubMed  Google Scholar 

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Acknowledgements

The authors acknowledge the Indian Council of Agricultural Research (ICAR) Consortia Research Platform on Vaccines and Diagnostics, Govt. of India, for the financial support to carry out this work. The authors also thank the shrimp farmers of Tamil Nadu and Andhra Pradesh for providing samples and information for this study.

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Correspondence to Sathish Kumar T.

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Section Editor: Christopher Whipps

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T, S., A, N., J, J. et al. Visual loop-mediated isothermal amplification (LAMP) for the rapid diagnosis of Enterocytozoon hepatopenaei (EHP) infection. Parasitol Res 117, 1485–1493 (2018). https://doi.org/10.1007/s00436-018-5828-4

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  • DOI: https://doi.org/10.1007/s00436-018-5828-4

Keywords

  • EHP
  • LAMP
  • Closed tube LAMP
  • SYBR green I dye
  • FTA cards