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

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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

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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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Authors and Affiliations

  1. ICAR-Central Institute of Brackishwater Aquaculture, #75 Santhome High Road, Raja Annamalai Puram, Chennai, 600028, India

    Sathish Kumar T, Navaneeth Krishnan A, Joseph Sahaya Rajan J, Makesh M, Jithendran K. P, Alavandi S. V & Vijayan K. K

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  1. Sathish Kumar T
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  2. Navaneeth Krishnan A
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  3. Joseph Sahaya Rajan J
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  4. Makesh M
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  5. Jithendran K. P
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  6. Alavandi S. V
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  7. Vijayan K. K
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Correspondence to Sathish Kumar T.

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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

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