Abstract
Colorimetric detection can be applied to differentiate between positive and negative conditions. It can be coupled with loop-mediated isothermal amplification to diagnose rumen fluke or paramphistome infection, also called colorimetric PAR-LAMP. This study conducted LAMP using three candidate indicator dyes, namely malachite green (MLG), methyl green (MTG), and neutral red (NTR), and the results were observed by the naked eye. The dye concentration was optimized to obtain the most pronounced positive–negative result discrimination. Subsequently, we conducted target sensitivity tests using the DNA of Fischoederius elongatus at different concentrations. To validate the detection accuracy, the result was confirmed by gel electrophoresis. The sensitivity test presented the lowest detectable DNA concentration or limit of detection (LOD), with 1 pg for MLG, 0.5 ng for MTG, and 50 pg for NTR. Different LODs revealed inhibition of LAMP reaction and reduced efficiency of result presentation for colorimetric-based detection, particularly NTR and MTG. For MLG-LAMP, we observed no cross-reaction of non-target DNA and improved reaction with the DNA of Fischoederius cobboldi and Calicophoron sp., with multi-detection. In addition, naked eye observation and agarose gel electrophoresis (AGE) evaluation of the MLG-LAMP results showed a moderate and strong agreement with LAMP-AGE and microscopic examinations. Based on our results, colorimetric PAR-LAMP is a rapid, comfortable, and point-of-care procedure for the diagnosis of paramphistome infection.
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All data supporting the findings of this study are available within the paper and its Supplementary Information; only agarose gel figures (in Supplementary File 3) will be made available on request.
Abbreviations
- AGE :
-
Agarose gel electrophoresis
- DSE :
-
Diagnostic sensitivity
- DSP :
-
Diagnostic specificity
- LAMP :
-
Loop-mediated isothermal amplification
- MLG :
-
Malachite green
- MTG :
-
Methyl green
- NTR :
-
Neutral red
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Acknowledgements
This project is funded by Srinakharinwirot University (307/2566, 211/2565), which provided research funding and granted us the use of several of their facilities.
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Srinakharinwirot University, 307/2566, 211/2565
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Thapana Chontananarth (TC) contributed, designed, and participated in all parts of the study. TC and Sirapat Nak-on (SN) collected and identified the samples. SN, Metawee Sabaijai (MS), Awika Raksaman (AR), Wasin Panich (WP), and Thanawan Tejangkura (TT) conducted the experiment in the laboratory and analyzed the data. All authors were working together in writing the manuscript. All authors have approved this manuscript.
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All animal experiments in this work were approved by the Institute of Animals for Scientific Purpose Development (IAD) and the Committee for Animal Care and Use, Srinakharinwirot University, Thailand.
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The primers in this study were described in form of approving patent number 2203001221 by department of intellectual property (DIP), Thailand.
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Nak-on, S., Sabaijai, M., Raksaman, A. et al. Visualization and development of colorimetric loop-mediated isothermal amplification for the rapid detection and diagnosis of paramphistome infection: colorimetric PAR-LAMP. Parasitol Res 123, 126 (2024). https://doi.org/10.1007/s00436-024-08150-z
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DOI: https://doi.org/10.1007/s00436-024-08150-z