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Application of a real-time fluorescence resonance energy transfer polymerase chain reaction assay with melting curve analysis for the detection of Paragonimus heterotremus eggs in the feces of experimentally infected cats.
J Vet Diagn Invest. 2013 Sep; 25(5):620-6.JV

Abstract

Paragonimus heterotremus is a medically important lung fluke that causes human and animal paragonimiasis in Southeast Asia, including Thailand. In the current study, a real-time fluorescence resonance energy transfer polymerase chain reaction (real-time FRET PCR) with melting curve analysis was developed and evaluated to detect P. heterotremus eggs in the feces of experimentally infected cats. The detection limit of this method for the P. heterotremus DNA sequence was 3 × 10(2) copies of the positive control plasmid and 10(-3) ng of P. heterotremus genomic DNA. The assay system could detect 10 eggs of P. heterotremus per gram of cat feces. No fluorescence signal was observed when DNA purified from 16 other organisms or genomic DNA from cats and human beings were tested. Real-time FRET PCR yielded positive results for all fecal samples from 17 P. heterotremus-infected cats and showed a negative relationship (r = -0.852, P < 0.001) between the number of parasite eggs in feces and the number of PCR cycles. The assay could detect genomic DNA from P. heterotremus, P. westermani, P. macrorchis, P. siamensis, P. harinasutai, and P. bangkokensis and can differentiate P. heterotremus from the other 5 species. The 6 Paragonimus species examined were divided into 4 groups by melting peak analysis. This assay can be useful for the detection of, and epidemiological studies on, P. heterotremus infection in endemic areas.

Authors+Show Affiliations

1Pewpan M. Intapan, Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand. pewpan@kku.ac.th.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

23897922

Citation

Tantrawatpan, Chairat, et al. "Application of a Real-time Fluorescence Resonance Energy Transfer Polymerase Chain Reaction Assay With Melting Curve Analysis for the Detection of Paragonimus Heterotremus Eggs in the Feces of Experimentally Infected Cats." Journal of Veterinary Diagnostic Investigation : Official Publication of the American Association of Veterinary Laboratory Diagnosticians, Inc, vol. 25, no. 5, 2013, pp. 620-6.
Tantrawatpan C, Intapan PM, Thanchomnang T, et al. Application of a real-time fluorescence resonance energy transfer polymerase chain reaction assay with melting curve analysis for the detection of Paragonimus heterotremus eggs in the feces of experimentally infected cats. J Vet Diagn Invest. 2013;25(5):620-6.
Tantrawatpan, C., Intapan, P. M., Thanchomnang, T., Sanpool, O., Janwan, P., Lulitanond, V., Anamnart, W., & Maleewong, W. (2013). Application of a real-time fluorescence resonance energy transfer polymerase chain reaction assay with melting curve analysis for the detection of Paragonimus heterotremus eggs in the feces of experimentally infected cats. Journal of Veterinary Diagnostic Investigation : Official Publication of the American Association of Veterinary Laboratory Diagnosticians, Inc, 25(5), 620-6. https://doi.org/10.1177/1040638713497944
Tantrawatpan C, et al. Application of a Real-time Fluorescence Resonance Energy Transfer Polymerase Chain Reaction Assay With Melting Curve Analysis for the Detection of Paragonimus Heterotremus Eggs in the Feces of Experimentally Infected Cats. J Vet Diagn Invest. 2013;25(5):620-6. PubMed PMID: 23897922.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Application of a real-time fluorescence resonance energy transfer polymerase chain reaction assay with melting curve analysis for the detection of Paragonimus heterotremus eggs in the feces of experimentally infected cats. AU - Tantrawatpan,Chairat, AU - Intapan,Pewpan M, AU - Thanchomnang,Tongjit, AU - Sanpool,Oranuch, AU - Janwan,Penchom, AU - Lulitanond,Viraphong, AU - Anamnart,Witthaya, AU - Maleewong,Wanchai, Y1 - 2013/07/29/ PY - 2013/7/31/entrez PY - 2013/7/31/pubmed PY - 2014/4/25/medline KW - Egg detection KW - feces KW - parasite KW - real-time fluorescence resonance energy transfer polymerase chain reaction KW - second internal transcribed spacer SP - 620 EP - 6 JF - Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc JO - J. Vet. Diagn. Invest. VL - 25 IS - 5 N2 - Paragonimus heterotremus is a medically important lung fluke that causes human and animal paragonimiasis in Southeast Asia, including Thailand. In the current study, a real-time fluorescence resonance energy transfer polymerase chain reaction (real-time FRET PCR) with melting curve analysis was developed and evaluated to detect P. heterotremus eggs in the feces of experimentally infected cats. The detection limit of this method for the P. heterotremus DNA sequence was 3 × 10(2) copies of the positive control plasmid and 10(-3) ng of P. heterotremus genomic DNA. The assay system could detect 10 eggs of P. heterotremus per gram of cat feces. No fluorescence signal was observed when DNA purified from 16 other organisms or genomic DNA from cats and human beings were tested. Real-time FRET PCR yielded positive results for all fecal samples from 17 P. heterotremus-infected cats and showed a negative relationship (r = -0.852, P < 0.001) between the number of parasite eggs in feces and the number of PCR cycles. The assay could detect genomic DNA from P. heterotremus, P. westermani, P. macrorchis, P. siamensis, P. harinasutai, and P. bangkokensis and can differentiate P. heterotremus from the other 5 species. The 6 Paragonimus species examined were divided into 4 groups by melting peak analysis. This assay can be useful for the detection of, and epidemiological studies on, P. heterotremus infection in endemic areas. SN - 1943-4936 UR - https://www.unboundmedicine.com/medline/citation/23897922/Application_of_a_real_time_fluorescence_resonance_energy_transfer_polymerase_chain_reaction_assay_with_melting_curve_analysis_for_the_detection_of_Paragonimus_heterotremus_eggs_in_the_feces_of_experimentally_infected_cats_ L2 - http://journals.sagepub.com/doi/full/10.1177/1040638713497944?url_ver=Z39.88-2003&amp;rfr_id=ori:rid:crossref.org&amp;rfr_dat=cr_pub=pubmed DB - PRIME DP - Unbound Medicine ER -