title: 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.
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.affiliation: 1Pewpan M. Intapan, Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand. pewpan@kku.ac.th.
authors.forename: Chairat
authors.initials: C
authors.lastname: Tantrawatpan
authors.forename: Pewpan M
authors.initials: PM
authors.lastname: Intapan
authors.forename: Tongjit
authors.initials: T
authors.lastname: Thanchomnang
authors.forename: Oranuch
authors.initials: O
authors.lastname: Sanpool
authors.forename: Penchom
authors.initials: P
authors.lastname: Janwan
authors.forename: Viraphong
authors.initials: V
authors.lastname: Lulitanond
authors.forename: Witthaya
authors.initials: W
authors.lastname: Anamnart
authors.forename: Wanchai
authors.initials: W
authors.lastname: Maleewong
mesh_terms: D000818:Animals; D002371:Cat Diseases / Q000175:diagnosis / Q000469:parasitology*; D002415:Cats; D018509:DNA, Helminth / Q000737:chemistry / Q000235:genetics; D021903:DNA, Ribosomal Spacer / Q000737:chemistry / Q000235:genetics; D005243:Feces / Q000469:parasitology; D031541:Fluorescence Resonance Energy Transfer / Q000662:veterinary; D057230:Limit of Detection; D008171:Lung Diseases / Q000175:diagnosis / Q000469:parasitology / Q000662:veterinary*; D010237:Paragonimiasis / Q000175:diagnosis / Q000469:parasitology / Q000662:veterinary*; D010238:Paragonimus / Q000235:genetics / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods / Q000592:standards / Q000662:veterinary*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013785:Thailand; D015047:Zoonoses / Q000469:parasitology*
chemical_list: D018509:DNA, Helminth; D021903:DNA, Ribosomal Spacer
keywords: Egg detection; feces; parasite; real-time fluorescence resonance energy transfer polymerase chain reaction; second internal transcribed spacer