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.
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.
['Tantrawatpan|Chairat|C|', 'Intapan|Pewpan M|PM|', 'Thanchomnang|Tongjit|T|', 'Sanpool|Oranuch|O|', 'Janwan|Penchom|P|', 'Lulitanond|Viraphong|V|', 'Anamnart|Witthaya|W|', 'Maleewong|Wanchai|W|']
D000818:Animals D002371:Cat Diseases D002415:Cats D018509:DNA, Helminth D021903:DNA, Ribosomal Spacer D005243:Feces D031541:Fluorescence Resonance Energy Transfer D057230:Limit of Detection D008171:Lung Diseases D010237:Paragonimiasis D010238:Paragonimus D060888:Real-Time Polymerase Chain Reaction D015203:Reproducibility of Results D012680:Sensitivity and Specificity D013785:Thailand D015047:Zoonoses
real-time fluorescence resonance energy transfer polymerase chain reaction feces parasite Egg detection second internal transcribed spacer