Tantrawatpan Chairat, Intapan Pewpan M, Thanchomnang Tongjit, Sanpool Oranuch, Janwan Penchom, Lulitanond Viraphong, Anamnart Witthaya, Maleewong Wanchai
1Pewpan M. Intapan, Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand.
J Vet Diagn Invest. 2013 Sep;25(5):620-6. doi: 10.1177/1040638713497944. Epub 2013 Jul 29.
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.
异盘并殖吸虫是一种具有重要医学意义的肺吸虫，可在包括泰国在内的东南亚地区引起人和动物的并殖吸虫病。在本研究中，开发并评估了一种结合熔解曲线分析的实时荧光共振能量转移聚合酶链反应（实时FRET PCR），以检测实验感染猫粪便中的异盘并殖吸虫卵。该方法对异盘并殖吸虫DNA序列的检测限为阳性对照质粒的3×10²拷贝和异盘并殖吸虫基因组DNA的10⁻³ ng。该检测系统能够检测每克猫粪便中10个异盘并殖吸虫卵。当检测从其他16种生物纯化的DNA或猫和人类的基因组DNA时，未观察到荧光信号。实时FRET PCR对17只感染异盘并殖吸虫的猫的所有粪便样本均产生阳性结果，并且粪便中寄生虫卵的数量与PCR循环数之间呈负相关（r = -0.852，P < 0.001）。该检测方法能够检测来自异盘并殖吸虫、卫氏并殖吸虫、大睾并殖吸虫、暹罗并殖吸虫、哈氏并殖吸虫和曼谷并殖吸虫的基因组DNA，并能将异盘并殖吸虫与其他5个物种区分开来。通过熔解峰分析，所检测的6种并殖吸虫物种被分为4组。该检测方法可用于流行地区异盘并殖吸虫感染的检测和流行病学研究。
