Pattaradilokrat Sittiporn, Tiyamanee Wisawa, Simpalipan Phumin, Kaewthamasorn Morakot, Saiwichai Tawee, Li Jian, Harnyuttanakorn Pongchai
Department of Biology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand.
Department of Biology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand.
Vet Parasitol. 2015 May 30;210(1-2):1-9. doi: 10.1016/j.vetpar.2015.03.023. Epub 2015 Apr 6.
Avian malaria is one of the most common veterinary problems in Southeast Asia. The standard molecular method for detection of the avian malaria parasite involves the phenol-chloroform extraction of parasite genomic (g)DNA followed by the amplification of parasite gDNA using polymerase chain reaction (PCR). However, the phenol-chloroform extraction method is time-consuming and requires large amounts of samples and toxic organic solvents, thereby limiting its applications for parasite detection in the field. This study aimed to compare the performance of chelex-100 resin and phenol/chloroform extraction methods for the extraction of Plasmodium gallinaceum gDNA from whole avian blood that had been dried on filter papers (a common field sampling method). The specificity and sensitivity of PCR assays for P. gallinaceum cytochrome B (cytb) and cytochrome oxidase subunit I (coxI) gene fragments (544 and 588bp, respectively) were determined, and found to be more sensitive with gDNA extracted by the chelex-100 resin method than with the phenol/chloroform method. These PCR assays were also performed to detect P. gallinaceum in 29 blood samples dried on filter papers from domestic chickens in a malaria endemic area, where the reliable identification of seven field isolates of P. gallinaceum was obtained with an accuracy of 100%. The analysis of cytb and coxI gene nucleotide sequences revealed the existence of at least two genetically distinct populations of P. gallinaceum in Thailand, both of which differed from the reference strain 8A of P. gallinaceum. In conclusion, the chelex-100 resin extraction method is a simple and sensitive method for isolating gDNA from whole avian blood dried on filter paper. Genomic DNA extracted by the chelex method could subsequently be applied for the PCR-based detection of P. gallinaceum and DNA sequencing. Our PCR assays provide a reliable diagnostic tool for molecular epidemiological studies of P. gallinaceum infections in domestic chickens and wild birds.
禽疟是东南亚最常见的兽医问题之一。检测禽疟原虫的标准分子方法包括用酚-氯仿提取寄生虫基因组（g）DNA，然后使用聚合酶链反应（PCR）扩增寄生虫gDNA。然而，酚-氯仿提取方法耗时，需要大量样本和有毒有机溶剂，从而限制了其在现场寄生虫检测中的应用。本研究旨在比较螯合树脂100和酚/氯仿提取方法从滤纸上干燥的全禽血中提取鸡疟原虫gDNA的性能（一种常见的现场采样方法）。测定了针对鸡疟原虫细胞色素B（cytb）和细胞色素氧化酶亚基I（coxI）基因片段（分别为544和588bp）的PCR检测的特异性和敏感性，发现用螯合树脂100方法提取的gDNA比酚/氯仿方法更敏感。还对疟疾流行地区29份家鸡滤纸上干燥的血样进行了这些PCR检测以检测鸡疟原虫，其中可靠鉴定出7株鸡疟原虫野外分离株，准确率达100%。对cytb和coxI基因核苷酸序列的分析表明，泰国至少存在两个遗传上不同的鸡疟原虫种群，两者均与鸡疟原虫参考菌株8A不同。总之，螯合树脂100提取方法是从滤纸上干燥的全禽血中分离gDNA的一种简单而灵敏的方法。用螯合方法提取的基因组DNA随后可用于基于PCR的鸡疟原虫检测和DNA测序。我们的PCR检测为家鸡和野鸟中鸡疟原虫感染的分子流行病学研究提供了一种可靠的诊断工具。
