虫卵基因序列分析鉴定斯氏并殖吸虫病

目的探索运用分子生物学技术分析虫卵基因序列鉴定并殖吸虫病。方法从云南省的并殖吸虫病流行区采集溪蟹,常规分离囊蚴,经形态学鉴定后感染实验终宿主家猫,从猫粪便中分离虫卵,对虫卵进行详细的形态学观察和鉴定。用匀浆器研磨虫卵提取基因组DNA,PCR扩增出虫卵中完整的核糖体基因第二间隔区（ITS2）和部分线粒体细胞色素C氧化酶亚单位I基因（COI）,测序获得该基因的核苷酸序列。将核苷酸序列输入GenBank中进行Blast比较,通过ITS2和COI的同源性来判断所感染的并殖吸虫种类。结果取自猫粪便中的虫卵形态符合并殖吸虫卵的形态特征。该虫卵的ITS2基因序列与GenBank中的斯氏并殖吸虫的基因序列同源性为99%,COI基因序列的同源性也高达98%。结果鉴定为斯氏并殖吸虫虫卵,证明家猫感染的是斯氏并殖吸虫病。结论通过并殖吸虫卵的基因序列分析,不仅可以快速诊断并殖吸虫病,而且还能准确地鉴定感染者所感染的并殖吸虫虫种。

Sequence analysis of egg DNA to identify the paragonimiasis caused by Paragonimus skrjabini

Objective To explore the use of molecular biology techniques to identify the paragonimiasis by analyzing the sequences of egg DNA.Methods Crabs were collected from the paragonimiasis epidemic area for the separation the metacercariae.The metacercariae were morphologically identified and used to infect cat.Eggs were obtained from the infected cat feces,and morphologically identified.DNA was extracted from the eggs and used for the amplification of ITS2 and COI by PCR.The amplified DNA was sequenced and blasted with the GenBank database.Results The morphology of eggs obtained from the infected cat feces is identical with the eggs of lung fluke.The sequence homology of ITS2 and COI PCR DNA fragment obtained from this test were 99% and 98%,respectively,of the ITS2 and COI sequences of Paragonimus skrjabini in GenBank database.So the species of this lung fluke eggs is P.skrjabini.Conclusion Using the analysis of the sequence of ITS2 and COI of egg DNA not only can diagnose paragonimiasis,but also can identify the species of Paragonimus quickly and accurately.This method can be applied to other disease diagnosis and pathogen identification.