摘要
目的研究用细胞色素C氧化酶1基因(cox1)及内转录间隔区1(ITS-1)特异性序列检测小鼠无虫病理组织中裂头蚴感染的可行性。方法 6~8周龄昆明小鼠20只,采用随机抽签法分为感染组(15只)和未感染组(5只),感染组小鼠经口感染蛇源裂头蚴(5条/只),未感染组不作任何处理。感染后第14天剖杀小鼠,取感染组小鼠皮下含裂头蚴肌肉组织(含虫组织)、去除裂头蚴肌肉组织(无虫组织)及未感染组小鼠相应部位肌肉,制作组织切片,苏木精-伊红染色法(HE)染色后观察。用改良的蛋白酶K消化法提取未染色肌肉组织切片的DNA,PCR扩增cox1(2对引物,对应cox1-1和cox1-2片段)和ITS-1序列。选取从感染组无虫组织DNA扩增的cox1-1进行克隆并测序,与Gen Bank中猬裂头蚴序列(KJ418421、KF990161、GQ999947)进行比对。以猪囊尾蚴感染的小鼠肌肉组织同法制作切片并提取DNA,评价cox1和ITS-1 PCR扩增的特异性。结果感染组含虫组织DNA经1次PCR即可扩增出414 bp(cox1-1)、151 bp(cox1-2)、822 bp(ITS-1)的条带。感染组无虫组织DNA第1次PCR未扩增出条带,以第1次PCR产物为模板再次PCR,扩增出相同大小的3条条带。未感染组DNA第1次和第2次PCR均未扩增出条带。序列比对结果显示,从感染组无虫组织DNA扩增的cox1-1序列与KJ418421、KF990161、GQ999947序列的同源性分别为98.2%、99.1%、99.1%。猪囊尾蚴感染组织DNA没有扩增出条带。结论以cox1和ITS-1序列为靶序列进行重复PCR,能鉴定小鼠无虫病理组织裂头蚴感染。
Objective To assess the feasibility of using specific sequences of cytochrome C oxidase 1 gene(cox1) and internal transcribed spacer 1(ITS-1) to detect plerocercoid infection in worm-free pathological tissue of mice. Methods Twenty Kunming mice aged 6-8 weeks were randomly divided into the infection group(15 mice)and the uninfected group(5 mice). Mice in the infected group were orally infected with plerocercoids from snake(5 plecerocoids/mouse), while the uninfected mice received no treatment. The mice were sacrificed on day 14 after infection. Subcutaneous muscle tissues containing plerocercoids(worm-containing tissue), worm-stripped tissues(wormfree tissue) and their correspondant of the uninfected group were taken and cut into sections. A portion of these sections were processed for hematoxylin-eosin(HE) staining, while the other portion was used for DNA extraction with a modified proteinase K digestion method. The cox1(using 2 pairs of primers, resulting in cox1-1 and cox1-2)and ITS-1(1 pair of primers) were amplified by PCR, and PCR products from DNA of the worm-free tissues in the infection group were cloned, sequenced and aligned with gene sequences of KJ418421, KF990161 and GQ999947 of plerocercoid. The same method was used to prepare muscle tissues from mice infected with cysticercus cellulosae from pigs and the DNA preparation. The species specificity of cox1 and ITS-1 was verified by PCR. Results PCR amplification on DNA of the worm-containing tissue produced specific bands of 822 bp(ITS-1), 414 bp(cox1-1),and 151 bp(cox1-2) after the first round of PCR. No bands were produced from DNA of the worm-free tissue under the same condition but a further round of PCR using the products of the first round resulted in three bands of the same sizes as above. No bands were seen in the uninfected group, either after the first or the second round of PCR. The cox1-1 sequence in the worm-free tissue shared 98.2%, 99.1% and 99.12% homology with KJ418421,KF990161 and GQ999947 of plerocercoid, respectively. No specific bands were amplified from DNA of cysticercus-infected tissue of pigs. Conclusion Repeated PCR amplification using cox1 and ITS-1 gene sequences as the target can identify plerocercoid infection in worm-free pathological tissues of mice.
作者
胡月
陈艳
李金福
蒙兴慧
刘巧霞
刘鉴
HU Yue;CHEN Yan;LI Jin-fu;MENG Xing-hui;LIU Qiao-xia;LIU Jian(Characteristic and Key Laboratory of Modern Pathogen Biology,Guizhou Medical University,Department of Education of Guizhou Province,Guiyang 550004,China)
出处
《中国寄生虫学与寄生虫病杂志》
CAS
CSCD
北大核心
2018年第4期357-360,365,共5页
Chinese Journal of Parasitology and Parasitic Diseases
基金
贵州省科技厅社会发展攻关项目[黔科合SY字(2014)3024号]~~