隐匿管状线虫分子鉴定和国内实验动物感染调查  

Prevalence and molecular identification of Syphacia obvelata infestation in specific pathogen-free and clean animals in China

作　　者：高正琴[1] 岳秉飞[1] 

机构地区：[1]中国食品药品检定研究院,北京100050

出　　处：《药物分析杂志》2016年第2期234-242,共9页Chinese Journal of Pharmaceutical Analysis

基　　金：国家科技支撑计划(2013BAK11B03)资助项目

摘　　要：目的:对无特定病原体(specific pathogen-free,SPF)和清洁级实验动物隐匿管状线虫进行分子鉴定和感染调查,为实验动物国家标准的修订提供参考依据。方法:4 649只SPF实验动物(包括小型猪25只,猴5只,兔40只,地鼠296只,豚鼠186只,大鼠438只,小鼠3 629只,鸡25只,鸭5只)和1 304只清洁级实验动物(包括兔3只,地鼠26只,豚鼠147只,大鼠229只,小鼠897只)来自全国不同的厂家。应用直接镜检实时动态显微视屏摄录技术,对实验动物的隐匿管状线虫进行筛查。应用多重聚合酶链式反应(PCR)和测序技术,鉴定分离自实验动物的隐匿管状线虫cox 1(细胞色素C过氧化物酶亚基1)、cyt b(细胞色素b)、nad 1(NADH脱氢酶亚单位1)、nad 5(NADH脱氢酶亚单位5)、rrn L(核糖体RNA大亚基)和28S r RNA(28S核糖体RNA)基因,从分子水平上确证隐匿管状线虫感染。结果:应用直接镜检实时动态显微视屏摄录技术,从实验动物中检出数量众多隐匿管状线虫的虫卵、幼虫和成虫。根据隐匿管状线虫的卵细胞、幼虫、雌雄成虫的大小和形态来鉴定虫种。应用多重PCR测序技术,能从分离自实验动物的单个隐匿管状线虫的虫卵、幼虫和成虫中鉴定出cox 1、cyt b、nad 1、nad 5、rrn L和28S r RNA基因,与其他不同种属的寄生虫无交叉反应。在研究中,通过对确定的阳性样本测序保证了引物的特异性,探究了隐匿管状线虫分离株间核苷酸的可变性,排除了可能由于鼠管状线虫和四翼无刺线虫造成的假阳性结果。应用直接镜检实时动态显微视屏摄录技术,从4 649份SPF实验动物和1304份清洁级实验动物样本中分别检出隐匿管状线虫阳性样本96份和35份。应用多重PCR和测序技术,鉴定证明这些阳性样本中确实含有隐匿管状线虫特异性的DNA。测序结果显示,自不同SPF实验动物和清洁级实验动物分离获得的隐匿管状线虫的cox 1、cyt b、nad 1、nad 5、rrn L和28 s r RObjective： To acquire the prevalence and molecular identification data on Syphacia obvelata infestation in specific pathogen-free （ SPF ） and clean animals, so as to provide references for the revision of national standard. Methods： Totally 4 649 SPF laboratory animals （ including 25 mini-pigs, 5 monkeys, 40 rabbits, 296 hamsters, 186 guinea pigs, 438 rats, 3 629 mice, 25 chickens and 5 ducks ） and 1 304 clean animals （ including 3 rabbits, 26 hamsters, 147 guinea pigs, 229 rats and 897 mice ） came from different manufactures in China. Direct microscopy real-time dynamic video recording technique was performed to screen for Syphacia obvelata in laboratory animals. A multiple polymerase chain reaction （ muhiple-PCR ） test was carried out based on the amplification of the conserved portions of the Syphacia obvelata cytochrome c oxidase subunit 1 （ cox 1 ）, cytochrome b （ cyt b ）, NADH dehydrogenase subunits 1 （nad 1 ）, NADH dehydrogenase subunits 5 （ nad 5 ）, large subunit ribosomal RNA （ rrn L ） and 28S ribosomal RNA （ 28S rRNA ） genes, and the molecular sequencing of the multiple-PCR amplicons confirmed infection with Syphacia obvelata. Results： Numerous Syphacia obvelata eggs, larvae and adults were detected in laboratory animals by using direct microscopy real-time dynamic video recording technique. Syphacia obvelata was speciated based on the morphology and size of ovum, larvae, female and male adult worm. Multiple-PCR and sequencing were performed to identify cox 1, cyt b, had 1, nad 5, rrn L and 28S rRNA genes of DNA extracted from the single egg, larva and adult parasite identified Syphacia obvelata. This approach allowed the specific identification with no amplicon being amplified from heterogeneous DNA samples, and sequencing had confirmed the identity of the sequences amplified. Molecular characterization by multiple-PCR amplification and sequencing of the cox 1, cyt b, nad 1, had 5, rrn L and 28S rRNA genes demonstrated the presence of Syphacia obvelata in l

关 键 词：隐匿管状线虫 分子鉴定 感染调查 直接镜检实时动态显微视屏摄录技术 多重聚合酶链式反应 测序 无特定病原体 清洁级动物 

分 类 号：R917[医药卫生—药物分析学]