隐匿管状线虫分子鉴定和国内实验动物感染调查

发布时间：2018-12-20 13:17

【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 RNA部分基因序列核苷酸相似性达100%。SPF实验动物和清洁级实验动物的隐匿管状线虫感染率分别为2.1%和2.7%。结论:应用直接镜检实时动态显微视屏摄录技术联合多重PCR测序技术能够快速精准检测鉴定出隐匿管状线虫。实验动物常会感染一些人兽共患寄生虫,隐匿管状线虫的人兽共患本质可以视作为公共卫生的一个预警。本研究首次对中国SPF和清洁级的实验动物隐匿管状线虫进行了分子鉴定和感染调查。
[Abstract]:Objective: to investigate the molecular identification and infection of non-specific pathogens (specific pathogen-free,SPF) and clean grade experimental animals, so as to provide reference for the revision of national standards for laboratory animals. Methods: a total of 4 649 SPF experimental animals (including 25 miniature pigs, 5 monkeys, 40 rabbits, 296 hamsters, 186 guinea pigs, 438 rats, 3 629 mice, 25 chickens and 5 ducks) and 1 304 clean grade experimental animals (including 3 rabbits) were used. There were 26 hamsters, 147 guinea pigs, 229 rats and 897 mice. Real-time dynamic microscopic video recording technique of direct microscopic examination was used to screen the hidden tubular nematodes in laboratory animals. Cox 1 (cytochrome C peroxidase subunit 1), cyt b (cytochrome b), nad 1) isolated from experimental animals was identified by multiplex polymerase chain reaction (PCR) and sequencing. Nad 5 (NADH dehydrogenase subunit 5), rrn L (ribosomal RNA large subunit) and 28S r RNA (28S ribosomal RNA) genes confirmed the occult tubular nematode infection at molecular level. Results: a large number of eggs, larvae and adults of tubular nematodes were detected from experimental animals by direct microscopic real-time dynamic microscopic video recording technique. The species were identified according to the size and morphology of egg cells, larva, female and male adults of ductile nematodes. Using multiple PCR sequencing techniques, the cox 1 cyt bad 1 nad 5 rn L and 28 S r RNA genes were identified from the eggs, larvae and adults of a single occult tubular nematode isolated from experimental animals. There was no cross reaction with other species of parasites. In the study, the specificity of primers was ensured by sequencing the positive samples, and the mutability of nucleotides among isolates was explored, and the false positive results caused by tubuloid nematode and non-nematode were excluded. Using direct microscopic real-time dynamic microscopic video recording technique, 96 and 35 positive samples of concealed tubular nematode were detected from 4 649 SPF laboratory animals and 1304 clean grade experimental animal samples, respectively. Multiple PCR and sequencing techniques were used to identify the specific DNA. in these positive samples. The results of sequencing showed that cox _ 1 cyt bad _ 1nad _ (1) nad _ (5) was isolated from different SPF laboratory animals and clean grade experimental animals. The nucleotide similarity of partial gene sequences of rrn L and 28 s r RNA was 2.1% and 2.7% in 100%.SPF laboratory animals and clean grade experimental animals, respectively. Conclusion: direct microscopic real-time dynamic microscopic video recording combined with multiple PCR sequencing techniques can be used to detect and identify occult tubular nematode quickly and accurately. Experimental animals are often infected with zoonotic parasites, and the nature of zoonosis, which conceals tubular nematodes, can be regarded as a public health warning. In this study, molecular identification and infection investigation of SPF and clean grade experimental animals were carried out for the first time.
【作者单位】：中国食品药品检定研究院;
【基金】：国家科技支撑计划(2013BAK11B03)资助项目
【分类号】：R383.1;R-33

【参考文献】