【摘要】：目的:對無特定病原體(specific pathogen-free,SPF)和清潔級實驗動物隱匿管狀線蟲進行分子鑒定和感染調(diào)查,為實驗動物國家標準的修訂提供參考依據(jù)。方法:4 649只SPF實驗動物(包括小型豬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,雞25只,鴨5只)和1 304只清潔級實驗動物(包括兔3只,地鼠26只,豚鼠147只,大鼠229只,小鼠897只)來自全國不同的廠家。應用直接鏡檢實時動態(tài)顯微視屏攝錄技術(shù),對實驗動物的隱匿管狀線蟲進行篩查。應用多重聚合酶鏈式反應(PCR)和測序技術(shù),鑒定分離自實驗動物的隱匿管狀線蟲cox 1(細胞色素C過氧化物酶亞基1)、cyt b(細胞色素b)、nad 1(NADH脫氫酶亞單位1)、nad 5(NADH脫氫酶亞單位5)、rrn L(核糖體RNA大亞基)和28S r RNA(28S核糖體RNA)基因,從分子水平上確證隱匿管狀線蟲感染。結(jié)果:應用直接鏡檢實時動態(tài)顯微視屏攝錄技術(shù),從實驗動物中檢出數(shù)量眾多隱匿管狀線蟲的蟲卵、幼蟲和成蟲。根據(jù)隱匿管狀線蟲的卵細胞、幼蟲、雌雄成蟲的大小和形態(tài)來鑒定蟲種。應用多重PCR測序技術(shù),能從分離自實驗動物的單個隱匿管狀線蟲的蟲卵、幼蟲和成蟲中鑒定出cox 1、cyt b、nad 1、nad 5、rrn L和28S r RNA基因,與其他不同種屬的寄生蟲無交叉反應。在研究中,通過對確定的陽性樣本測序保證了引物的特異性,探究了隱匿管狀線蟲分離株間核苷酸的可變性,排除了可能由于鼠管狀線蟲和四翼無刺線蟲造成的假陽性結(jié)果。應用直接鏡檢實時動態(tài)顯微視屏攝錄技術(shù),從4 649份SPF實驗動物和1304份清潔級實驗動物樣本中分別檢出隱匿管狀線蟲陽性樣本96份和35份。應用多重PCR和測序技術(shù),鑒定證明這些陽性樣本中確實含有隱匿管狀線蟲特異性的DNA。測序結(jié)果顯示,自不同SPF實驗動物和清潔級實驗動物分離獲得的隱匿管狀線蟲的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達100%。SPF實驗動物和清潔級實驗動物的隱匿管狀線蟲感染率分別為2.1%和2.7%。結(jié)論:應用直接鏡檢實時動態(tài)顯微視屏攝錄技術(shù)聯(lián)合多重PCR測序技術(shù)能夠快速精準檢測鑒定出隱匿管狀線蟲。實驗動物常會感染一些人獸共患寄生蟲,隱匿管狀線蟲的人獸共患本質(zhì)可以視作為公共衛(wèi)生的一個預警。本研究首次對中國SPF和清潔級的實驗動物隱匿管狀線蟲進行了分子鑒定和感染調(diào)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.
【作者單位】： 中國食品藥品檢定研究院;
【基金】：國家科技支撐計劃(2013BAK11B03)資助項目
【分類號】：R383.1;R-33

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