首页> 外文会议>International Colloquium on Paratuberculosis >Definitive identification of single versus mixed mycobacterial infection(s) in red deer (Census elaphus) by combined duplex upstream-p34:f57 amplification and Hpy188l enzymatic restriction of duplex amplicons
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Definitive identification of single versus mixed mycobacterial infection(s) in red deer (Census elaphus) by combined duplex upstream-p34:f57 amplification and Hpy188l enzymatic restriction of duplex amplicons

机译:通过组合双相上游-P34,对红鹿(人口普查eLaphus)中单一与混合分枝杆菌感染的明确鉴定 - P34:F57扩增和双相扩增的HPY188L酶限制

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Severe emaciation and mortalities, suggestive of mycobacterial infections, were recently reported in both adult and young wild red deer (Cervus elaphus) in the south-eastern part of Belgium. In deer, tuberculous lesions are not pathognomonic of Mycobacterium bovis (Mbo), due to gross and microscopic similarities with lesions caused by Mycobacterium avium subspecies paratuberculosis (Map) or Mycobacterium avium subspecies avium (Maa). The aim of this study was to improve the molecular species-specific identification of Mbo, Maa, and Map in deer mycobacterial infections. DNA banding patterns were assessed prior and after Hpy188l restriction of f57:usptream (us)-p34 duplex amplicons. As reported, the duplex f57:us-p34 PCR duplex differentiated Mbo from Map and Maa infections, whereas the restriction step differentiated single Map and Maa from mixed Map/Maa infections. The endonuclease Hpy188l cleaves DNA between nucleotides N and G in the unique TCNGA sequence. This restriction site was found at position 138 upstream the us-p34 initiation codon in all Maa strains tested, regardless of their origin and the IS901PCR results. In contrast, the restriction site was abrogated in all Map strains tested, regardless of their origin, the Mycobactin J dependency and the IS900 PCR results. Consequently, the two-step strategy, i.e. duplex us-p34:f57 PCR and Hpy188l restriction, allowed to exclude Mbo infection and to identify single (Map and Maa) or mixed (Map/Maa) infections in wild red deer in Belgium. Accordingly, we propose to integrate, in a functional molecular definition of Map, the absence of the Hpy188l restriction site in the us-p34 amplicon.
机译:严重的消瘦和死亡率,分枝杆菌感染的提示,在成人和在比利时的东南部年轻狂野的马鹿(马鹿)最近报道。在鹿,结核性病变不特异病征性的牛分枝杆菌(MBO),由于与病变肉眼和显微镜的相似性引起的鸟分枝杆菌副结核亚种(MAP)或鸟分枝杆菌亚种杆菌(MAA)。这项研究的目的是提高鹿分枝杆菌感染MBO,MAA和地图的分子物种特异性的识别。 DNA条带图案被预先和后F57的Hpy188l限制评估:usptream(我们)-p34双工扩增子。据报道,双工F57:我们-P34双重PCR从地图和MAA感染分化MBO,而限制步从混合地图/ MAA感染分化一张地图和MAA。核苷酸N和G的在独特TCNGA序列的核酸内切裂解Hpy188l DNA。此限制性位点在138位置上发现上游的US-P34起始密码子在所有菌株马阿测试,无论其来源和IS901PCR结果。相比之下,限制性位废除在所有测试的地图株,不论其出身,对分支杆菌生长素Ĵ依赖和IS900 PCR结果。因此,两步走战略,即复式我们-P34:F57 PCR和Hpy188l限制,允许排除姆博感染,并确定单(地图和MAA),或在比利时野生马鹿混合(图/ MAA)感染。因此,我们建议进行整合,在地图中,美P34扩增子缺少Hpy188l酶切位点的功能分子的定义。

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