Development of a framework for genotyping bovine-derived Cryptosporidium parvum, using a multilocus fragment typing tool.
Development of a framework for genotyping bovine-derived Cryptosporidium parvum, using a multilocus fragment typing tool.
复制标题
DOI：
10.1186/s13071-015-1107-8
复制
发表时间：
2015-10-01
影响因子：
3.2
通讯作者：
Katzer F
中科院分区：
文献类型：
--
作者：
Hotchkiss EJ;Gilray JA;Brennan ML;Christley RM;Morrison LJ;Jonsson NN;Innes EA;Katzer F
需要一种综合的C.到目前为止，还没有充分的歧视性计划得到兽医或公共卫生研究人员的充分验证或广泛采用。多位点片段分型（MLFT）可以提供良好的区分，并且相对快速和便宜。MLFT工具在其分型能力、特异性、精密度（重复性和再现性）、准确度和基因型区分牛源性隐孢子虫的能力方面进行了评估。为了达成共识，基于其在先前研究中的成功应用，选择了六种标记物：MM 5，MM 18，MM 19，TP 14，MS 1和MS 9。等位基因根据扩增的重复区域的片段大小进行分配，如通过毛细管电泳所确定的。此外，扩增GP 60基因的一个区域并测序以确定gp 60亚型，并将其添加到6个标记的等位基因谱中以确定多位点基因型（MLG）。MLFT工具施加到140 ℃。2004年在柴郡（主要是产奶动物）和2011年在阿伯丁郡/凯斯内斯（肉牛）进行的两项英国小牛横断面研究中采集的细小病毒样本。可分型性为84%。引物未扩增在牛中经常检测到的供试非细小病毒种属。在可重复性方面，运行内和运行间片段大小几乎没有变化。实验室之间，片段大小不同，但等位基因调用是可重复的。MLFT与单独的gp 60测序（SID 0.44）相比具有良好的区分能力（Simpson多样性指数，SID为0.92）。一些标记比其他标记提供更多信息，MS 1和MS 9在测试样品中证明是单等位基因的。现在需要进一步的实验室间试验，包括人源性C。小样本，允许朝着一个综合的，标准化的分型方案，使源属性和确定牲畜在未来的人类C。小的
There is a need for an integrated genotyping approach for C. parvum; no sufficiently discriminatory scheme to date has been fully validated or widely adopted by veterinary or public health researchers. Multilocus fragment typing (MLFT) can provide good differentiation and is relatively quick and cheap to perform. A MLFT tool was assessed in terms of its typeability, specificity, precision (repeatability and reproducibility), accuracy and ability to genotypically discriminate bovine-derived Cryptosporidium parvum. With the aim of working towards a consensus, six markers were selected for inclusion based on their successful application in previous studies: MM5, MM18, MM19, TP14, MS1 and MS9. Alleles were assigned according to the fragment sizes of repeat regions amplified, as determined by capillary electrophoresis. In addition, a region of the GP60 gene was amplified and sequenced to determine gp60 subtype and this was added to the allelic profiles of the 6 markers to determine the multilocus genotype (MLG). The MLFT tool was applied to 140 C. parvum samples collected in two cross-sectional studies of UK calves, conducted in Cheshire in 2004 (principally dairy animals) and Aberdeenshire/Caithness in 2011 (beef animals). Typeability was 84 %. The primers did not amplify tested non-parvum species frequently detected in cattle. In terms of repeatability, within- and between-run fragment sizes showed little variability. Between laboratories, fragment sizes differed but allele calling was reproducible. The MLFT had good discriminatory ability (Simpson’s Index of Diversity, SID, was 0.92), compared to gp60 sequencing alone (SID 0.44). Some markers were more informative than others, with MS1 and MS9 proving monoallelic in tested samples. Further inter-laboratory trials are now warranted with the inclusion of human-derived C. parvum samples, allowing progress towards an integrated, standardised typing scheme to enable source attribution and to determine the role of livestock in future outbreaks of human C. parvum.