Assessment of three methods for multilocus fragment typing of Cryptosporidium parvum from domestic ruminants in north west Spain
Assessment of three methods for multilocus fragment typing of Cryptosporidium parvum from domestic ruminants in north west Spain
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DOI：
10.1016/j.vetpar.2011.11.039
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发表时间：
2012-05-25
影响因子：
2.6
通讯作者：
Chalmers, R. M.
中科院分区：
文献类型：
--
作者：
Diaz, P.;Hadfield, S. J.;Chalmers, R. M.
采用毛细管电泳（CE）、高分辨平板凝胶电泳和测序3种不同的方法对微小隐孢子虫（Cryptosporidium parvum）微卫星区ML 1和MI2的27个犊牛分离株和14个羔羊分离株进行PCR片段大小分析。为了评估这种原生动物在国内反刍动物在西班牙西北部的遗传变异，结合60 kDa的糖蛋白（GP 60）基因的序列分析，建立一个多位点的类型和分析农场和主机species.CE显示更大的整体分型能力（T），判别力和易用性比平板凝胶电泳和测序都受到影响的PCR口吃，特别是在ML 2。与测序相比，CE片段大小始终长4 bp，测序被认为是等位基因大小测定的金标准，但其可分型性最低;因此对CE大小进行了调整。在ML 1基因座上仅鉴定出三个等位基因（ML 1 -238、ML 1 -229和ML 1 -226）。ML 2位点的多态性较强，共检测到8个等位基因（ML 2 -235、ML 2 - 233、ML 2 -231、ML 2 - 229、ML 2 - 227、ML 2 -225、ML 2 -201和ML 2 -176）。在所有三个基因座（T = 0.90）分型的小孢子虫分离物：9个多基因座类型（MLTs）进行了鉴定。3位点分型法的区分力为0.83。尽管研究了更多的小牛分离株，但在小牛分离株（7个MLT）中观察到的遗传变异性大于羔羊分离株（4个MLT）。牛MLT以MLT 1（ML 1 -238，ML 2 -231，GP 60亚型IIaA 15 G2 R1）为主，羔羊以MLT 3（ML 1 -238，ML 2 -227，GP 60亚型IIaA 16 G3 R1）为主。CE似乎是一个经济和快速的选择进行微卫星片段大小分析提供良好的可分型性，歧视和易用性，但可能需要校准测序标准。(C)2011 Elsevier B. V.保留所有权利。
The performance of three different methods, capillary electrophoresis (CE), high resolution slab-gel electrophoresis and sequencing, for PCR fragment size analysis of two Cryptosporidium parvum microsatellite regions, ML1 and MI2, was investigated by analysing 27 isolates from calves and 14 from lambs. To assess genetic variability of this protozoan in domestic ruminants in north west Spain, results were combined with sequence analysis of the 60 kDa glycoprotein (GP60) gene creating a multilocus type and analysed by farm and host species.CE showed greater overall typability (T), discriminatory power and ease of use than slabgel electrophoresis and sequencing which were both affected by PCR stutter, especially at ML2. CE fragment sizes were consistently 4 bp longer compared to sequencing which is considered the gold standard for allele sizing but which gave the lowest typability; CE sizes were therefore adjusted. Only three alleles were identified at the ML1 locus (ML1-238, ML1-229 and ML1-226). The ML2 locus was more polymorphic and eight alleles were found (ML2-235, ML2-233, ML2-231, ML2-229, ML2-227, ML2-225, ML2-201 and ML2-176).Adjusted ML1 and ML2 CE fragment sizes were combined with GP60 subtype for 37 of the 41 C. parvum isolates which were typable at all three loci (T = 0.90): nine multilocus types (MLTs) were identified. The discriminatory power of the 3-locus typing method was 0.83. Greater genetic variability was observed in calf isolates (7 MLTs) than in those from lambs (4 MLTs) although more calf isolates were studied. The most common MLT in cattle was MLT1 (ML1-238, ML2-231, GP60 subtype IIaA15G2R1), while MLT3 (ML1-238, ML2-227, GP60 IIaA16G3R1) was predominant in lambs.Our findings demonstrate that high discrimination can be achieved by means of multilocus typing. CE appears to be an economic and rapid option for performing microsatellite fragment size analysis offering good typability, discrimination and ease of use but may require calibration to sequenced standards. (C) 2011 Elsevier B.V. All rights reserved.