Use of a bioinformatic-assisted primer design strategy to establish a new nested PCR-based method for Cryptosporidium.
Use of a bioinformatic-assisted primer design strategy to establish a new nested PCR-based method for Cryptosporidium.
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DOI：
10.1186/s13071-017-2462-4
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发表时间：
2017-10-23
影响因子：
3.2
通讯作者：
Gasser RB
中科院分区：
文献类型：
--
作者：
Koehler AV;Korhonen PK;Hall RS;Young ND;Wang T;Haydon SR;Gasser RB
准确追踪集水区粪便、水和/或土壤样本中的隐孢子虫对于制定管理隐孢子虫病向人类传播的潜在风险的策略至关重要。多种 PCR 检测均用于此目的。尽管一些检测可以对动物粪便样本中的隐孢子虫 DNA 进行特异性扩增，但有些检测却不能。事实上，我们观察到核糖体RNA基因（SSU）小亚基中一些寡核苷酸引物的非特异性，这给粪便样本中隐孢子虫物种和基因型（类群）的鉴定和分类带来了障碍。使用一种新颖的生物信息学方法，我们探索了所有可用的隐孢子虫基因组序列，以获得新的、具有诊断信息的多拷贝区域，以在核核糖体RNA基因（LSU）的大亚基中专门设计寡核苷酸引物，作为用于识别和/或分类隐孢子虫类群的有效的基于巢式PCR的测序方法的基础。这种新建立的 PCR 具有很高的分析特异性和灵敏度，现已与各同事开发的其他检测方法一起在我们的实验室中常规使用。虽然这里使用的当前生物信息学工作流程是针对隐孢子虫核 DNA 中引物的特定设计，但这种方法应该广泛适用于许多其他微生物。本文的在线版本 (10.1186/s13071-017-2462-4) 包含补充材料，可供授权用户使用。
The accurate tracking of Cryptosporidium in faecal, water and/or soil samples in water catchment areas is central to developing strategies to manage the potential risk of cryptosporidiosis transmission to humans. Various PCR assays are used for this purpose. Although some assays achieve specific amplification from Cryptosporidium DNA in animal faecal samples, some do not. Indeed, we have observed non-specificity of some oligonucleotide primers in the small subunit of nuclear ribosomal RNA gene (SSU), which has presented an obstacle to the identification and classification of Cryptosporidium species and genotypes (taxa) from faecal samples. Using a novel bioinformatic approach, we explored all available Cryptosporidium genome sequences for new and diagnostically-informative, multi-copy regions to specifically design oligonucleotide primers in the large subunit of nuclear ribosomal RNA gene (LSU) as a basis for an effective nested PCR-based sequencing method for the identification and/or classification of Cryptosporidium taxa. This newly established PCR, which has high analytical specificity and sensitivity, is now in routine use in our laboratory, together with other assays developed by various colleagues. Although the present bioinformatic workflow used here was for the specific design of primers in nuclear DNA of Cryptosporidium, this approach should be broadly applicable to many other microorganisms. The online version of this article (10.1186/s13071-017-2462-4) contains supplementary material, which is available to authorized users.
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影响因子：
3.2
作者：
Koehler AV;Haydon SR;Jex AR;Gasser RB
通讯作者：
Gasser RB
影响因子：
1.7
作者：
Jurka, J;Kapitonov, VV;Walichiewicz, J
通讯作者：
Walichiewicz, J
影响因子：
2.2
作者：
Ki, Jang-Seu;Han, Myung-Soo
通讯作者：
Han, Myung-Soo
影响因子：
2.2
作者：
Dalén, L;Götherström, A;Angerbjörn, A
通讯作者：
Angerbjörn, A
影响因子：
4.4
作者：
Ryan, U;Xiao, LH;Pavlasek, I
通讯作者：
Pavlasek, I