PathogenMip Assay: A Multiplex Pathogen Detection Assay
PathogenMip Assay: A Multiplex Pathogen Detection Assay
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DOI：
10.1371/journal.pone.0000223
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发表时间：
2007-02-21
期刊：
影响因子：
3.7
通讯作者：
Pourmand, Nader
中科院分区：
文献类型：
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作者：
Akhras, Michael S.;Thiyagarajan, Sreedevi;Pourmand, Nader
分子倒置探针（MIP）检测此前已应用于大规模人类SNP检测。在这里，我们描述了PathogenMip检测，一个完整的协议，探针生产和应用方法，病原体检测。我们已经证明了这种检测的实用性，最初的一组24个探针靶向与宫颈癌进展相关的最临床相关的HPV基因型。探针构建是基于一种新颖的，具有成本效益的，基于连接酶的协议。通过在平行实验中进行焦磷酸测序和微阵列芯片检测来验证该测定。HPV质粒用于验证测定的灵敏度和选择性。此外，采用PathogenMip Assay结果对原发性肿瘤的20份基因组DNA提取物进行基因分型，结果与使用基于L1的HPV基因分型方案进行的常规测序结果100%一致。PathogenMip检测是一种广泛使用的方案，用于生产和使用高度区分的探针，在病原体基因分型方面具有经过实验验证的结果，可潜在地应用于任何微生物的检测和表征。
The Molecular Inversion Probe (MIP) assay has been previously applied to a large-scale human SNP detection. Here we describe the PathogenMip Assay, a complete protocol for probe production and applied approaches to pathogen detection. We have demonstrated the utility of this assay with an initial set of 24 probes targeting the most clinically relevant HPV genotypes associated with cervical cancer progression. Probe construction was based on a novel, cost-effective, ligase-based protocol. The assay was validated by performing pyrosequencing and Microarray chip detection in parallel experiments. HPV plasmids were used to validate sensitivity and selectivity of the assay. In addition, 20 genomic DNA extracts from primary tumors were genotyped with the PathogenMip Assay results and were in 100% agreement with conventional sequencing using an L1-based HPV genotyping protocol. The PathogenMip Assay is a widely accessible protocol for producing and using highly discriminating probes, with experimentally validated results in pathogen genotyping, which could potentially be applied to the detection and characterization of any microbe.