Detection and genotyping by real-time PCR/RFLP analyses of Giardia duodenalis from human faeces
Detection and genotyping by real-time PCR/RFLP analyses of Giardia duodenalis from human faeces
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DOI：
10.1099/jmm.0.05193-0
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发表时间：
2003-08-01
影响因子：
3
通讯作者：
McLauchlin, J
中科院分区：
文献类型：
--
作者：
Amar, CFL;Dear, PH;McLauchlin, J
建立了一种检测和区分人体粪便中贾第鞭毛虫聚集体A和B的巢式PCR方法（TPILC-PCR）。该测定包括初始的多重基于阻断的扩增。随后使用LightCycler和SYBR绿色I对集合体A和B进行两次单独的特异性实时PCR测定，以通过熔点分析鉴定PCR产物。利用RFLP技术对G. Pastialis组合A组I和II。实时巢式PCR检测了纯化的滋养体、隐孢子虫卵囊、含多种潜在病原体（包括G.粪便涂片和细菌悬液。该方法特异性强、灵敏度高、重复性好、快速。
A nested PCR assay (TPILC-PCR) was developed to detect and distinguish between Giardia duodenalis assemblages A and B from human faeces by analysis of the triose phosphate isomerase gene (tpi). The assay comprised an initial multiplexed block-based amplification. This was followed by two separate real-time PCR assays specific for assemblages A and B using a LightCycler and SYBR Green I to identify PCR products by melting-point analysis. RFLP analysis was applied to distinguish G. duodenalis assemblage A groups I and II. The real-time nested PCR was evaluated using DNA extracted from purified giardial trophozoites, Cryptosporidium oocysts, whole faeces containing a range of potential pathogens (including G. duodenalis), faecal smears and bacterial suspensions. The assay was specific, sensitive, reproducible and rapid.