Development and evaluation of a real-time PCR assay for quantification of Giardia and Cryptosporidium in sewage samples
Development and evaluation of a real-time PCR assay for quantification of Giardia and Cryptosporidium in sewage samples
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DOI：
10.1007/s00253-010-2984-6
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发表时间：
2011-02-01
影响因子：
5
通讯作者：
Canigral, Irene
中科院分区：
文献类型：
--
作者：
Alonso, Jose L.;Amoros, Inmaculada;Canigral, Irene
隐孢子虫和贾第鞭毛虫是世界范围内人类腹泻病的主要原因，也是原生动物水传播疾病的主要原因。两个DNA TaqMan PCR为基础的贾第虫和隐孢子虫的方法，分别靶向β-贾第虫基因的74-bp序列和COWP隐孢子虫基因的151-bp序列，被用作模型，比较两种不同的LNA/DNA TaqMan探针，以提高检测限的实时PCR检测。LNA探针是最敏感的，导致C（t）值比DNA贾第虫TaqMan探针低0.96至1.57，比DNA隐孢子虫TaqMan探针低0.56至2.21。用LNA取代的TaqMan贾第虫和隐孢子虫探针的评估导致实时PCR曲线具有比常规DNA TaqMan探针更早的C（t）值。总之，LNA探针可以用于更灵敏的检测限。
Cryptosporidium and Giardia are major causes of diarrheal disease in humans worldwide and are major causes of protozoan waterborne diseases. Two DNA TaqMan PCR-based Giardia and Cryptosporidium methods targeting a 74-bp sequence of the beta-giardin Giardia gene and a 151-bp sequence of the COWP Cryptosporidium gene, respectively, were used as models to compare two different LNA/DNA TaqMan probes to improve the detection limit in a real-time PCR assay. The LNA probes were the most sensitive resulting in 0.96 to 1.57 lower C (t) values than a DNA Giardia TaqMan probe and 0.56 to 2.21 lower than a DNA Cryptosporidium TaqMan probe. Evaluation of TaqMan Giardia and Cryptosporidium probes with LNA substitutions resulted in real-time PCR curves with an earlier C (t) values than conventional DNA TaqMan probes. In conclusion, the LNA probes could be useful for more sensitive detection limits.