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Detection and identification of Giardia species using real-time PCR and sequencing
Journal of Microbiological Methods ( IF 1.7 ) Pub Date : 2021-07-14 , DOI: 10.1016/j.mimet.2021.106279
N Jothikumar 1 , Jennifer L Murphy 1 , Vincent R Hill 1
Affiliation  

We report a specific region of Giardia spp. 18S ribosomal RNA (18S rDNA) that serves as an ideal target for quantitative PCR (qPCR) detection and sequencing to identify Giardia species, including the clinically-relevant G. duodenalis, in clinical and environmental samples. The presence of multiple copies of the 18S rDNA gene and variations in the selected 18S genomic region enabled the development of a rapid, sensitive qPCR screening method for the detection of Giardia spp. The analytical sensitivity of the Giardia qPCR assay was determined to be a cyst equivalent of 0.4 G. duodenalis cysts per PCR reaction. Amplicon sequencing of the PCR product confirmed Giardia spp. detection and among the 35 sequences obtained, 31, 3 and 1 isolates were classified as belonging to G. duodenalis, G. microti and G. muris, respectively. The TaqMan assay reported here may be useful for the detection of low levels of Giardia in clinical and environmental samples, and further enables the effective use of direct sequencing of the PCR product for Giardia confirmation and to identify major species of Giardia, including G. duodenalis.



中文翻译:

使用实时 PCR 和测序检测和鉴定贾第鞭毛虫物种

我们报告了贾第鞭毛虫属的特定区域。18S 核糖体 RNA (18S rDNA) 作为定量 PCR (qPCR) 检测和测序的理想靶标,可在临床和环境样本中鉴定贾第鞭毛虫属,包括临床相关的十二指肠杆菌。18S rDNA 基因的多个拷贝的存在和选定的 18S 基因组区域中的变异使得能够开发一种快速、灵敏的 qPCR 筛选方法来检测贾第鞭毛虫属。贾第鞭毛虫qPCR 测定的分析灵敏度被确定为相当于 0.4  G的囊肿。每个 PCR 反应的十二指肠囊肿。PCR产物的扩增子测序证实贾第虫属 检测得到的35个序列中,分别有31个、3个和1个分离株被归类为G. duodenalis、G. microtiG. muris。此处报道的 TaqMan 检测可能有助于检测临床和环境样本中的低水平贾第鞭毛虫,并进一步使 PCR 产物的直接测序有效地用于贾第鞭毛虫确认和鉴定贾第鞭毛虫的主要物种,包括十二指肠贾第鞭毛虫.

更新日期:2021-07-14
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