Quantification of Cryptosporidium parvum in natural soil matrices and soil solutions using qPCR

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• 作者：Emre Koken^a ; Christophe J.G. Darnault^a ; ^{darnault@uic.edu} ; Astrid R. Jacobson^b ; David Powelson^b ; William Hendrickson^c
• 关键词：BSA ; (bovine serum albumin) ; Ct ; (threshold cycle) ; MgCl2 ; (magnesium chloride) ; PR ; (percent recovery) ; qPCR ; (quantitative polymerase chain reaction) ; SDS ; (sodium dodecyl sulfate) ; TE ; (Tris-EDTA or Tris-ethylene diamine tetraacetic acid buffer)
• 刊名：Journal of Microbiological Methods
• 出版年：2013
• 出版时间：15 February, 2013
• 年：2013
• 卷：92
• 期：2
• 页码：135-144
• 全文大小：856 K

文摘

Traditional microscopy methods for the detection and quantification of Cryptosporidium parvum in soil matrices are time-consuming, labor-intensive, and lack sensitivity and specificity. This research focused on developing a qPCR protocol for the sensitive and specific detection and quantification of C. parvum in natural soil matrices and soil-water extracts. The physico-chemical parameters ¡ª lysis media, number of thermal shocks and thawing temperatures ¡ª controlling DNA extraction efficiency were investigated. Experimental results identified oocyst age as a critical parameter affecting oocyst disruption and quantification. The most efficient oocyst disruption method for C. parvum oocysts regardless of their age was established as 5 thermal shocks with thawing at 65 ¡ãC in Tris-EDTA (TE) buffer. In addition to the purification columns used to remove PCR inhibitors present in environmental matrices, a combination of 3 mM MgCl₂ and 600 ng/¦Ìl BSA yielded the highest amplicon yield for both young and aged oocysts. Sucrose flotation was determined to be a better oocyst isolation method than two-phase flotation. The optimized parameters for DNA extraction and the qPCR assay resulted in very specific and sensitive detection of C. parvum. Minimum detection limits were 0.667 for young C. parvum oocysts and 6.67 for aged C. parvum oocysts per PCR reaction. The accuracy of the detections and quantifications was 0.999. Protocol performance was tested in contrasting soil samples and soil-water extract samples on the basis of percentage of recovery (PR) values. Depending on the number of oocysts used to inoculate the samples, the average PR values ranged from 7.2 to 43.5 % , 29.3-52.5 % , and 11.5-60.8 % for Trenton, Greenson, and Sparta soil-water extracts, respectively, and 12.1-77 % for DI water. PR values ranged from 4.3 % to 107.8 % for Trenton, Greenson and Sparta soil samples.