USEPA Method 1623 is the standard method in the United States for the detection of Cryptosporidium in water samples, but quantitative real-time polymerase chain reaction (qPCR) is an alternative technique that has been successfully used to detect Cryptosporidium in aqueous matrices. This study examined various modifications to a commercial nucleic acid extraction procedure in order to enhance PCR detection sensitivity for Cryptosporidium. An alternative DNA extraction buffer allowed for qPCR detection at lower seed levels than a commercial extraction kit buffer. In addition, the use of a second spin column cycle produced significantly better detection (P = 0.031), and the volume of Tris–EDTA buffer significantly affected crossing threshold values (P = 0.001). The improved extraction procedure was evaluated using 10 L of tap water samples processed by ultrafiltration, centrifugation and immunomagnetic separation. Mean recovery for the sample processing method was determined to be 41% using microscopy and 49% by real-time PCR (P = 0.013). The results of this study demonstrate that real-time PCR can be an effective alternative for detecting and quantifying Cryptosporidium parvum in drinking water samples.
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Research Article|
July 01 2015
Evaluation of alternative DNA extraction processes and real-time PCR for detecting Cryptosporidium parvum in drinking water Available to Purchase
Gina H. Kimble;
1Charlotte Water, 4222 Westmont Drive, Charlotte, NC 28217, USA and Department of Civil and Environmental Engineering, University of North Carolina at Charlotte, 9201 University City Blvd, Charlotte, NC 28223, USA
E-mail: [email protected]
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Vincent R. Hill;
Vincent R. Hill
2Water, Sanitation and Hygiene Laboratory Team, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, 1600 Clifton Road NE, Atlanta, GA 30329, USA
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James E. Amburgey
James E. Amburgey
3Department of Civil and Environmental Engineering, University of North Carolina at Charlotte, 9201 University City Blvd, Charlotte, NC 28223, USA
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Water Supply (2015) 15 (6): 1295–1303.
Article history
Received:
January 08 2015
Accepted:
June 18 2015
Citation
Gina H. Kimble, Vincent R. Hill, James E. Amburgey; Evaluation of alternative DNA extraction processes and real-time PCR for detecting Cryptosporidium parvum in drinking water. Water Supply 1 December 2015; 15 (6): 1295–1303. doi: https://doi.org/10.2166/ws.2015.096
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