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United States Department of Agriculture

Agricultural Research Service

Title: Rapid Extracton of DNA from Escherichia Coli and Cryptosporidium Parvum for Use in Pcr

Authors
item Higgins, James
item Jenkins, Mark
item Shelton, Daniel
item Fayer, Ronald
item Karns, Jeffrey

Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: August 23, 2001
Publication Date: November 1, 2001
Citation: Higgins, J.A., Jenkins, M.C., Shelton, D.R., Fayer, R., Karns, J.S. 2001. Rapid extracton of dna from escherichia coli and cryptosporidium parvum for use in pcr. Applied and Environmental Microbiology. 67:5321-5324.

Interpretive Summary: Different methods of DNA extraction/ isolation from both Cryptosporidium parvum oocysts and E.coli colonies were examined. Each method was evaluated by its ability to yield quality DNA, necessary for satisfactory PCR results. The Xtra Amp tubes were found to be the easiest to use for both Cryptosporidium oocysts and E.coli cultures but the most versatile method used was the Instagene matrix.

Technical Abstract: The Xtra AmpTM tube, IsocodeTM paper, InstageneTM matrix, and PrepManTM matrix methods were evaluated for their ability to rapidly extract PCR- quality DNA from cultures and colonies of E. coli O157:H7. When assayed using conventional and real time PCR for the lac Z and eae genes, all methods provided satisfactory DNA from pure E. coli cultures and colonies, and overnight coliform cultures from water samples. However, cruder cultures from manure/broth mixes required the use of the Qiagen QIAampTM DNA Stool Mini Kit, to adequately remove PCR inhibitor(s). The Xtra Amp and Instagene methods were also useful for extracting DNA from < 200 oocysts of Cryptosporidium parvum for analysis by nested PCR for the 18S rRNA, Cp11, and Cp41 genes. The Instagene matrix, and nested 18S rRNA PCR, proved to be useful for detecting C. parvum oocysts that had been recovered from 10 L of source or finished water by continuous flow centrifugation followed by immunomagnetic separation. With the exception of the Stool kit, all of the methods are less than $2.00 per sample and 10 samples can be processed in under 45 minutes.

Last Modified: 7/22/2014
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