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Title:   Author  item
                                            Higgins, Jamesitem
                                            Jenkins, Markitem
                                            Shelton, Danielitem
                                            Fayer, Ronalditem
                                            Karns, Jeffrey
  Submitted to: Applied and Environmental Microbiology
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 8/23/2001
                                    Publication  Date: 11/1/2001
                                    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.


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