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United States Department of Agriculture

Agricultural Research Service

Research Project: MICROBIAL ECOLOGY AND SAFETY OF FRESH ON-FARM ORGANICALLY GROWN PRODUCE Title: PMA treatment is an effective means to reduce false positive PCR testing results

Authors
item Farahi, Farnoosh
item Nou, Xiangwu

Submitted to: BARC Poster Day
Publication Type: Abstract Only
Publication Acceptance Date: March 16, 2009
Publication Date: April 15, 2009
Citation: Farahi, F., Nou, X. 2009. PMA treatment is an effective means to reduce false positive PCR testing results. BARC Poster Day. 4/15/2009.

Technical Abstract: Traditional and real time PCR are widely used in detecting bacterial pathogens in various food matrix and environmental samples. Sometimes a positive detection using PCR can not be confirmed by subsequent culture isolation of the targeted pathogen, resulting in a potential “false positive.” False positive testing results can be caused by several factors, including PCR specificity and the sensitivity of the culture isolation for confirmation. A main cause of false positive PCR testing results is the presence of DNA from killed target cells or DNA from non-cellular sources, such as bacterial phages in the case of Escherichia coli O157:H7 testing. Propidium monoazide (PMA) is a light activated DNA crosslinker that can penetrate the cytoplasmic membrane of damaged cells but not that of healthy cells. It has been used to distinguish living cells from killed cells. The use of PMA to reduce potential false positive testing results is evaluated. E. coli O157:H7 cells were inactivated using heat, ethanol, or chlorine. Bacterial chromosomal DNA was isolated with and without PMA treatment, and traditional PCR was used to amplify target genes. PMA treatment had no effect on the purification and subsequent PCR for healthy bacterial cells but it prevented DNA isolation from thermally or chemically inactivated bacterial cells. When healthy and killed cells or previously isolated DNA were used to inoculate soil or fresh produce, only the target gene for the healthy cells can be detected by traditional PCR. These data suggest that PMA treatment is an effective means to eliminate false positive PCR testing results caused by the presence of target DNA from dead cells or non-cellular sources.

Last Modified: 9/2/2014
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