Inhibition of PCR by components of food samples, microbial diagnostic assays and DNA-extraction solutions

Rossen, L.; Norskov, P.; Holmstrøm, Kim; Rasmussen, Ole F.

doi:10.1016/0168-1605(92)90017-w

Cited by 710 publications

(448 citation statements)

References 9 publications

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“…Similar protocols have previously been used with good results in other studies (Zhou et al, 1996;Schabereiter-Gurtner et al, 2001). Cocoa pulp contains several potentially PCR-inhibiting compounds, such as polysaccharides, enzymes and other proteins that have to be removed during DNA extraction and purification (Pettipher, 1986;Rossen et al, 1992;Wilson, 1997). This was done by adding cetyltrimethyl ammonium bromide (CTAB) to DNA extraction buffer II.…”

Section: Development Of a Dgge Protocol For The Study Of Yeast Populamentioning

confidence: 89%

Yeast populations associated with Ghanaian cocoa fermentations analysed using denaturing gradient gel electrophoresis (DGGE)

Nielsen¹,

Hønholt²,

Tano‐Debrah

et al. 2005

Yeast

118

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The yeast populations associated with the fermentation of Ghanaian cocoa were investigated using denaturing gradient gel electrophoresis (DGGE). Samples were collected at 12-24 h intervals from heap and tray fermentations, at three different fermentation sites and different periods during the season. Eukaryotic universal primers were used to amplify a fragment of the 26S rRNA gene. The DGGE profiles were relatively complex, underlining that the fermentation of cocoa is a complex microbial process. The identities of selected fragments in the denaturing gels were revealed by sequencing. Hanseniaspora guilliermondii, Candida krusei and Pichia membranifaciens were detected from most fermentations, indicating their possible important role in the fermentation of Ghanaian cocoa. Saccharomyces cerevisiae and Candida zemplinina were almost exclusively detected during tray fermentations. The developed DGGE protocol was compared with traditional culture-based isolations. The results were comparable but slightly different, as one yeast species (C. zemplinina) was only detected using DGGE. On the other hand, Trichosporon asahii yielded only faint bands in the denaturing gels, despite the fact that it was detected using culturebased methods. Analysis of pure cultures showed that the targeted region of the 26S rRNA gene was poorly amplified in T. asahii, whereas all other investigated isolates were amplified efficiently using the chosen PCR approach. Cluster analysis revealed that the DGGE profiles clustered according to fermentation method and fermentation site. Furthermore, clustering according to progress in the fermentation was observed. The DGGE technique therefore seems to offer a relatively fast and reliable method for studying yeast population dynamics during cocoa fermentations.

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Section: Development Of a Dgge Protocol For The Study Of Yeast Populamentioning

confidence: 89%

Yeast populations associated with Ghanaian cocoa fermentations analysed using denaturing gradient gel electrophoresis (DGGE)

Nielsen¹,

Hønholt²,

Tano‐Debrah

et al. 2005

Yeast

118

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“…Moist snuff contains pasteurized tobacco, sodium chloride, sodium carbonate, and propylene glycol. Salts releasing Na + ions could affect PCR [26,31]. Glycols could provide a favorable environment for amplification [32,33], but if the concentration is too high they can inhibit the PCR [31].…”

Section: Discussionmentioning

confidence: 99%

“…Salts releasing Na + ions could affect PCR [26,31]. Glycols could provide a favorable environment for amplification [32,33], but if the concentration is too high they can inhibit the PCR [31]. Thus, ExTaq Hot Start may be better at resisting raw tobacco and PicoMaxx High Fidelity may be better at resisting its various combustion products.…”

Section: Discussionmentioning

confidence: 99%

Synergy between DNA polymerases increases polymerase chain reaction inhibitor tolerance in forensic DNA analysis

Hedman

Nordgaard

Dufva

et al. 2010

Analytical Biochemistry

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a b s t r a c tThe success rate of diagnostic polymerase chain reaction (PCR) analysis is lowered by inhibitory substances present in the samples. Recently, we showed that tolerance to PCR inhibitors in crime scene saliva stains can be improved by replacing the standard DNA polymerase AmpliTaq Gold with alternative DNA polymerase-buffer systems (Hedman et al., BioTechniques 47 (2009) 951-958). Here we show that blending inhibitor-resistant DNA polymerase-buffer systems further increases the success rate of PCR for various types of real crime scene samples showing inhibition. For 34 of 42 ''inhibited" crime scene stains, the DNA profile quality was significantly improved using a DNA polymerase blend of ExTaq Hot Start and PicoMaxx High Fidelity compared with AmpliTaq Gold. The significance of the results was confirmed by analysis of variance. The blend performed as well as, or better than, the alternative DNA polymerases used separately for all tested sample types. When used separately, the performance of the DNA polymerases varied depending on the nature of the sample. The superiority of the blend is discussed in terms of complementary effects and synergy between the DNA polymerase-buffer systems.

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“…Furthermore, the immunofluorescence test gives cross reactions with Gram-negative bacteria, including other species of Chlamydia, thus increasing the number of falsepositive cases [7]. PCR gives high specificity and sensibility [7,12,20,[21][22][23][24], however in some situations, such as blood samples with high concentrations of proteins and lipids, the sensibility of the reaction can be lowered due to inhibition of PCR [25]. In any case, a high prevalence of C. trachomatis was found in both studies.…”

Section: Discussionmentioning

confidence: 99%

<![CDATA[<b>Detection of <i>Chlamydia trachomatis</i> in endocervical smears of sexually active women in Manaus-AM, Brazil, by PCR</b>]]>

Santos¹,

Teixeira²,

Vicente³

et al. 2003

Braz J Infect Dis

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Chlamydia trachomatis is now one of the most prevalent bacteria found in classic sexually transmissible diseases (STD), and as such, constitutes a serious public health problem. We examined the prevalence of Chlamydia trachomatis, by polymerase chain reaction (PCR), in 121 sexually active women who sought treatment for STD in the Alfredo da Matta Institute of Dermatology and Venerology and the Institute of Tropical Medicine of Amazonas in Manaus, Brazil. These women were examined by a specific PCR for the chlamydial plasmid, and the nature of the amplicon was determined by restriction analysis and DNA sequencing. The PCR diagnosis revealed a prevalence of 20.7% infected women.

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Inhibition of PCR by components of food samples, microbial diagnostic assays and DNA-extraction solutions

Cited by 710 publications

References 9 publications

Yeast populations associated with Ghanaian cocoa fermentations analysed using denaturing gradient gel electrophoresis (DGGE)

Yeast populations associated with Ghanaian cocoa fermentations analysed using denaturing gradient gel electrophoresis (DGGE)

Synergy between DNA polymerases increases polymerase chain reaction inhibitor tolerance in forensic DNA analysis

<![CDATA[<b>Detection of <i>Chlamydia trachomatis</i> in endocervical smears of sexually active women in Manaus-AM, Brazil, by PCR</b>]]>

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