Inhibitory Effects of Collagen on the PCR for Detection of
            <i>Clostridium perfringens</i>

Kim, Sangburm; Labbé, Ronald G.; Ryu, Sangryeol

doi:10.1128/aem.66.3.1213-1215.2000

Cited by 41 publications

(23 citation statements)

References 15 publications

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“…Testing the various polymerases in the presence of the other defined inhibitors provided additional insight into the mechanisms through which they decrease PCR efficiency. Inhibition by type I collagen has been reported with food samples (20) and skeletal remains (36), although the mechanism of inhibition is undetermined. Kim et al.…”

Section: Discussionmentioning

confidence: 99%

Polymerase Resistance to Polymerase Chain Reaction Inhibitors in Bone*

Eilert

Foran

2009

Journal of Forensic Sciences

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Amplification of DNA from aged or degraded skeletal remains can be a challenging task, in part due to naturally occurring inhibitors of the polymerase chain reaction. PCR inhibitors may act by inactivating a polymerase itself, or compete with or bind other reaction components, although various polymerases may be differentially susceptible to such insult. In this study, ten thermostable polymerases from six bacterial species were examined for their ability to amplify DNA in the presence of bone-derived or individual PCR inhibitors. Two polymerases, one from Thermus aquaticus and one from Thermus thermophilus, showed lower susceptibility to inhibition from bone, while polymerases from Thermus flavus were highly susceptible. Addition of bovine serum albumin improved the activity of most of the enzymes. Taken together, the results indicate that thermostable DNA polymerases have different susceptibility to bone-derived PCR inhibitors, and that those most often used in forensic laboratories may not be optimal when working with DNA from skeletal remains.

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Section: Discussionmentioning

confidence: 99%

Polymerase Resistance to Polymerase Chain Reaction Inhibitors in Bone*

Eilert

Foran

2009

Journal of Forensic Sciences

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“…Generally, current molecular methods designed for the detection of common pathogens in foods include an enrichment step that serves two purposes: to dilute out the interfering substances in the food matrix and to provide some assurance that the DNA sequences being detected are viable cells (12). In addition to these purposes, an enrichment culture step for the detection of C. perfringens strains induces the production of several enzymes that might reduce known inhibitors (e.g., collagen molecules) of PCR (5). An enrichment culture step also enables molecular assays to detect spores (16).…”

Section: Discussionmentioning

confidence: 99%

Detection of Enterotoxigenic Clostridium perfringens in Meat Samples by Using Molecular Methods

Kaneko

Miyamoto

Mimura

et al. 2011

Appl Environ Microbiol

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To prevent food-borne bacterial diseases and to trace bacterial contamination events to foods, microbial source tracking (MST) methods provide important epidemiological information. To apply molecular methods to MST, it is necessary not only to amplify bacterial cells to detection limit levels but also to prepare DNA with reduced inhibitory compounds and contamination. Isolates carrying the Clostridium perfringens enterotoxin gene (cpe) on the chromosome or a plasmid rank among the most important food-borne pathogens. Previous surveys indicated that cpe-positive C. perfringens isolates are present in only ϳ5% of nonoutbreak food samples and then only at low numbers, usually less than 3 cells/g. In this study, four molecular assays for the detection of cpe-positive C. perfringens isolates, i.e., ordinary PCR, nested PCR, real-time PCR, and loop-mediated isothermal amplification (LAMP), were developed and evaluated for their reliability using purified DNA. For use in the artificial contamination of meat samples, DNA templates were prepared by three different commercial DNA preparation kits. The four molecular assays always detected cpe when >10 3 cells/g of cpe-positive C. perfringens were present, using any kit. Of three tested commercial DNA preparation kits, the InstaGene matrix kit appeared to be most suitable for the testing of a large number of samples. By using the InstaGene matrix kit, the four molecular assays efficiently detected cpe using DNA prepared from enrichment culture specimens of meat samples contaminated with low numbers of cpe-positive C. perfringens vegetative cells or spores. Overall, the current study developed molecular assay protocols for MST to detect the contamination of foods with low numbers of cells, and at a low frequency, of cpe-positive C. perfringens isolates.Clostridium perfringens is an important pathogen of human gastrointestinal (GI) tract diseases such as food poisoning, antibiotic-associated diarrhea, and sporadic diarrhea as well as nosocomial diarrheal disease outbreaks (6, 10). The most important toxin made by this bacterium when it causes human GI tract diseases is Clostridium perfringens enterotoxin (CPE) (10,14). Although C. perfringens is a ubiquitous bacterium in the environment, only a small subpopulation of this bacterium, usually less than 5%, harbors the CPE gene (cpe) (10). Probably because of this rarity, previous surveys identifying cpepositive C. perfringens isolates in food, human feces, and the environment have reported various results (1,4,8,9,11,16). Moreover, the number of contaminated C. perfringens cells (cpe-positive and cpe-negative strains) present in most nonoutbreak food samples has been reported to be fewer than 3 using the most-probable-number method (9, 11, 16). Therefore, to prevent food poisoning and nosocomial outbreaks, a method able to detect cpe-positive strains with high sensitivity and applicability for the testing of a large number of samples is necessary for use in epidemiological surveys.Microbial source tracking (MST) methods allow...

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“…However, P. abyssi pol D exhibited higher sensitivity to humic acid and urea than Taq DNA pol. In a separate study, Vent™ DNA pol showed a high tolerance against blood components, but amplification by Pwo DNA pol was inhibited by blood components (Al-Soud and Radstrom 2001) and collagen (Kim et al 2000).…”

Section: Pcr In the Presence Of Inhibitorsmentioning

confidence: 95%

Archaeal DNA polymerases in biotechnology

Zhang

Kang

et al. 2015

Appl Microbiol Biotechnol

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DNA polymerase (pol) is a ubiquitous enzyme that synthesizes DNA strands in all living cells. In vitro, DNA pol is used for DNA manipulation, including cloning, PCR, site-directed mutagenesis, sequencing, and several other applications. Family B archaeal DNA pols have been widely used for molecular biological methods. Biochemical and structural studies reveal that each archaeal DNA pol has different characteristics with respect to fidelity, processivity and thermostability. Due to their high fidelity and strong thermostability, family B archaeal DNA pols have the extensive application on high-fidelity PCR, DNA sequencing, and site-directed mutagenesis while family Y archaeal DNA pols have the potential for error-prone PCR and random mutagenesis because of their low fidelity and strong thermostability. This information combined with mutational analysis has been used to construct novel DNA pols with altered properties that enhance their use as biotechnological reagents. In this review, we focus on the development and use of family B archaeal DNA pols.

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Inhibitory Effects of Collagen on the PCR for Detection of Clostridium perfringens

Cited by 41 publications

References 15 publications

Polymerase Resistance to Polymerase Chain Reaction Inhibitors in Bone*

Polymerase Resistance to Polymerase Chain Reaction Inhibitors in Bone*

Detection of Enterotoxigenic Clostridium perfringens in Meat Samples by Using Molecular Methods

Archaeal DNA polymerases in biotechnology

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