The construction and use of a PCR internal control

Sachadyn, Paweł; Kur, J

doi:10.1006/mcpr.1998.0170

Cited by 53 publications

(51 citation statements)

References 5 publications

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“…Primers for the construction of the IPC contained the autolysin primer sequences external to sequences specific for a 264 bp portion of bacteriophage lambda DNA (Table 3). This gave a 302 bp fragment that could be amplified by the lytA primers, a similar construct to that used in previous studies (Povlsen et al, 1998;Sachadyn & Kur, 1998). The IPC fragment was cloned into plasmids using the TOPO TA cloning kit (Invitrogen/Life Technologies), which were then linearized by restriction with XbaI (Roche Diagnostics) and subsequently spiked into the master mix of all LightCycler reactions at a level of approximately 22 copies per reaction.…”

Section: Methodsmentioning

confidence: 99%

Autolysin-targeted LightCycler assay including internal process control for detection of Streptococcus pneumoniae DNA in clinical samples

Sheppard

Harrison

Morris

et al. 2004

Journal of Medical Microbiology

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The development and clinical evaluation of a LightCycler PCR assay, including an internal process control (IPC), to detect the Streptococcus pneumoniae autolysin gene in clinical samples is reported. The assay was developed to provide a second target for use in conjunction with existing pneumolysin PCR assays to increase the reliability of non-culture PCR diagnosis of pneumococcal infection. Primers amplify a 173 bp fragment of the autolysin gene (lytA), which is detected by fluorescence-labelled hybridization probes. An IPC was designed to check for the presence of PCR inhibitors and loss of assay sensitivity. The IPC product was amplified by the lytA primers and detected by a second set of hybridization probes. The analytical specificity of the autolysin PCR assay was 100 % against 39 other bacterial species tested; these included related streptococci and other organisms. The assay, which could reliably detect 50 fg purified pneumococcal DNA per reaction, was capable of distinguishing between S. pneumoniae and atypical Streptococcus mitis and Streptococcus oralis strains known to contain the lytA gene. Using DNA extracts from a panel of EDTA bloods from patients with blood-culture-confirmed pneumococcal infection, the autolysin PCR had a sensitivity of 42 . 9 %, which was similar to a previously reported TaqMan pneumolysin PCR (43 . 8 %) run in parallel. Total agreement was shown between the autolysin assay and the pneumolysin TaqMan assay when used to test 23 culture-negative clinical samples, of which eight were positive by PCR, adding valuable clinical information. A specific autolysin-based LightCycler assay has been developed to complement pneumolysin PCR for the detection of S. pneumoniae in clinical samples. This should be a particularly useful tool for the rapid and sensitive diagnosis of pneumococcal meningitis, even after an antibiotic has been administered. However, poor sensitivity on blood samples limits its usefulness in other bacteraemic infections.

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Section: Methodsmentioning

confidence: 99%

Autolysin-targeted LightCycler assay including internal process control for detection of Streptococcus pneumoniae DNA in clinical samples

Sheppard

Harrison

Morris

et al. 2004

Journal of Medical Microbiology

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Section: Introductionmentioning

confidence: 98%

“…In addition, with an increased reliance on PCR-based diagnostic tests for pathogenic microorganisms, internal standards are useful to obtain reliable diagnosis and to avoid false negative results (Brightwell et al 1998). MPCR is ideally suited for incorporation of an internal standard (Sachadyn & Kur 1998); successful amplification of the internal standard confirms that the quality of DNA isolated from a sample will support amplification and that no inhibitors or technical failures were encountered.The aim of this study was to modify an existing MPCR to include an internal standard for DNA quality control and to apply this modified MPCR in a large-scale field trial to assess its utility versus the conventional diagnostic techniques of histological examination and the RMFT assay. …”

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confidence: 98%

Application of a multiplex PCR for the detection of protozoan pathogens of the eastern oyster Crassostrea virginica in field samples

Russell¹,

Frasca²,

Sunila³

et al. 2004

Dis. Aquat. Org.

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Populations of eastern oysters Crassostrea virginica along the east coast of North America have repeatedly experienced epizootic mass mortality due to infections by protozoan parasites, and molecular diagnostic methodologies are fast becoming more widely available for the diagnosis of protozoan diseases of oysters. In this study we applied a modified version of an existing multiplex polymerase chain reaction (PCR) for detection of the eastern oyster parasites Haplosporidium nelsoni, H. costale and Perkinsus marinus from field-collected samples. We incorporated primers for DNA quality control based on the large subunit ribosomal RNA (LSU rRNA) gene of C. virginica. The multiplex PCR (MPCR) simultaneously amplified genomic DNA of C. virginica, and cloned DNA of H. nelsoni, P. marinus and H. costale. In field trial applications, we compared the performance of the MPCR to that of the conventional diagnostic techniques of histopathological tissue examination and the Ray/Mackin fluid thioglycollate medium (RMFT) assay. A total of 530 oysters were sampled from 18 sites at 12 locations along the east coast of the United States from the Gulf of Mexico to southern New England. The modified MPCR detected 21% oysters with H. nelsoni, 2% oysters with H. costale, and 40% oysters with P. marinus infections. In comparison, histopathological examination detected H. nelsoni and H. costale infections in 6 and 0.8% oysters, respectively, and the RMFT assay detected P. marinus infection in 31% oysters. The MPCR is a more sensitive diagnostic assay for detection of H. nelsoni, H. costale, and P. marinus, and incorporation of an oyster quality control product limits false negative results. KEY WORDS: Diagnostics · Eastern oyster · Haplosporidium costale · Haplosporidium nelsoni · Multiplex PCR · Perkinsus marinus · Quality control Resale or republication not permitted without written consent of the publisherDis Aquat Org 59: [85][86][87][88][89][90][91] 2004 These methods are time-consuming and expensive, and histomorphological similarities between plasmodia of H. nelsoni and H. costale make diagnosis of mixed infections in the absence of species-defining spores difficult (Andrews & Castagna 1978). In the case of Perkinsus marinus, the Ray/Mackin fluid thioglycollate (RMFT) assay is useful (Ray 1952(Ray , 1966. However, the assay fails to differentiate between the many different types of Perkinsus species found to infect mollusks worldwide (Robledo et al. 2000). It has been shown that 2 Perkinsus species may simultaneously affect the same individual host (Kotob et al. 1999, Coss et al. 2001. In addition, the RMFT assay has reportedly failed to detect infections below 1000 P. marinus parasites per gram wet tissue (Bushek et al. 1994).Polymerase chain reaction (PCR) and DNA probe methodologies have been developed and used successfully for rapid detection of Haplosporidium nelsoni , Stokes & Burreson 2001, H. costale (Ko et al. 1995, and Perkinsus marinus (Marsh et al. 1995, Robledo et al. 1998. PCR is ideal for specificall...

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“…For a size-dependent discrimination by gel electrophoresis of target and IAC nucleic acids, chimerical IACs may be produced by deleting, or inserting sequences between the recognition primer sites (Abdulmawjood et al, 2002;Cubero et al, 2002;Müller et al, 1998;Sachadyn and Kur, 1998). A smaller size IAC can be constructed by deleting an internal sequence fragment.…”

Section: Construction Of An Iacmentioning

confidence: 99%

Molecular methodology in Food Microbiology diagnostics: trends and current challenges

Rodrı́guez-Lázaro

Hernández

2006

13th World Congress of Food Science &Amp; Technology

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Foodborne diseases are among the most serious public health concerns worldwide. Consequently, the application of microbiological controls within the quality assessment programs in the food industry is a premise to minimize the risk of infection for the consumer. Classical microbiological methods involve, in general, the use of appropriate pre-enrichment and enrichment, isolation on selective media, and subsequent confirmation using morphological, biochemical and/or serological tests. They are laborious, time consuming and not always reliable. A number of alternative, rapid and sensitive molecular methods for the detection, identification and quantification of foodborne pathogens have been developed to overcome these drawbacks. However, until now, they have shown low practical implementation for monitoring and microbiological control. Amplification techniques have a number of advantages including improved speed, excellent detection limit, specificity, sensitivity and potential for quantification as well as easy automation. Major drawbacks are the cost of the analysis and the qualified personnel needed to carry out the experiments, plus the lack of standardisation of protocols approved by normalisation bodies. Moreover, the cost of reagents is decreasing and more qualified people are available to perform this kind of analysis. Efforts on standardisation and normalisation of these techniques are a key priority.The molecular methods, in conclusion, are a promising alternative that can substitute or complement the current reference methods in food microbiology diagnostics. More suitable and reliable results can be achieved in terms of speed and precision, and can detect and enumerate specifically viable microorganisms.

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The construction and use of a PCR internal control

Cited by 53 publications

References 5 publications

Autolysin-targeted LightCycler assay including internal process control for detection of Streptococcus pneumoniae DNA in clinical samples

Autolysin-targeted LightCycler assay including internal process control for detection of Streptococcus pneumoniae DNA in clinical samples

Application of a multiplex PCR for the detection of protozoan pathogens of the eastern oyster Crassostrea virginica in field samples

Molecular methodology in Food Microbiology diagnostics: trends and current challenges

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