Nucleic Acid Detection Methods

Wiedbrauk, Danny L.; Drevon, Ann M.

doi:10.1016/b978-012748920-9/50002-9

Cited by 5 publications

(2 citation statements)

References 27 publications

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“…The PCR thermal profile consisted of an initial incubation of 3 min at 94°C; followed by 40 cycles of 15 s at 94°C, 20 s at 60°C, and 30 s at 72°C; and finally a 10-min hold at 72°C. The B. pertussis product was captured in an avidin-coated enzyme-linked immunosorbent assay plate and subsequently detected by enzymatic reaction with a probe specific to the IS481 product (34). The probe was labeled with digoxigenin.…”

Section: Methodsmentioning

confidence: 99%

Evaluation of Real-Time PCR for Detection of and Discrimination between Bordetella pertussis , Bordetella parapertussis , and Bordetella holmesii for Clinical Diagnosis

Templeton¹,

Scheltinga²,

Zee³

et al. 2003

J Clin Microbiol

109

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PCR is increasingly being used as a diagnostic test for the detection of Bordetella pertussis and Bordetella parapertussis DNA, as it has improved sensitivity and specificity in comparison to conventional techniques. The assay described here uses the two insertion sequences IS481 and IS1001 for B. pertussis and B. parapertussis, respectively, with detection by molecular beacons. The real-time PCR for IS481 detects both B. pertussis and Bordetella holmesii, and the real-time PCR for IS1001 detects both B. parapertussis and B. holmesii. By performing both assays discrimination between B. pertussis and B. parapertussis can be obtained. The sensitivity was 1 to 10 CFU/ml for B. pertussis, 10 CFU/ml for B. parapertussis, and 10 CFU/ml for B. holmesii in both assays. The clinical sensitivity of the B. pertussis assay was not affected by duplexing with an internal control PCR. Real-time PCR, conventional PCR, and culture were performed on 57 clinical samples. Eight of the 57 (14%) were found positive by culture, 19 of 57 (33%) were found positive by conventional PCR, and 22 of 57 (39%) were found positive by real-time PCR. One sample was inhibitory. When the B. pertussis assay was compared with a clinical standard for B. pertussis infection, sensitivity was 38, 83, and 100% and specificity was 100, 97, and 97% for culture, conventional PCR, and real-time PCR, respectively. The real-time PCR designed for B. pertussis and B. parapertussis provides sensitive and specific diagnosis of B. pertussis and B. parapertussis infections and is therefore suitable for implementation in the diagnostic laboratory.

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Section: Methodsmentioning

confidence: 99%

Evaluation of Real-Time PCR for Detection of and Discrimination between Bordetella pertussis , Bordetella parapertussis , and Bordetella holmesii for Clinical Diagnosis

Templeton¹,

Scheltinga²,

Zee³

et al. 2003

J Clin Microbiol

109

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“…Molecular methods such as quantitative polymerase chain reaction (qPCR) offer increasingly sensitive techniques to detect small numbers of viruses but can be inhibited or enhanced by airborne contaminants such as humic acids, metals, proteins and other free nucleic acids[1]. Methods developed to overcome inhibitor effects include increasing template concentrations[2], adding internal standards[3], diluting environmental samples[4, 5], using alternative DNA polymerases[4, 6], and adding proteins post-extraction[7]. However, due to the complexity of the contaminant mixtures and their degree of influence on microorganisms, there is no general method that can be used to eliminate their effects in all cases[1, 5, 8] and optimized protocols have to be evaluated for each application.…”

Section: Introductionmentioning

confidence: 99%

An optimized method to detect influenza virus and human rhinovirus from exhaled breath and the airborne environment

et al. 2009

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Respiratory viruses are difficult to characterize in the airborne environment due to their low concentration and the presence of a wide range of inhibitors. As a first step in studying airborne viruses, we optimized molecular biology methods to quantify influenza viruses and human rhinovirus. Quantitative PCR was used as an endpoint to evaluate RNA extraction techniques and reverse transcription protocols. We found that a Trizol-chloroform extraction and MultiScribe™ RT increased virus detection 10-fold compared to methods used in published field studies of airborne respiratory viruses. Virus was recovered without inhibition from samples contaminated with up to 50 μg/sample of particulate matter. The methods developed can be used in studies of airborne respiratory viruses.

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PCR Methods in Foods

Maurer¹

2006

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Nucleic Acid Detection Methods

Cited by 5 publications

References 27 publications

Evaluation of Real-Time PCR for Detection of and Discrimination between Bordetella pertussis , Bordetella parapertussis , and Bordetella holmesii for Clinical Diagnosis

Evaluation of Real-Time PCR for Detection of and Discrimination between Bordetella pertussis , Bordetella parapertussis , and Bordetella holmesii for Clinical Diagnosis

An optimized method to detect influenza virus and human rhinovirus from exhaled breath and the airborne environment

PCR Methods in Foods

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