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High frequency of false-positive signals in a real-time PCR-based "Plus/Minus" assay.

Artikel i vetenskaplig tidskrift
Författare Forough Nowrouzian
Ingegerd Adlerberth
Agnes E Wold
Publicerad i APMIS : acta pathologica, microbiologica, et immunologica Scandinavica
Volym 117
Nummer/häfte 1
Sidor 68-72
ISSN 1600-0463
Publiceringsår 2009
Publicerad vid Institutionen för biomedicin, avdelningen för infektionssjukdomar
Sidor 68-72
Språk en
Länkar dx.doi.org/10.1111/j.1600-0463.2008...
Ämnesord DNA Primers, DNA, Bacterial, analysis, DNA, Viral, analysis, Escherichia coli Proteins, genetics, False Positive Reactions, Fluorescent Dyes, Genes, Bacterial, Humans, Infant, Organic Chemicals, Polymerase Chain Reaction, methods
Ämneskategorier Mikrobiologi inom det medicinska området

Sammanfattning

Molecular biological methods using real-time polymerase chain reaction (RT-PCR) for detection of bacterial and viral genes in different environments have been developed into assays from different commercial sources. Applied Biosystems include and support two applications with their TaqMan instrument: the "Plus/Minus" and the "Allelic Discrimination" assays. These approaches are RT-PCR based, use short primers and fluorescent-labeled TaqMan probes and include three processes: a pre-read run, a PCR-amplification run, and a post-read run. In the "Plus/Minus" assay, samples and controls (distilled water) are loaded into the instrument, which calculates a positive or a negative outcome based on differences in signals between samples and the controls. When testing the "Plus/Minus" assay for detection of usp genes encoding a uropathogenic specific protein in Escherichia coli, an inordinately high proportion of false-positive signals was observed. This was shown to be due to a serious methodological deficiency. Our observations indicate that an adequate no-template control closely matching the target samples in all aspects, including amount of DNA, is required to establish a correct threshold in the pre-read run that forms the basis for further calculations in the post-read run of the "Plus/Minus" assay.

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