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Real-time polymerase chain reaction followed by fast sequencing allows rapid genotyping of microbial pathogens
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
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2011 (English)In: Scandinavian Journal of Infectious Diseases, ISSN 0036-5548, E-ISSN 1651-1980, Vol. 43, no 2, 95-99 p.Article in journal (Refereed) Published
Abstract [en]

In this study we describe a novel protocol for rapid molecular analysis of patient samples using a combination of real-time polymerase chain reaction (PCR) and Sanger sequencing. This would normally take 2 working days in the diagnostic laboratory, but using this protocol the process can be completed within 3 h using equipment normally found in the laboratory. The innovative steps in this protocol are the sequencing of the product generated in the diagnostic real-time PCR, addition of a sequencing tail to the PCR primer, which increases the quality of the sequence without loss of sensitivity or specificity, and optimization of the hands-on and instrument steps using modern reagents. The read length of the sequencing step is routinely 250 nucleotides, which is substantially longer than existing rapid sequencing methods, increasing the chances of covering several genetic markers within 1 analysis. As proof of the concept, we used the detection and genotyping of the intestinal parasite Giardia lamblia, but the protocol can be applied to any PCR and sequence-based analysis.

Place, publisher, year, edition, pages
2011. Vol. 43, no 2, 95-99 p.
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Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-147752DOI: 10.3109/00365548.2010.524661ISI: 000286564900003PubMedID: 20950215OAI: oai:DiVA.org:uu-147752DiVA: diva2:401104
Available from: 2011-03-01 Created: 2011-02-28 Last updated: 2017-12-11Bibliographically approved

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