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Design of a single‐tube, endpoint, linear‐after‐the‐exponential‐ PCR assay for 17 pathogens associated with sepsis
Author(s) -
Rice L.M.,
Reis A.H.,
Ronish B.,
CarverBrown R.K.,
Czajka J.W.,
Gentile N.,
Kost G.,
Wangh L.J.
Publication year - 2013
Publication title -
journal of applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 1364-5072
DOI - 10.1111/jam.12061
Subject(s) - sepsis , microbiology and biotechnology , biology , exponential function , computational biology , immunology , mathematics , mathematical analysis
Aims The goal of this study was to construct a single‐tube multiplex molecular diagnostic assay using linear‐after‐the‐exponential ( LATE )‐ PCR for the detection of 17 microbial pathogens commonly associated with septicaemia. Methods and Results The assay described here detects 17 pathogens associated with sepsis via amplification and analysis of gene‐specific sequences. The pathogens and their targeted genes were: K lebsiella spp. ( pho E); A cinetobacter baumannii ( gyr B); S taphylococcus aureus ( spa ); E nterobacter spp. ( thd F); P seudomonas aeruginosa ( tox A); coagulase‐negative staphylococci ( tuf ), E nterococcus spp. ( tuf ); C andida spp. (P450). A sequence from an unidentified gene in L actococcus lactis , served as a positive control for assay function. LATE ‐ PCR was used to generate single‐stranded amplicons that were analysed at endpoint over a wide range of temperatures in four fluorescent colours. Each target was detected by its pattern of hybridization to a sequence‐specific low‐temperature fluorescent probe derived from molecular beacons. Conclusions All 17 microbial targets were detected in samples containing low numbers of pathogen genomes in the presence of high levels of human genomic DNA . Significance and Impact of the Study This assay used new technology to achieve an advance in the field of molecular diagnostics: a single‐tube assay for detection of pathogens commonly responsible for septicaemia.