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Verification of monoplex and multiplex linear‐after‐the‐exponential PCR gene‐specific sepsis assays using clinical isolates
Author(s) -
Gentile N.L.,
Dillier A.M.,
Williams G.V.,
Ackers J.,
Reis A.H.,
Rice L.M.,
Wangh L.J.,
Czajka J.W.,
Kost G.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.12062
Subject(s) - library science , clinical microbiology , multiplex , medical laboratory , medicine , biology , bioinformatics , pathology , computer science , microbiology and biotechnology
Aims To verify monoplex and multiplex gene‐specific linear‐after‐the‐exponential polymerase chain reaction ( LATE ‐ PCR ) assays for identifying 17 microbial pathogens (i.e., K lebsiella sp., A cinetobacter baumannii , S taphylococcus aureus , E nterobacter sp., P seudomonas aeruginosa , coagulase negative staphylococci, E nterococcus sp., C andida sp.) commonly associated with septicaemia using clinical isolates. Methods and Results Clinical isolates of each target pathogen were collected from the U niversity of C alifornia, D avis M edical C enter ( UCDMC ) microbiology laboratory. Five microlitres (μl) of each culture suspension (1 × 10 8 CFU ml −1 ) were added to 20 μl of monoplex mastermix. DNA extracted from clinical isolates was tested in multiplex. Monoplex assays demonstrated 100% sensitivity at this input level, except E nterobacter cloacae (2·7%), A c. baumannii (57%) and P s. aeruginosa (97·8%). All clinical isolates were positive in multiplex, with the exception of two A c. baumannii , two K lebsiella oxytoca and two C andida parapsilosis isolates. Conclusions Sixteen pathogens can be identified by monoplex LATE ‐ PCR assays with sensitivities ≥97·8%. The multiplex assay demonstrated 91·4% sensitivity when tested with DNA extracted from 70 different target strains. Significance and Impact of the Study This study demonstrates the potential of LATE ‐ PCR to serve as an adjunct to culture if the reagents are optimized for sensitivity. Results warrant further testing through analytical and clinical validation of the multiplex assay.