Direct droplet digital PCR (dddPCR) for species specific, accurate and precise quantification of bacteria in mixed samples

Here we demonstrate a direct droplet digital PCR assay (dddPCR) for simultaneous identification and quantification of bacteria in a bacterial mixture. We encapsulate bacterial cells in droplets together with PCR reagents to amplify specific target genes. Our method introduces accurate and precise qu...

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Bibliographic Details
Published inAnalytical methods Vol. 11; no. 44; pp. 573 - 5735
Main Authors Pacocha, Natalia, Scheler, Ott, Nowak, Mikolaj Marcin, Derzsi, Ladislav, Cichy, Joanna, Garstecki, Piotr
Format Journal Article
LanguageEnglish
Published Cambridge Royal Society of Chemistry 2019
Subjects
Bacteria
Deoxyribonucleic acid
DNA
Droplets
Microfluidics
Polymerase chain reaction
Purification
Quantitation
Reagents
Workload
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Summary:Here we demonstrate a direct droplet digital PCR assay (dddPCR) for simultaneous identification and quantification of bacteria in a bacterial mixture. We encapsulate bacterial cells in droplets together with PCR reagents to amplify specific target genes. Our method introduces accurate and precise quantitation of bacteria in a bacterial mixture by digital PCR, while eliminating the errors and workload associated with extraction and purification of DNA. The method thus combines the precision and specificity of digital PCR with robustness of a direct PCR. We also show that dddPCR is more tolerant to inhibition by non-specific bacteria as compared to traditional direct real-time PCR. Here we demonstrate a direct droplet digital PCR assay (dddPCR) for simultaneous identification and quantification of bacteria in a bacterial mixture.
Bibliography:10.1039/c9ay01874c
Electronic supplementary information (ESI) available: Schematics of microfluidic chips, experiments showing influence of different PCR conditions on qPCR and dddPCR, and detailed explanation about experimental conditions. See DOI
ISSN:1759-9660
1759-9679
DOI:10.1039/c9ay01874c