Comparison of Quantitative PCR and Droplet Digital PCR Multiplex Assays for Two Genera of Bloom-Forming Cyanobacteria, Cylindrospermopsis and Microcystis

• Published in: Applied and Environmental Microbiology 2015-08, Vol.81 (15), p.5203-5211
• Main Authors: Te, Shu Harn, Chen, Enid Yingru, Gin, Karina Yew-Hoong
• Format: Article
• Language: English
• Subjects: Bacteria; Bioassays; Comparative analysis; Cost-Benefit Analysis; Cyanobacteria; Cylindrospermopsis; Cylindrospermopsis - classification; Cylindrospermopsis - genetics; Cylindrospermopsis - isolation & purification; DNA Primers - genetics; Methods; Microbiological Techniques - economics; Microbiological Techniques - methods; Microbiology; Microcystis; Microcystis - classification; Microcystis - genetics; Microcystis - isolation & purification; Multiplex Polymerase Chain Reaction - economics; Multiplex Polymerase Chain Reaction - methods; Oligonucleotide Probes - genetics; Polymerase chain reaction; Public health; Real-Time Polymerase Chain Reaction - economics; Real-Time Polymerase Chain Reaction - methods; Sensitivity and Specificity; Water quality
• ISSN: 0099-2240; 1098-5336; 1098-6596
• DOI: 10.1128/aem.00931-15

The increasing occurrence of harmful cyanobacterial blooms, often linked to deteriorated water quality and adverse public health effects, has become a worldwide concern in recent decades. The use of molecular techniques such as real-time quantitative PCR (qPCR) has become increasingly popular in the detection and monitoring of harmful cyanobacterial species. Multiplex qPCR assays that quantify several toxigenic cyanobacterial species have been established previously; however, there is no molecular assay that detects several bloom-forming species simultaneously. Microcystis and Cylindrospermopsis are the two most commonly found genera and are known to be able to produce microcystin and cylindrospermopsin hepatotoxins. In this study, we designed primers and probes which enable quantification of these genera based on the RNA polymerase C1 gene for Cylindrospermopsis species and the c-phycocyanin beta subunit-like gene for Microcystis species. Duplex assays were developed for two molecular techniques-qPCR and droplet digital PCR (ddPCR). After optimization, both qPCR and ddPCR assays have high linearity and quantitative correlations for standards. Comparisons of the two techniques showed that qPCR has higher sensitivity, a wider linear dynamic range, and shorter analysis time and that it was more cost-effective, making it a suitable method for initial screening. However, the ddPCR approach has lower variability and was able to handle the PCR inhibition and competitive effects found in duplex assays, thus providing more precise and accurate analysis for bloom samples.