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

Abstract

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. C yanobacteria, also known as blue-green algae, constitute the most notorious phylum of phytoplankton capable of forming harmful blooms in freshwater aquatic ecosystems (1). Their presence in freshwater and brackish and coastal marine waters is of particular interest because of their massive accumulation and proliferation into nuisance blooms in nutrient-enriched water. These cyanobacterial blooms are the cause for a multitude of water quality concerns, due to their potential to produce secondary metabolites, some of which are toxins and compounds that compromise taste and odor. Cyanotoxins have been linked to human and animal illness and death and pose serious health hazards to communities which use surface waters and reservoirs as potable waters (2). Hence, the detection of cyanobacteria is crucial for reliable and prudent water management. The use of efficient detection methods in routine monitoring of waters is necessary to safeguard precious water resources.Microscopic counting combined with chemical detection of cyanotoxins in water samples is the conventional method to evaluate harmful cyanobacterial blooms (3). However, morphological identification is prone to limitations such as being time-consuming and unable to distinguish between toxic and nontoxic species and prone to misinterpretation when limited morphological differences are available (4, 5). In light of these discrepancies, DNAbased detection methods are becoming increasingly popular be...

Assessment of the real-time PCR and different digital PCR platforms for DNA quantification

“…For routine diagnosis of microorganisms and viruses, qPCR has been shown to be the most cost-effective and time-effective platform, which is in agreement with other studies [ 38 , 39 ]. The minor differences in estimations of cost-effectiveness and time-effectiveness for qPCR and the QX100 system between the reports mentioned and our calculations are probably due to different prices for consumables, labour fees and indirect costs between countries, and different optimization of analysis protocols in terms of time.…”

A qPCR-Based Tool to Diagnose the Presence of Harmful Cyanobacteria and Cyanotoxins in Drinking Water Sources

“…The failure of the duplex system may be caused by differences in primer concentration, probe concentration, polymerase/dNTP/buffer concentration, annealing/extension time and amount of template DNA [71,72,73,74]. Similarly, previous studies have also shown that the amount of template DNA affects its detection in multiplexed systems [51,58,59,60], where the detection of low concentration genes was inhibited by the high concentration ones. In this study, the Ct values for the rpo C1 gene in these three uniplex-based samples were 9–10 cycles smaller than those for the pks gene, suggesting that abundance of the pks gene in the sample was much lower (Table 1).…”

Is qPCR a Reliable Indicator of Cyanotoxin Risk in Freshwater?

“…The disadvantages of multiplex qPCR are related to its laborious optimization steps. These steps are required because of the competition between primers or between targets, cross-oligo interactions and difficulties in quantifying the gene targets in complex samples containing predominantly background DNA [ 96 , 97 ]. The determination of absolute 16S rRNA gene copy numbers can also be problematic with qPCR because of the variability in the number of these genetic loci in the genomes of different cyanobacteria species.…”