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Standardization of qPCR and RT-qPCR detection of microcystin producers for early warning of microcystin production
Citation:
Sienkiewicz, N., I. Struewing, AND J. Lu. Standardization of qPCR and RT-qPCR detection of microcystin producers for early warning of microcystin production. 12th International Conference on Toxic Cyanobacteria, Toledo, OH, May 22 - 27, 2022.
Impact/Purpose:
1) Public concern over cyanobacterial blooms has increased due to their higher frequency of occurrences and their potential ecological and health impacts. 2) To form a more robust detection system with high sensitivity and accuracy based on highly specific assays, there are some conditions and challenges with PCR methods that need to be defined and validated. 3) This information can be used to aid in toxin measurements and aid in making management decisions related to harmful algal blooms. 4) OW, regional offices, scientists and water quality managers could be interested in the results.
Description:
The frequent occurrence of cyanobacterial harmful algal blooms (CyanoHAB) has become a significant threat to human and ecological health by their toxin products. Microcystin is also the most common cyanotoxin produced, with Microcystis and Planktothrix being the major toxin producing cyanobacterial genera during CyanoHABs. Previously, we have demonstrated that qPCR and RT-qPCR can be used as an early warning detection system for microcystin production in fresh surface waters. However, to form a more robust detection system with high sensitivity and accuracy based on highly specific assays, there are some conditions and challenges with PCR methods that need to be defined and validated. The qPCR and RT-qPCR templates derived from the sample matrices can range from a low level (undetectable) to an extremely high level (up to 1010 L-1) of targets and biomass and thus can increase inhibition and decrease detection limit at one of or both the RT and PCR steps of the assays. To create a more robust qPCR-based early warning system, two commonly used quantitative PCR systems (hydrolysis probe-based TaqMan qPCR and fluorescent dye-based SYBR Green qPCR) were evaluated for their specificity, sensitivity, efficiency, and accuracy. Three potential inhibitory scenarios for these PCR assays and corresponding inhibitory relief strategies were evaluated for their effectiveness. The resulting qPCR procedures were then further evaluated to monitor the occurrences of microcystin producers during the early bloom stages of HABs.
URLs/Downloads:
https://www.bgsu.edu/bowen-thompson-student-union/conference-and-event-services/international-conference-on-toxic-cyanobacteria.html