Rapid on-site multiplex assays for total and toxigenic Microcystis using real-time PCR with microwave cell disruption

A. Michinaka, H. K. Yen, Y. T. Chiu, H. W. Tsao, Tsair-Fuh Lin

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

A quantitative real-time polymerase chain reaction (qPCR) is a robust means by which to monitor toxin-producing cyanobacteria. However, qPCR usually requires DNA extraction, which is a time-consuming, labor-intensive pretreatment. To be able to quickly determine the potential of cyanotoxin contamination in the field, a rapid pretreatment method for DNA extraction and a portable qPCR device are needed. In this study, we applied a microwave-based method for the qPCR pretreatment and a multicolor portable qPCR with UPL and TaqMan probes to quantify toxigenic and total Microcystis. The method was tested using laboratory cultures of toxigenic Microcystis aeruginosa PCC7820. The qPCR results showed the cycle thresholds value (Ct value) correlated well with cell numbers, with detection limit at about 1,000 cells/ml. This scheme was applied in 22 environmental samples from six drinking water reservoirs (DWRs) in Taiwan. Although the results for qPCR were about four times higher than those of microscopic observation, good correlation between qPCR and microscope methods were obtained (r-square: 0.79, P < 0.01). The ratios of toxigenic Microcystis to total Microcystis in two reservoirs, Sin-Shan Reservoir and Shih-men Reservoir, were less than 10%. In three other reservoirs, Ren-Yi-Tan Reservoir, Nan-Hua Reservoir and Bao-Shan Reservoir, much higher (>46.1%) ratios were obtained. The scheme may assist quick assessment of the risk associated with toxic cyanobacteria in DWRs.

Original languageEnglish
Pages (from-to)1247-1252
Number of pages6
JournalWater Science and Technology
Volume66
Issue number6
DOIs
Publication statusPublished - 2012 Oct 12

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Polymerase chain reaction
polymerase chain reaction
Assays
Microwaves
assay
Potable water
cyanobacterium
DNA
drinking water
microwave
toxin
Microscopes
Contamination
labor
probe
Personnel
method

All Science Journal Classification (ASJC) codes

  • Environmental Engineering
  • Water Science and Technology

Cite this

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title = "Rapid on-site multiplex assays for total and toxigenic Microcystis using real-time PCR with microwave cell disruption",
abstract = "A quantitative real-time polymerase chain reaction (qPCR) is a robust means by which to monitor toxin-producing cyanobacteria. However, qPCR usually requires DNA extraction, which is a time-consuming, labor-intensive pretreatment. To be able to quickly determine the potential of cyanotoxin contamination in the field, a rapid pretreatment method for DNA extraction and a portable qPCR device are needed. In this study, we applied a microwave-based method for the qPCR pretreatment and a multicolor portable qPCR with UPL and TaqMan probes to quantify toxigenic and total Microcystis. The method was tested using laboratory cultures of toxigenic Microcystis aeruginosa PCC7820. The qPCR results showed the cycle thresholds value (Ct value) correlated well with cell numbers, with detection limit at about 1,000 cells/ml. This scheme was applied in 22 environmental samples from six drinking water reservoirs (DWRs) in Taiwan. Although the results for qPCR were about four times higher than those of microscopic observation, good correlation between qPCR and microscope methods were obtained (r-square: 0.79, P < 0.01). The ratios of toxigenic Microcystis to total Microcystis in two reservoirs, Sin-Shan Reservoir and Shih-men Reservoir, were less than 10{\%}. In three other reservoirs, Ren-Yi-Tan Reservoir, Nan-Hua Reservoir and Bao-Shan Reservoir, much higher (>46.1{\%}) ratios were obtained. The scheme may assist quick assessment of the risk associated with toxic cyanobacteria in DWRs.",
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Rapid on-site multiplex assays for total and toxigenic Microcystis using real-time PCR with microwave cell disruption. / Michinaka, A.; Yen, H. K.; Chiu, Y. T.; Tsao, H. W.; Lin, Tsair-Fuh.

In: Water Science and Technology, Vol. 66, No. 6, 12.10.2012, p. 1247-1252.

Research output: Contribution to journalArticle

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T1 - Rapid on-site multiplex assays for total and toxigenic Microcystis using real-time PCR with microwave cell disruption

AU - Michinaka, A.

AU - Yen, H. K.

AU - Chiu, Y. T.

AU - Tsao, H. W.

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