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A PCR Method for Rapid and Specific Detection of Freshwater Toxigenic Microcystis spp. Based on. Microcystin Synthetase C (mcyC) Gene. Jian Yuan1 ...

A PCR Method for Rapid and Specific Detection of Freshwater Toxigenic Microcystis spp. Based on Microcystin Synthetase C (mcyC) Gene Jian Yuan1, Hyun-Joong Kim2, Steve Ensley1, Baoqing Guo1, Paula Imerman1, Chris Filstrup3, Kyoung-Jin Yoon1 1. Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, Iowa. 50011. 2. Department of Food Science and Human Nutrition, College of Agriculture and Life Sciences, Iowa State University, Ames, Iowa. 50011. 3. Department of Ecology, Evolution and Organismal Biology, College of Agriculture and Life Sciences, Iowa State University, Ames, Iowa. 50011.

Introduction

The numbers of positives (Microcystis or MC related) generated from field samples are summarized as follows:

Microcystis is a cyanobacterial genus including several toxigenic freshwater species, which can form harmful algal blooms (HABs) and produce a potent hepatotoxin named microcystin (MC) that can cause skin irritation and hepatic malfunction to exposed animals and human. With the increasing reports concerning MC intoxication, the accurate and rapid detection of toxic Microcystis spp. in natural water is vital for early and timely warning for MC before blooms occur. In this study, a molecular detection method based on PCR was established.

16S rRNA gene

Microscopy

mcyC gene

LC/MS

60

56

18

17

The MC production (presence and concentration) was not correlated with the biomass of Microcystis, suggesting the inability of microscopy to discriminate toxic and non-toxic strains (Fig. 5). 10

2

y = 0.015 ·x + 0.223, R = 0.17

Fig. 2. Specificity of the 16S rRNA (top) and mcyC (bottom) gene primers for Microcystis spp. and toxigenic strains. Lane 1 - 9 represent M. aeruginosa LB 2385, M. aeruginosa LB 2388, M. aeruginosa LB 2386, Nodularia spumigena B 2091, Ocsillatoria sp. 29135, Nostoc sp. 27896, Anabaena sp. 29211, Cynlindrospermum lichenoforme 29412 and negative control.

concentration (ppb)

8

6

4

2

0

0

50

100

150

200

250

300

biomass (mg/L)

Fig. 5. Regression curve showing the lack of correlations between biomasses of Microcystis (X axis) and concentration of MCs (Y axis) in water samples from 4 farm ponds Fig. 1. Microcystin concentration in lakes tested (Courtesy of U. S. Geological Survey)

The breakdown of mcyC gene vs. MC detection in the field samples is summarized in Fig. 6.

Materials and methods • Designed specific primers for Microcystis spp. based on 16S rRNA and mcyC genes. • Established and validated regular PCR assays using known cyanobacteria strains (Table 1), assessed the limit of detection (LOD) and preliminarily evaluated their utility to environmental samples. • Periodically collected water samples (bloom and open water per pond) between June and October in 2015 from 4 farm ponds in the US Midwest (N = 60). • Tested the field samples by the PCR assays, cyanobacteria microscopy and LC/MS analysis for MC. Table 1. Cyanobacteria strains used in this study Strain names

Archive #

Source

MC toxicity

Microcystis aeruginosa

LB 2385

UTEX

Y

Microcystis aeruginosa

LB 2388

UTEX

Y

Microcystis aeruginosa

LB 2386

UTEX

N

Nodulatoria spumigena

B 2091

UTEX

Y

Ocsillatoria sp.

29135

ATCC

Y

Nostoc sp.

27896

ATCC

Y

Anabaena sp.

29211

ATCC

Y

Cynlindrospermum lichenoforme

29412

ATCC

N

Fig. 3. LODs of PCR for M. aeruginosa cells (top) and mcyC gene copies (bottom) per reaction. Lane 1-9 on the top panel represent 1.44 × 106 to 10-1 M. aeruginosa cells by 10-fold serial dilution and negative control. Lane 1-6 on the bottom panel represent 5.6 × 104 to 100 copies of mcyC gene by 10-fold serial dilution and negative control.

Results The designed primer sets for 16S rRNA and mcyC gene were demonstrated to be specific for the targeted M. aeruginosa strains (Fig. 2). Estimated LODs of PCR assay for M. aeruginosa and mcyC gene were 144 cells and 56 copies per PCR reaction, respectively (Fig. 3). The utility of the PCR method for testing sample matrix mimicking environmental water samples was successfully proven. (Fig. 4).

Fig. 4. Utility of PCR assays for environmental samples. Lane 1 - 9 represent spiked samples of M. aeruginosa LB 2385, M. aeruginosa LB 2388, M. aeruginosa LB 2386, Nodularia spumigena B 2091, Ocsillatoria sp. 29135, Nostoc sp. 27896, Anabaena sp. 29211, Cynlindrospermum lichenoforme 29412 and negative control.

Fig. 6. The detection of mcyC gene and/or microcystins in water samples from 4 farm ponds. The number in each parentheses is the number of samples under each category.

Discussion and conclusion Conventional microscopy overestimated the presence of toxigenic Microcystis spp. as it could not differentiate toxin and non-toxic strains. While MCs were detected by analytic chemistry (e.g., LC/MS), the source of MCs could not be determined as several cyanobacteria (e.g., Microcystis, Nostoc and Oscillatoria) can produce MCs. In contrast, the newly developed PCR method specifically detected the presence of toxigenic Microcystis sp. with better sensitivity than chemical analysis, demonstrating that the method can be used for rapid and accurate detection of toxinproducing Microcystis spp. in samples from ponds and lakes. The same concept could be applicable to detection of other toxigenic cyanobacteria. A great degree of PCR inhibition was observed when testing field samples. Further refinement in sample pretreatment and extraction protocol to obtain a higher yield of pure DNA may improve test sensitivity. A semi-automated sample processing and a real-time PCR should be considered when high-throughput testing is desired.

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