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Development and standardisation of the PhytoxigeneTM

CyanoDTec Test: A rapid molecular assay for the routine monitoring and differentiation of toxin producing 

cyanobacterial blooms

Authors: Mark Van Asten1,2, Jason N Woodhouse2, Leonardo Pinheiro3 , Kerry R Emslie3 and Brett A Neilan2

1 Diagnostic Technology Pty Ltd2 School of Biotechnology and Biomolecular Sciences, UNSW Australia

3 National Measurement Institute (NMI), Australia

2015 Water Microbiology Conference: May 18‐22, Chapel Hill, NC

Toxin Species Distribution and challenge 

• Cyanobacteria can be readily differentiated into three phylogenetically distinct orders

– Nostocales

– Oscillatorales

– Chroococales

• For each of the major cyanobacterial toxins, biosynthetic pathways can be found in representatives from each order

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Specialised Metabolite Biosynthesis

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76

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Module 1(PKS)

Module 2(PKS)

Module 3(PKS)

Module 4(PKS)

Module 5(NRPS)

Module 6(NRPS)

Module 7(NRPS)

+ substrate 3

+ substrate 2

+ substrate 4

+ substrate 5

+ substrate 6

+ substrate 7

cyclisation

cyclised compound

Substrate: amino acidsSubstrate: carboxyacyl‐CoA

Non‐Ribosomal Peptide Synthetase (NRPS)Polyketide synthase (PKS)

Enzyme: protein that acts as a biological catalystModule: set of catalytic centres that facilitates a set of unique reactionsDomain: individual catalytic centre that facilitates a distinct reaction 

Discovery of CyanotoxinGene Clusters

• Microcystin• Tillett et al, 2000

• Nodularin• Moffitt and Neilan, 2004

• Cylindrospermopsin• Mihali et al, 2008

• Saxitoxin• Kellmann et al, 2008

Neilan Laboratory of Microbial & Molecular Diversity

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Microcystin Biosynthesis

Microcystin biosynthesis

• Microcystins are a class of cyclic hepapetidesfeaturing the Adda (PKS) side chain– Variations can be introduced by presence/absence of tailoring enzymes (ie mcyL)

– Variations can also be introduced by the relaxed specificity of the mcyB1 (Leu) and mcyC (Arg) adenylation domains 

• Microcystin‐LR (Leu/Arg)

• Microcystin‐RR (Arg/Arg)

• Microcystin‐YR (Tyr/Arg)

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~ 4.5 kb

mcyH I F E J D G A B C atn1

Microcystis aeruginosa PCC7806

dnaN G F E D A B C uma1-6mcy J-H

Anabaena sp. 90

Planktothrix agardhii CYA126

mcyT D E G H A B C J ORF1

Nodularia spumigena NSOR10

ORF6-8 I H G F E D C A B ORF1-5

KEY:Nonribosomal peptide synthetase(NRPS) genes

Polyketide synthase (PKS) genes

Tailoring genes

Putative transposases

flanking genes

microcystin gene clusters

Species Strain Hepatoxic Hepatoxin Other Cyanotoxins OriginChroococcalesMicrocystis aeruginosa PCC7806 + microcystin - The NetherlandsMicrocystis aeruginosa PCC7005 + microcystin - ScotlandMicrocystis sp. UTEX 2667 + microcystin - United StatesMicrocystis sp. UTEX 2664 + microcystin - United StatesMicrocystis viridis NIES 102 + microcystin - JapanMicrocystis wesenbergii NIES 107 + microcystin - JapanMicrocystis aeruginosa HUB53 - - - GermanyMicrocystis aeruginosa UWOCC C1 - - - United StatesMicrocystis aeruginosa UWOCC C4 - - United StatesMicrocystis aeruginosa UWOCC P3 - - United StatesMicrocystis sp. UWOCC Bauld B - - - AustraliaSynechocystis sp. PCC6803 - - - United States

NostocalesAnabaena sp. strain 202A2 + microcystin - FinlandAnabaena circinalis NIES 19 - - saxitoxin EnglandCylindrospermopsis sp. strain T3 - - cylindropermopsis AustraliaNodularia harveyana PCC7804 + nodularin FranceNodularia harveyana PCC73104 - - - CanadaNodularia spumigena NROS 10 + nodularia - FinlandNodularia spumigena. BY1 + nodularia - Baltic SeaNodularia spumigena. HEM + nodularia - Baltic SeaNostoc sp. strain 152 + microcystin - JapanNostoc sp. PCC7120 - - - United StatesNostoc sp. PCC73120 - - - Australia

OscillatorialesLyngbya sp. BAN 01 - - n/a AustraliaOscillatoria sp. strain18R + microcystin - FinlandOscillatoria sp. strain 195 + microcystin - FinlandPhormidium tenue - - - n/a JapanPhormidium sp. isolate 2-26b + microcystin n/a United StatesPhormidium sp. isolate 1-6c + microcystin n/a United StatesPhormidium sp. isolate 4-19b + microcystin n/a United States

Detection of cyclic peptide hepatotoxins in three orders of cyanobacteria (Chroococcales, Nostocales, Oscillatoriales)

AMT gene of the microcystin and nodularin synthetases (mcyE, ndaF)

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180000 2000 4000 6000 8000 10000 12000 14000 16000

sxtAsxtB sxtD sxtE sxtF sxtG sxtH sxtIJ sxtK sxtL sxtM sxtNsxtC sxtN

Saxitoxin production in Cynobacteria

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Cylindrospermopsis

Anabaena

From gene to toxin...microcystin

HOMe

HNH

O

H

Me

N

O

H N

MeCOOHH

O CH2

NH

O

NH

MeH

H

HN

OCOOHH

MeH

O

HN

NH

NH2HN

H

O

Me

Me

GK DHIJ

Me H Me

CF B1 A1GP EP A2 B2EK

1 23

4

5

6

7

PK1

PK2-4

T T T

The Phytoxigene test is based on simple paradigm; Like any biological product, toxins have a genetic pathway, if the gene is present then toxin can be produced

Conversely if gene not present, no toxin can be produced

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Challenges of cyanobacteria monitoring include:Identification of cyanobacteria, is it toxic or non‐toxic? 

Is there a potential to become Toxic?

Warragamba Dam August 2007

Understanding the biological process for toxin production and the  gene sequences responsible enables the 

development of standardised molecular tests for the; 

• Detection of toxin producing cyanobacteria, irrespective of species and order

• Monitoring and measurement to allow for specific targets and guidelines

• Quantification of the potential for a toxic event

• Predictive surveillance tool to add to the monitoring process, pre and post bloom

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Development of CyanoDTec: A rapid molecular assay for the routine 

monitoring of toxic cyanobacterial blooms

Key role of NMI• Develop primary measurement methods, reference 

materials and infrastructure to allow traceable measurements to be performed across all areas of 

science.

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Certified Reference Material

• Homogeneous and stable‐ subsamples, time

• Specified properties – define the measurement

• Fit for purpose – context, accuracy needed

• Authoritative body – independent, qualified

• Property values – numbers

• Uncertainty‐ confidence interval (accuracy)

• Traceability ‐ units

• Characterised by carrying out measurements using two or more independent reference methods.

• ISO Guide 34:2009, General requirements for the competence of reference material producers.

AN

Nn

n, amount of substance (mole)

Calibrant: dNMP reference materials 

N, number of moleculesNA, Avogadro’s constant (6.0221479 x 1023)

No calibrant

Isotope dilution mass spectrometry (IDMS)Weighing

Digital PCRCounting

NMI methods used for DNA quantification

dAMP dGMP

TMP

dCMP

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Quantification of CyanoDTec DNA Reference Material

Independent test run 1 2 3 4PCR Assay sxtA cyrA 16S rRNA mcyEThreshold setting Auto Auto Auto Manual

Replicate 1 1.39E+09 1.41E+09 1.37E+09 1.39E+09Replicate 2 1.46E+09 1.37E+09 1.38E+09 1.35E+09Replicate 3 1.45E+09 1.40E+09 1.39E+09 1.39E+09Replicate 4 1.47E+09 1.37E+09 1.38E+09 1.39E+09Replicate 5 1.40E+09 1.42E+09 1.34E+09 1.38E+09Replicate 6 1.33E+09 1.43E+09 1.37E+09 1.37E+09Replicate 7 1.35E+09 1.41E+09 1.37E+09 1.38E+09Replicate 8

Data for technical replicatesNumber of technical replicates 7 7 7 7Concentration copies/µL 1.41E+09 1.40E+09 1.37E+09 1.38E+09Standard deviation (SD) copies/µL 5.58E+07 2.48E+07 1.62E+07 1.36E+07Relative standard deviation (RSD) % 3.97 1.77 1.18 0.99Grand Mean concentration copies/µL 1.39E+09Expanded Uncertainty copies/µL 4.30E+07Relative expanded uncertainty % 3.1

16S rRNA sxtA cyrA mcyE/nadF

Quantification by digital PCR confirms 1:1 copy number ratio for the four cyanobacteria target sequences 

Preparation of CyanoDTec DNA RM working standards 

Batch of DNA standards at approximate concentrations of 0, 20, 200, 2000 and 20,000 copies per µL in each vial

Sample NA011 dilution

NA012 dilution

NA013 dilution

NA014 dilution

NA015 dilution

Threshold setting Auto Auto Auto Auto Auto

Replicate 1

copies/µL

0.00E+00 1.97E+01 1.89E+02 1.96E+03 1.97E+04

Replicate 2 0.00E+00 2.16E+01 2.00E+02 1.93E+03 1.95E+04

Replicate 3 0.00E+00 2.07E+01 1.99E+02 2.01E+03 1.96E+04

Replicate 4 0.00E+00 1.96E+01 1.87E+02 1.96E+03 1.98E+04

Replicate 5 0.00E+00 1.85E+01 1.99E+02 2.04E+03 1.99E+04

Number of technical replicates 5 5 5 5 5

Concentration copies/µL 0.00E+00 2.00E+01 1.95E+02 1.98E+03 1.97E+04

Standard deviation (SD) copies/µL 0.00E+00 1.16E+00 6.44E+00 4.58E+01 1.36E+02

Relative standard deviation (RSD) % 0.00 5.82 3.30 2.32 0.69

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Amplification plots of reference material

16S rRNA target assays using BioGX 8 master mix

y = -3.3561x + 38.296

R2 = 0.9992

0

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0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0log DNA RM copy number

Ct

va

lue

s

sxtA target assays using BioGX 8 master mix

y = -3.4412x + 39.939

R2 = 0.9966

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0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0log DNA RM copy number

Ct

valu

es

cyrA target assays using BioGX 8 master mix

y = -3.3627x + 40.468

R2 = 0.9956

0

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0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0log DNA RM copy number

Ct

valu

es

mcyE target assays using BioGX 8 master mix

y = -3.3278x + 41.358

R2 = 0.9912

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0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0log DNA RM copy number

Ct

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16S rRNA

sxtA cyrA mcyE

Amplification efficiencies of 95 to 99%

Highly accurate Reference Materials (CyanoNAS) have been produced to verify and/or validate methodologies for detecting toxin producing cyanobacteria using DNA‐based technology.

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CyanoDTec Test Specifics• Simultaneously identifies and quantifies the presence of 

total cyanobacteria along with 3 genes responsible for 4 different toxin production;

– Cyanobacteria species, 16S rRNA gene

– Microcystin & Nodularin, mcyE gene

– Cylindrospermopsin, cyrA gene

– Saxitoxin, sxtA gene

• Test completed within 3 hours

• Scalable 

• Cost effective

• Specific, no gene no toxin

CyanoDTecMethodology

• Prepare Sample (std extraction protocols)

• Re‐hydrolyse bead/enzyme mix in kit

• Add 5ul of sample to 20ul of enzyme mix into reaction rube

• Run PCR

• TOTAL time 2‐3 hours

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Assay Design and Setupparallel or sequential options

Sample/Standards/Controls

Internal Amplification Control/ 16S rRNA

Toxin Gene Targetsmcy/nda, cyr, sxt

Total Cyanobacteria Assay Toxin gene Assay

Quantifies total Cyanobacteria, determines  if dilution is needed or if toxin gene test required

Quantifies and identifies the presence of a toxin gene  specific to microcystin, nodularin, cylindrospermopsin and saxitoxinin cyanobacteria

• standardised cyanobacterial toxin gene assay

• Improves understanding and insights of bloom 

– Dynamics

– Toxic capacity/potential

• Offers direct relationship between gene level and toxin capacity and production for water management and establishment of toxin gene level guidelines

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ACKNOWLEDGEMENTS

• Lee Bowling

National Measurement Institute, Department of Industry

Brett Neilan

Jason Woodhouse

Yvette Gaweda

Leo Pinheiro

Kerry Emlsie

Cheryl Lim