qPCR, ELISA, and the HAB Workflow

Andrew Bair

Laboratory Scientist,

Ohio Environmental Protection Agency

Implementation of common techniques into the regulatory framework

HAB Overview Harmful Algal Blooms (HABs) are caused by the

unchecked proliferation of algae, some of which can produce harmful toxins.

Primarily driven by bioavailable phosphorus, HABs have been on the uptick for over a decade.

Source: NOAA Bulletin, June 2016

HAB Overview

the production of neurotoxins (MYC, STX, CYN)

human illness or death via consumption of seafood contaminated by toxic algae

mechanical damage to other organisms, such as disruption of epithelial gill tissues in fish

oxygen depletion of the water column (hypoxia or anoxia)

HAB Overview In 2013, DES analyzed 421 HAB samples. In 2015, DES analyzed 2,511 HAB samples, an almost 6x

increase in two years.

Satellite image of Lake Erie exhibiting a cyanobacterial (‘blue green algae’) bloom.

HAB Overview

With an ever-increasing sample load, OEPA requires an efficient workflow; one which cuts down on unnecessary analysis and focuses on problem areas.

Consequently, OEPA has settled on a two-pronged approach: qPCR for routine screening and prediction of potential toxin-producing blooms, and ELISA for confirmation quantification.

qPCR Quantitative Real-Time Polymerase Chain Reaction

It (PCR) is one of the most powerful technologies in molecular biology. It can be used to amplify target DNA millions of times.

qPCR

Three Steps of the PCR Cycle:

1) Denaturation95°

2) Annealing60°

3) Extension60°

qPCR1) Denaturation - In this step the hydrogen bonds which hold together the complementary strands of DNA are broken by heat.

qPCR2) Annealing - The primers are annealed, or attached, to the denatured DNA template.

Primer 101

DNA Polymerase, the enzyme used to amplify DNA in PCR, cannot bind to single-stranded DNA.A primer is a short strand of nucleic acid sequences (generally about 10 base pairs) that serves as a starting point for DNA synthesis.

qPCR3) Extension – At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding nucleotides that are complementary to the template

250 mL Sample

Vacuum filtration manifold, 50mL of sample

Vacuum filtrate and keep the filter

Fold filter up, aseptically and place into a Bead Lysis Tube

1.5mL Bead Lysis Tube

Bead Beat for 120 seconds, then centrifuge for 60 seconds

Transfer supernatant to microcentrifuge tube. Sample is ready for storage or for plating.

96-well Optical PlateThermocycler

How Do We Prepare Samples?

qPCR

Two different Master Mixes

Total Cyanobacteria Assay (16s) Determines the gene copies of

total Cyanobateria Contains IAC

Total Cyanotoxin Assay Microcystins/Nodularin Cylidrospermopsin Saxitoxin

qPCR

In typical PCR, the result is a ‘band’ of genetic material on a gel. Comparison of the intensity of this band to a known standard can give ‘semi-quantitative’ results

qPCR entails data collection during amplification, as opposed to after the reaction is complete.

qPCR

ELISA Enzyme-Linked Immunosorbent Assay Carried out on a pre-coated, 96-well microtiter plate

ELISA Samples are frozen and thawed in a dry ice/ethanol bath

three times Cells are lysed, toxin is released Samples are filtered to remove any detritus

ELISA Several different types Direct Indirect Sandwich (Direct or Indirect) Competitive – can be combined with any of the other three

ELISA Focus on direct/competitive – used

for Saxitoxin, Cylindrospermopsin Wells are pre-coated with

secondary antibody (anti-rabbit) Sample, CYN-HRP conjugate, and

Rabbit-anti-CYN antibody are added

Sample and CYN-HRP conjugate compete to bind with Rabbit-anti-CYN antibody

These newly formed complexes bind to secondary antibody coating

Color solution is added; only creates signal with CYN-HRP Conjugate complexes

ELISA

As the color solution creates a signal only with the HRP-Conjugate complex, wells which contain samples high in CYN will not exhibit color

Signal is inversely proportional to sample CYN concentration

Advantages of ELISA Specific, quantitative results Does not require

radioisotopes Relatively inexpensive

equipment when compared to LC-MS/MS

Requires analyst expertise Time-Consuming

Advantages of qPCR

Allows us to quantify four parameters simultaneously

Can be used to determine the ability of a bloom to produce actual cyanotoxin

Less expensive than running ELISA for 4 parameters

Less time-consuming: 5 to 6 hr. for 20 samples at 4 parameters

Data Implementation Ohio Public Water Systems are required to routinely

sample source water

1 qPCR sample every 2 weeks

125 PWS ≈ 62 samples/week

More data is better

Data Implementation Therefore, qPCR is used as a screening procedure, and to

determine a baseline for each PWS This will give us an idea of typical gene counts found in

each reservoir and help us react accordingly

Data Implementation

When a toxin-producing gene is found in significant numbers, this triggers a sampling event for said parameter. (CYN, STX, MCY)

This prevents unnecessary ELISA , yet allows us to stay ahead of potential blooms – gene quantification is often a precursor to toxin production

Summary qPCR quantifies gene copies/µL and is used as a routine

monitoring and screening procedure Cheaper, less time consuming, predictive power Any positive result triggers a sampling for the positive

parameter (CYN, MYC) ELISA is then used to confirm and quantify toxin

production

THANK YOU!

To the OEPA, OAWWA, and you.