qpcr, ELISA, and the HAB Workflow

Transcription

1 qpcr, ELISA, and the HAB Workflow Implementation of common techniques into the regulatory framework Andrew Bair Laboratory Scientist, Ohio Environmental Protection Agency

2 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

3 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)

4 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.

5 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.

6 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.

7 qpcr Three Steps of the PCR Cycle: 1) Denaturation 95 2) Annealing 60 3) Extension 60

8 qpcr 1) Denaturation - In this step the hydrogen bonds which hold together the complementary strands of DNA are broken by heat.

9 qpcr 2) Annealing - The primers are annealed, or attached, to the denatured DNA template.

10 Primer 101 DNA Polymerase, the enzyme used to amplify DNA in PCR, cannot bind to singlestranded 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.

11 qpcr 3) 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

12 How Do We Prepare Samples? 250 ml Sample Vacuum filtrate and keep the filter Fold filter up, aseptically and place into a Bead Lysis Tube 1.5mL Bead Lysis Tube Vacuum filtration manifold, 50mL of sample Bead Beat for 120 seconds, then centrifuge for 60 seconds Thermocycler 96-well Optical Plate Transfer supernatant to microcentrifuge tube. Sample is ready for storage or for plating.

13 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

14 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.

15 qpcr

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

17 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

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

19 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

20 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

21 Advantages of ELISA Specific, quantitative results Does not require radioisotopes Relatively inexpensive equipment when compared to LC-MS/MS Requires analyst expertise Time-Consuming

22 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

23 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

24 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

25 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

26 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

27 THANK YOU! To the OEPA, OAWWA, and you.