FUNGAL DIAGNOSTICS

PROBLEM

In November of 2019, Dr. Johannes Penner, a conservation research scientist at the University of Freiburg reached out to the team and proposed a problem to address given our diagnostic history.

Since the mid-1900s, a strange flesh-eating fungus has wiped out over 90 species and caused declines in the population of at least 501 amphibian species. Similarly, white-nose syndrome – another fungal disease – has left millions of bats dead in its path. This decline, apart from causing a devastating loss in biodiversity, poses multiple threats to the environment due to the significant role these animals play as pollinators, biodiversity indicators, and seed-dispersing foresters.

Using current methods, after capturing the animal in the wild, analysis with either PCR or qPCR is latent – finding the patient again is impossible. The wet lab procedures are also riddled with complex steps that only trained personnel can engage with. Testing at ports, which are the primary mode of spread given international trade, has been impossible since results are quite latent and expensive. Dr. Penner indicated that a rapid field-use diagnostic device similar to our past solutions would be a “game-changer” in monitoring amphibian populations, allowing conservationists, local agencies, etc. to act immediately.

PROJECT

NYUAD iGEM is developing a fully-fledged rapid, portable, cheap, easy to use, field fungal diagnostic device. Our project tackles different components that go into developing a point-of-care diagnostic device: sample collection, extraction & purification, amplification, detection, reaction medium, reporting, sensing, and data management. The end product is targeted to be used for monitoring the spread of infectious animal diseases such as chytridiomycosis which has been the main driver behind the amphibian decline.

Click on the tabs above to learn more about each component.

ACKNOWLEDGEMENTS

Team

biology
Adrian Villanueva
Michelle Hughes
Yujeong Oh
Sakura Grant
Zerina Rahic
Marko Susic

engineering
Yaman Garg
Roba Olana
Abhay Menon
Steffanie Dias
Maya Fayed
Ayesha Sameer
Zurab Jashi
Dimitrios Mastrogiannis

design
Quim Paredes
Mariam ElSahhar

computer science
Prajjwal Bhattarai
Safal Shrestha

Advisors

Ibrahim Chehade
Esteban Slavemini
Stephanne Boissinot
Justin Wilcox
Sandra Goutte
Youssef Idaghdour

Alan Healy
Andras Gyorgy
Rafael Song
Zoltan Derzsi
Mohammad Qasaimeh

SAMPLE COLLECTION

Our detection pathway relies upon the recognition of a DNA marker found only in the chytrid fungi. The pathway detects DNA mixtures that contains the Internal Transcribed Spacer (ITS) region. Used as a barcode region for fungal detection, the ITS region is highly conserved and nonpathogenic, making it safe to work with.

Our DNA marker: the ITS region

To collect the DNA, we would swab the skin of the amphibians to collect fungal spores (which then yield DNA), a standard practice for current diagnostic methods.

eDNA has recently proven to be effective as well, which is the sampling of amphibian habitats such as ponds and lakes, using filters to collect spores from the water. We also plan to have solutions for that down the road, but the focus of the current project is still on swabbing.

eDNA Sample Collection

EXTRACTION & PURIFICATION

The next step in the diagnostic pathway is to extract and purify the DNA from the fungal spores. This is a major challenge for nucleic acid based point of care diagnostics and we performed extensive literature research to boil down our available options to:

1. Bead Beating- Mechanical lysis using rapidly moving glass beads
2. Combination of Bead Beating+ Sonication+ Atomization described by Lim et al.
3. Detergent-based Lysis Buffer + Silica Matrix based purification
4. Detergent-based Lysis Buffer + Magnetic Beads based purification
5. Whatman FTA Paper

OmniLyse: A Portable Bead Beating Solution

Detergent-based Lysis Buffer + Silica Matrix based purification.

We are designing experiments to prototype and test all these options, combining them with what we call “reaction medium”, which is the platform for hosting the reactions in the device. Here, our options for reaction medium range from microfluidic chips, paper-based biosensors to innovative solutions that we’re in the process of brainstorming such as robotic automation of manual pipetting and modified PCR tubes.

Paper-based Biosensor

Microfluidic Chips

Modified PCR tubes

AMPLIFICATION

We have proof of concept that RPA (recombinase polymerase amplification) works to amplify the ITS region specifically of our target fungi. We are currently testing and comparing RPA to the LAMP (loop-mediated isothermal amplification) method to determine the optimal pathway. Both employ quick, isothermal techniques suitable for a field-detection device.

How RPA works:
[1] Recombinase and primer complex forms
[2] Complex binds to DNA and D-Loop forms
[3] SSB stabilizes single strand of D-Loop
[4] Newly synthesized strand displaces parental strand
[5] 2 identical strands are produced, process repeats

Mechanism of the RPA amplification

RPA Specificity test on Bsal DNA with Bd Primers and Bsal Primers

Our RPA reaction worked on our Bsal gBlock with Bsal primers. Meanwhile, when using our Bd primers with the Bsal gBlock, we did not get amplification. This shows that our RPA reaction with the corresponding primers are functional.

RPA Specificity test on Bd DNA with Bd Primers and Bsal Primers

Similarly. Our RPA reaction worked on our Bd gBlock with Bd primers. Meanwhile, when using our Bsal primers with the Bd gBlock, we did not get amplification. This means that our RPA primers are specific towards the target region it is intended to amplify

DETECTION

The CRISPR/Cas12a system in action

To increase specificity, we targeted our amplified product with CRISPR/Cas12a, derived from the Lachnospiracae Bacterium, which achieves attomolar sensitivity. Combining our RPA amplification with the Cas12a system is called DETECTR.

DETECTR: Proof of Concept on Bd gBlock

With our purified Bd gBlock, we have proved that our DETECTR protocol is functional. Figure 1 shows our positive result. With the corresponding primers and guide RNA for Bsal, we are confident that our DETECTR protocol will work on our Bsal ITS region. Alternatively, CRISPR/Cas13a system was found to be more sensitive in literature. Going forward with our project, we plan on testing the CRISPR/Cas13a system against our target ITS regions to identify the optimal detection pipeline.

REPORTING

When our CRISPR/Cas enzyme is activated, a means of detecting this activation is required to communicate the result to a user. We currently have two options that are under testing that will bridge CRISPR detection and user interaction: a quenched fluorescence reporter and a lateral flow assay.

Quenched Fluorescence Reporter

When our CRISPR/Cas system is activated, our quenched fluorescence single stranded DNA reporter is cleaved, which gives off a fluorescence signal that can be quantitatively detected through our sensing method.

Mechanism of our Quenched Fluorescence Reporter

Lateral Flow Assay

Meanwhile, our lateral flow immunoassay can also be used to detect the activation of our CRISPR/Cas system. This assay will take place in a strip with a test and control line. In essence, the absence of a test line is a negative result, while the presence of a test line is a positive result. This detection is achieved by using a dual labelled FAM-biotin single-stranded DNA reporter molecule. In the presence of the target DNA, our CRISPR/Cas system cleaves the reporter molecule, which is visualized by a positive result in the test line.

Lateral Flow Assay Mechanism

SENSING

To sense the fluorescence and LFA signals and convert them into quantified results, we identified the following options based on our engineering constraints and biological reactions. We have already designed experiments to test these options and are currently in the process of testing them:

Fluorescence

For excitation we have the options of an LED or a Laser, and we are testing both to find the best one. For detection, we are using a photodiode based module. To make the device cost effective, we are testing if we can avoid using expensive filters by using statistical techniques such as one-tailed t-tests, and we will be comparing this with performance using a conventional lens filter.

Fluorescence Sensing

LFA

We are using smartphones to take images of the LFA strip, and by capturing the intensity of coloration on the testline using computer vision and machine learning methods, we plan to get reliable quantitative results.

Using smartphones to quantify LFA

DATA

We are currently in the process of implementing a comprehensive, scalable database system with the primary objective of facilitating rapid analysis and dissemination of diverse data types are central to effective disease management. This data can be used to construct mathematical models that may provide valuable insight into the spread of a disease and boost conservation efforts.

The diagnostic results are stored in a cloud database. Any third party access to the database can be done using REST API, that can integrate with any surveillance systems. Alternatively, we will also implement native drivers for a few programming languages; most notably Python and R, with the intention of facilitating any analysis of the data.

We are also planning to make our sample and diagnostic data available to open-source, academic datasets like GEOME and AmphibianDiseasePortal.

Team:NYU Abu Dhabi/Poster