The main aim of Chytritect is to reduce the tedious amount of time and work it takes to diagnose amphibians with Chytridiomycosis in the field. Here we prove our ability to achieve this goal.
To obtain samples for tests, E.coli cells were inoculated with the target DNA of Bsal and placed in an incubator for 18-24 hours. Extraction of DNA from samples was then performed using the QIAprep Miniprep Kit protocol to obtain the pure DNA sample.
The extraction of the DNA component of Chytritect makes use of a microfluidic chip which uses surface chemistry to extract the DNA. Chytritect was able to successfully extract DNA with about 85% efficiency within a time of about 30 minutes.
For detection of Bsal, PCR was performed to amplify the target DNA. CRISPR/cas12a complex was pipetted into amplified DNA and the sample was checked every 5minutes for 40mins under UV light. CRISPR/cas12a was able to successfully detect the presence of Bsal as shown in the image below.
Figure 1: Detection results with CRISPR/cas12a- Sample with Bsal on the left and control on the right
To confirm that CRISPR/cas12a was detecting the correct target DNA, Gel electrophoresis was performed on the amplified DNA samples. As shown by the image below, Gel electrophoresis confirmed that the sample being detected by CRISPR/cas12a was indeed the Bsal target DNA.
Figure 2: Gel electrophoresis on target DNA sample.
The detection component of Chytritect uses a concentrator chip that causes the sample to concentrate at its center, allowing Chytritect to bypass the PCR step and thereby reducing largely the entire required for diagnoses. With the sample centered at the center of the concentrator chip, CRISPR/cas12a complex is then added to the sample to detect the presence of Bsal. Our results show rapid detection on the concentrator chip within 4 minutes.
To determine the limiting number of DNA copies per microlitre at which Bsal can be detected in the lab, the experiments were performed on samples with different copy numbers ranging from 10^10 copies/µl to 1 copy/µl. The result showed that the limit of detection was at 10^3 DNA copies per microlitre(see Figure 3).
Figure 3: Results showing the limit of detection at 10^3 copies per microlitre.
As summarized in the results page, we found the limit of detection of the concentrator chip module to be as low as 1 copy per microlitre. This represents a highly sensitive device which can differentiate between a low fungal load and a negative sample.
To find out the concentration of starting DNA in a swab we performed the following experiment.
This showed us that the initial DNA concentration before passing it through the chip will have an average of 520 ng/ul. Using results from our efficiency tests and our limit of detection tests, we can conclude that the initial concentration is sufficient for downstream detection of Bd/Bsal.
