Infection from Bd or Bsal in amphibians may present with pathological dermal effects with symptoms including seizures and redness. However, amphibians rarely present visible symptoms and in most cases, post mortem examination allows proper diagnosis of the infection. Furthermore, the current diagnostic methods are very dependent on laboratory equipment, time-consuming, and expensive. To diagnose the disease, the researcher first has to swab the surface of the amphibian and carefully store the swab in a tube. Then, he will send the tube to a laboratory that tests for these infections. An example is San Diego Zoo Wildlife Alliance Amphibian Disease Laboratory, which tests the sample for “Combination Taqman PCR for Bd and Bsal” with $35. They recommend samples to be kept cool and “placed in a freezer (ultracold is ideal, but not absolutely necessary for short-term storage)” and to “avoid high-temperature extremes and direct sunlight” when sending samples. Furthermore, frozen samples have to be shipped by overnight couriers, such as Fed Ex, UPS, and DHL, on dry ice. Some labs even mention the following: “Laboratories that perform molecular diagnostic testing for amphibian infectious diseases have become very busy and it can sometimes take several weeks to months to get results.” Undergoing these processes are costly and time-consuming, which significantly limit the diagnosis and make it more difficult to prevent the spread of infection and isolate the infected amphibian. For these reasons, utilizing our rapid detection device for on-site managers will greatly aid in preventing the infection in a more cost-effective and time-effective manner.
We have developed a rapid, point-of-care, field fungal diagnostic device along with integrated data management solutions for effective biosecurity measures. This means that environmental conservationists or local agencies involved in monitoring amphibian populations are our end-users:
Our product will become available as a diagnostic tool kit. The tool kit will include a sterilized swab and a testing solution that contains CRISPR and a concentrator chip. The users will use the sterilized swab to gently swab the ventral surfaces of the skin around 20 times. Target areas include the pelvic patch (5 passes with the swab), ventral thighs (5 passes each side with the swab), and toe webbing (5 passes on each foot). Then, the user will put the swab onto the testing kit and wait for ___ minutes to receive results. The tool kit will be made user-friendly by providing detailed step-by-step procedures in utilizing the diagnostic kit.
As the concentrator chip we are designing is not reusable, users will have a recyclable biohazard bag within the toolkit in which they can store the chip till they are able to dispose of it responsibly.
The samples may be easily contaminated as it is being collected in a field setting. While this will cause background noise in the concentrator chip, a positive sample will still be detectable. Further tests need to be carried out to determine the extent to which impurities can affect the overall detection of a sample in the concentrator chip.
When the sample is collected and placed into our diagnostic device, a CRISPR system with single-guide RNA will recognize the target gene and cut it. Then, the CRISPR system will cut any single-stranded nucleic acids nearby; since we are adding single-stranded DNA/RNA that has a fluorescent reporter system, the CAS enzyme will cut this molecule with the reporter and emit fluorescence. However, if the concentration of the sample is low and the CRISPR system with guide RNA couldn’t identify the gene of interest, there will be no cutting of the gene and reporter quencher, giving negative results.
Despite the accuracy with which the gene scissors locates its target site on the DNA, it misses sometimes. For example, the shorter the recognition sequence, the greater the probability that such a sequence will arise several times in the genome and Cas will cut the DNA in several locations. In addition, CRISPR-Cas12 sometimes makes mistakes and docks at other sites in addition to its target sequence.
The device can be utilized at room temperature and some Cas12 enzymes have been reported to have higher activity when the temperature is above 28°C. However, proteins usually need specific physical and chemical conditions, such as temperature and pH to work effectively and may denature and degrade if not handled correctly. Furthermore, the contents within our diagnostic device may degrade over time and will not generate accurate results. This should be noted and expiry dates should be clearly stated.
Malzahn, A. A., Tang, X., Lee, K., Ren, Q., Sretenovic, S., Zhang, Y., ... & Qi, Y. (2019). Application of CRISPR-Cas12a temperature sensitivity for improved genome editing in rice, maize, and Arabidopsis. BMC biology, 17(1), 1-14.
