This page details the safety and security considerations associated with our project. All experiments were designed to minimize risks to personnel, the environment, and society, in accordance with relevant biosafety and ethical guidelines.
In the spirit of synthetic biology instead of directly working with dangerous live pathogens (namely Chlamydia, Gonorrhea, Trichomonas, Syphilis, HPV16 and HPV18), we opted to use their DNA fragments to develop our test. The biological risk of using DNA fragments is according to NIH guidelines[1] very low. In the future, the goal would be to run tests with probes from patients with the actual pathogens, but due to limited time we didn’t plan for a clinical study.
We worked with non-pathogenic E. coli strains for cloning and Cas protein development. Although these strains are considered safe, all biohazard waste was autoclaved to prevent release of genetic material. During lab work, gloves were never used on external surfaces such as door handles, and extra care was taken when handling beakers in and out of incubators to avoid breakage and thus the environmental contamination.
According to the SDSs for TwistAmp reagents, the buffers are generally low-to-moderate hazard, but may cause skin or eye irritation, if in contact. For this reason, those were handled with gloves, lab coats and spills were cleaned with Ethanol.
DNA dye for gel (in our case ROTI® Gel Stain) carries toxicity/mutagenicity concerns. To address this we always handled the gels by wearing gloves, as advised to us from our lab. We chose this option rather than ethidium bromide, which is even more mutagenic.
Liquid Nitrogen for RPA strips and to transport Cas proteins in between laboratories: this poses a serious risk of getting burned. As a measure of security, we never used it on our own but always supervised and used appropriate clothing, such as thick gloves and glasses. We also were careful not to take the lift while transporting material with liquid nitrogen to avoid the risk of getting suffocated.
Centrifuges may be a risk as imbalances can cause breakage. Our lab supervisors taught us early on how to properly use centrifuges and to stop immediately in case of concerning sound produced by the machine.
UV transilluminator for gels implies an UV radiation exposure risk. We handled this risk by correctly using the machine, which was taught to us by our instructors.
Heating blocks were used for Cas proteins development with the high temperature of 95°C. We carefully handled it by taking hot tubes out with tweezers.
Potential risks of misuse and inaccurate results were evaluated and mitigated where possible.
The problem of false-negatives and false-positives is clearly explained by Rumbold B., Wenham C. & Wilson, J: "As with any diagnostic, kits may generate false positives or false negatives – potentially leading users to make decisions based on inaccurate information. With self-tests, risks are compounded by the increased likelihood of individuals failing to conduct the test properly, misunderstanding test results or misunderstanding the reported accuracy of those results."[2] Due to time constraints, we didn’t arrive at our final prototype and thus didn’t need to take measures against this problem. However, if we were to arrive at that point, we would limit this risk with two actions. First of all, we would include a control line in the test that confirms its functionality. Secondly, in the self-test’s instructions, we would inform people and make them conscious about this risk, by stating that false-positive or false-negative results may occur and provide the percentage of accuracy.
Amplification methods like RPA are highly sensitive, so contamination between experiments could cause false results.[3] To avoid false results, we always tried to include a negative control with the experiment run. In addition, to diminish the risk of contamination and getting false results, we worked under clean hoods with lab coats and gloves. RPA reagents were stored in a different freezer than the primers and DNA. Lastly, our prototype minimizes this risk in general because both of our detection methods (CRISPR-Cas and DNA-hybridization) act as a double-check of the sequence.