Viruses are microbes that infect cells through modes of endocytic mechanisms or direct fusion to the plasma membrane (Cohen, 2016). Upon cell invasion, viruses exploit the cell’s cellular process to create new virus particles through replication of its genetic information and production of viral proteins (Baron et al., 1996). They proceed to disseminate to target organs which may be sufficient to cause disease and symptoms and sites that release them back into the environment (Baron et al., 1996).
Team:TAS Taipei/Description
Project Description
Background
What are Viruses?
Virus Related Infection and Deaths
Figure 1: Influenza Cases Worldwide March 2020 (WHO | Influenza Update - 362, n.d.).
Seasonal flus account for approximately 3 to 5 million cases and 250 to 500 thousands deaths worldwide annually (Influenza (Seasonal), n.d.). Pandemics such as the ongoing SARS-CoV2 pandemic has already caused 30.9 million cases and 958 thousand deaths, and the numbers continue to rise (WHO Coronavirus Disease (COVID-19) Dashboard, n.d.). The great quantity of people subjected to these diseases makes diagnostic measures crucial to installing preventive and therapeutic action.
Figure 2: Worldwide SARS-CoV-2 Cases Up to Oct 23rd, 2020 (Microsoft Bing COVID-19 Tracker, n.d.).
Current Solutions
Sample Collection and Purification
In order to test for a viral infection, a sample must be collected and at times isolated. Currently, there are two main methods for viral collection: through nasopharyngeal swabs or saliva. For nasopharyngeal swabs, a swab is inserted into the nostril until it reaches the pharynx region. It is left there for a few seconds to absorb secretions and removed while slowly rotating to obtain cells (Marty et al., 2020). This is usually done by a medical personnel. Saliva on the other hand is usually self collected by spitting into a sterile container with saline (Jamal et al., n.d.). Various articles have shown that saliva collection is highly sensitive and yields similar test outcomes, making it a noninvasive reliable alternative to swabs (Greenwood, 2020; Iwasaki et al., 2020; Jamal et al., n.d.; Nasopharyngeal Swab vs. Saliva for COVID-19 Diagnosis, 2020).
Figure 3: Nasopharyngeal Swab from Miraclean Technology (Nasopharyngeal Sampling Swabs, n.d.).
Most viral extraction or purification techniques center around commercial RNA extraction kits. They usually involve using lysis buffers, wash buffers, elution buffers, membrane columns, and centrifugation to ultimately extract in a lengthy process (Tan & Yiap, 2009). These are more often for nucleic acid tests and aren’t usually required in rapid test settings.
Nucleic Acid Tests
RT-PCR
The gold standard of viral detection is reverse transcriptase polymerase chain reaction (RT-PCR)(CDC, 2020). This methodology allows for high accuracy in sensitivity, but due to the need of varying temperatures and thus a thermocycler, not only is this option expensive but requires skilled technicians to run tests and interpret the data (Corman et al., 2020; da Costa Lima et al., 2013; False-Negative Rate of RT-PCR SARS-CoV-2 Tests, n.d.; Tahamtan & Ardebili, 2020, 2020).RT-PCR however still has limited specificity due to the reliance of reverse transcriptase, which has no proofreading capacity, to form cDNA sequences (Alhassan et al., 2015). Approximately 1 error is inputted for every 1700 polymerized nucleotides but can reach to as high as 1 error per 70 polymerized nucleotides. RT-PCR compared to other methods of detection is relatively accurate (Bhagavan & Ha, 2015, p. 22; Roberts et al., 1988). However, the lowest error rate is 21% after 8 days of virus exposure and can go as high as 67% within the first 5 days after virus exposure (False-Negative Rate of RT-PCR SARS-CoV-2 Tests, n.d.).
Figure 4: RT-PCR process involving DNA replication and fluorescent readout.
Isothermal Amplification - Lamp
A rising method of nucleic acid amplification is isothermal amplification, which allows the amplification reaction to run at a constant temperature. One isothermal technique is loop mediated isothermal amplification (LAMP). LAMP utilizes 4-6 sets of primers to amplify many regions of the target sequence to ultimately create a loop structure that can be used as a backbone for further exponential amplification (Biolabs, n.d.). Real time fluorescent detection through probes, lateral flow, or DNA electrophoresis can then be used to examine the amplified or unamplified product (Biolabs, n.d.). However, one issue with LAMP is its need to run at 65oC (Ge et al., 2017). This requires the use of an incubator or thermocycler, which defeats the purpose of using LAMP instead of RT-PCR since speicific instrumentation or even the same machinery not so accessible may have to be used. LAMP also relies on reverse transcriptase for RNA virus detection, which limits the specificity of the assay due to the low fidelity of reverse transcriptase (Roberts et al., 1988).
Protein/Antibody Tests
Lateral Flow Immunoassays
A form of rapid test is through lateral flow immunoassays. On the top of the strip is usually an antibody whereas the bottom of the strip usually contains gold nanoparticles with either antibodies or viral antigens expressed on it (Koczula & Gallotta, 2016; Sajid et al., 2015). If viral antigens on a virus particle are being detected, the antibody nanoparticle that captures the antigen carries it to the detecting antibody (Koczula & Gallotta, 2016; Sajid et al., 2015). If human antibodies are being detected, then the viral antigen nanoparticle will capture the antibody and the detecting antibody will capture the nanoparticle antigens (Koczula & Gallotta, 2016; Sajid et al., 2015). Whatever the case, the presence of virus particles with the expressed antigens and human antibodies in response to virus exposure will cause specific lines to appear after a reaction occurs from the detecting antibody (Koczula & Gallotta, 2016).
These rapid tests do not require sample preparation and provide fast results. However, due to the lack of signal amplification of any kind, it's usually low in sensitivity and highly inaccurate (Gordon & Michel, 2008; Rapid Influenza Diagnostic Tests | CDC, 2019). Antibody testing often leads to a delayed diagnosis, as it takes approximately 2 weeks for humans to mount an immune response (Lippi et al., 2020).
Blood Serology
Blood serology tests usually use the bioanalytical technique indirect ELISA. Plating the ELISA wells with viral antigens, a sample with human antibodies mounted in response to the virus will bind to the viral antigen. A secondary conjugated antibody can then be used to provide a color or fluorescent readout. These ELISA plates can be scanned with a reader to quantify these values to be interpreted for results. However, the use of ELISA plates runs the risk of cross contamination and also requires a technician to follow a specific procedure to undertake the process (ELISA Troubleshooting Tips | Abcam, n.d.; Troubleshooting Guides - ELISA, n.d.). As stated, there is also a risk of delayed diagnosis.
Other
Viral Cell Cultures
A method that is neither nucleic acid testing nor protein and antibody testing is viral cell culture (CDC, 2020). Through growth mediums such as RetroNectin, medical personnel attempt to culture viruses from isolated samples. In other cases, viruses are first used to transfection mammalian cell lines to allow for expansion before culture (Harcourt et al., n.d.). The growth of a virus doesn’t completely identify the type of virus a patient has however. To identify it, DNA sequencing, IF staining, electron microscopy, and other methods are employed (Harcourt et al., n.d.; Hematian et al., 2016). As one can expect, this process can take long, sometimes up to 30 days (Harcourt et al., n.d.). It also poses significant dangers to the individuals operating the test, if not handled correctly (Virus Contaminations of Cell Cultures – A Biotechnological View, n.d.).
Our Goal
Theory
Figure 5: The rolling circle amplification RCA process on a molecular level.
To address some of the issues with the aforementioned viral detection tests. We employed an isothermal methodology known as padlock probe rolling circle amplification. The two ends of the padlock probe are designed to bind to a specific complementary target sequence. Upon hybridization, a ligase, usually T4 ligase, can ligate the ends of the padlock probe due to a 5’ phosphorylation on the padlock probe and the presence of ATP as a cofactor (Hamidi & Ghourchian, 2015). The result is a circularized padlock probe, which can now act as a template for DNA amplification (Hamidi & Perreault, 2019). If the padlock probe is not complementary to the target sequence, ligation should not occur (Gu et al., 2018). Without a circularized padlock probe, continual DNA amplification will not be able to occur and thus the readout will show a negative result.
The subsequent methods of amplification varies. Some involve the use of the 3’ to 5’ exonuclease activity of phi29 DNA polymerase to cut the target sequence until the ligation junction is reached, which enables DNA synthesis to proceed (Li et al., 2017). Others use an exonuclease or RNase to achieve the same function or direct annealing of a forward primer to the circularized backbone (Joffroy et al., 2018; RNase H-Assisted RNA-Primed Rolling Circle Amplification for Targeted RNA Sequence Detection | Scientific Reports, n.d.). Whatever the case, the DNA polymerase of choice, usually Bst DNA polymerase or phi29 DNA polymerase, has an existing DNA strand to synthesize DNA strands in circles around the padlock probe through its helicase activity (Joffroy et al., 2018). A second reverse primer that binds to the newly synthesized DNA strands is sometimes used to further increase DNA synthesis by introducing a branching-like amplification (Hamidi & Ghourchian, 2015, p. 1; Hamidi & Perreault, 2019, 2019; Larsson et al., 2004). Finally, the readout usually uses fluorescent probes that recognize the synthesized strand and is thus quantifiable (Hamidi & Ghourchian, 2015, p. 1; Larsson et al., 2004).
Modifications
To maximize the Rolling Circle Amplification technique, we made modifications to increase accuracy through sensitivity and specificity and add additional functions that will increase usability.
The modifications we included involves a colorimetric readout. We made use of the fact that for every nucleotide incorporated in DNA amplification, there is a release of H+ and PPi. Thus, with a pH indicator we can visualize a color change readout through a change in hydrogen ion concentration. But this also requires us to take out all buffers in the solutions of the reaction that might neutralize the hydrogen ions produced. Thus, our reaction was carried out with specially made solutions without any buffers such as Tris-HCl.
Another modification is our use of SplintR Ligase (PBCV-1 DNA Ligase and Chlorella virus DNA Ligase) to allow the ligation of our DNA padlock probe and viral RNA target. This enables high specificity through direct RNA targeting. But to prevent RNA degradation, we used Protector Rnase Inhibitors to neutralize the activity of RNases that cleaves RNA. We also used phi29 DNA polymerase along with splintR ligase, as both enzymes operate at optimal settings near room temperature (30°C and 25°C respectively) and does in fact allow a room temperature reaction as experimentally proven.
To enable the detachment of the target sequence to the circularized padlock probe in a simpler method, we designed universal primers that would bind to the backbone (non-target region) of the padlock probe. As a result, the helicase activity of the polymerase itself would remove the target sequence and allow for amplification to continue. An additional reverse primer that binds to the newly synthesized DNA strands is used to introduce a branching-like amplification. These methods ultimately help improve signal amplification which helps provide readouts even during low viral concentrations, increasing the sensitivity of our test. Studies have shown that the exonuclease activity of phi29 DNA polymerase can involve not only single stranded sequences but double stranded complementary sequences, we added phosphorothioate bonds to disable phi29 DNA polymerase from digesting it (Li et al., 2017).
All of these traits work together to form our simple, fast, and accurate universal viral detection platform.
Figure 6: Flowchart of our Viral Spiral Project for Viral Diagnosis
The Bigger Picture
Our RCA colorimetric experiments ultimately serve as the viral diagnosis portion of our project. But to truly bring it to life, we have to incorporate portions such as our modeling hardware piece, where we presented a theory and 2 in 1 saliva collection and purification device design to allow nucleic acid isolation. Using our software to quantify our qualitative color readouts into pH values and inputting the rate of pH change into our model, we determined the RNA concentration of our reactions. With RNA concentration, the viral load of the patient can be reported. Although viral load does not correlate with the severity of the viral disease, it can be a good indicator of the time since viral exposure and the level of infectivity of a sick individual. From a human practices standpoint, this can present ways for individuals to seek immediate medical attention for the benefit of the individual and population. This ultimately allows us to implement our project as a viral diagnostic home test kit that is not only highly usable, but provides useful information and helpful implications to anyone being tested.
References
Alhassan, A., Li, Z., Poole, C. B., & Carlow, C. K. S. (2015). Expanding the MDx toolbox for filarial diagnosis and surveillance. Trends in Parasitology, 31(8), 391–400. https://doi.org/10.1016/j.pt.2015.04.006
Baron, S., Fons, M., & Albrecht, T. (1996). Viral Pathogenesis. In Medical Microbiology. 4th edition. University of Texas Medical Branch at Galveston. https://www.ncbi.nlm.nih.gov/books/NBK8149/
Bhagavan, N. V., & Ha, C.-E. (2015). Chapter 22—DNA Replication, Repair, and Mutagenesis. In N. V. Bhagavan & C.-E. Ha (Eds.), Essentials of Medical Biochemistry (Second Edition) (pp. 401–417). Academic Press. https://doi.org/10.1016/B978-0-12-416687-5.00022-1
Biolabs, N. E. (n.d.). Loop-Mediated Isothermal Amplification | NEB. Retrieved September 21, 2020, from https://www.neb.com/applications/dna-amplification-pcr-and-qpcr/isothermal-amplification/loop-mediated-isothermal-amplification-lamp
CDC. (2020, February 11). Information for Laboratories about Coronavirus (COVID-19). Centers for Disease Control and Prevention. https://www.cdc.gov/coronavirus/2019-ncov/lab/grows-virus-cell-culture.html
Cohen, F. S. (2016). How Viruses Invade Cells. Biophysical Journal, 110(5), 1028. https://doi.org/10.1016/j.bpj.2016.02.006
False-Negative Rate of RT-PCR SARS-CoV-2 Tests. (n.d.). American College of Cardiology. Retrieved June 5, 2020, from http%3a%2f%2fwww.acc.org%2flatest-in-cardiology%2fjournal-scans%2f2020%2f05%2f18%2f13%2f42%2fvariation-in-false-negative-rate-of-reverse
Ge, Y., Zhou, Q., Zhao, K., Chi, Y., Liu, B., Min, X., Shi, Z., Zou, B., & Cui, L. (2017). Detection of influenza viruses by coupling multiplex reverse-transcription loop-mediated isothermal amplification with cascade invasive reaction using nanoparticles as a sensor. International Journal of Nanomedicine, Volume 12, 2645–2656. https://doi.org/10.2147/IJN.S132670
Gordon, J., & Michel, G. (2008). Analytical Sensitivity Limits for Lateral Flow Immunoassays. Clinical Chemistry, 54(7), 1250–1251. https://doi.org/10.1373/clinchem.2007.102491
Greenwood, M. (2020, August 15). Yale’s rapid COVID-19 saliva test receives FDA emergency use authorization. YaleNews. https://news.yale.edu/2020/08/15/yales-rapid-covid-19-saliva-test-receives-fda-emergency-use-authorization
Gu, L., Yan, W., Liu, L., Wang, S., Zhang, X., & Lyu, M. (2018). Research Progress on Rolling Circle Amplification (RCA)-Based Biomedical Sensing. Pharmaceuticals, 11(2), 35. https://doi.org/10.3390/ph11020035
Hamidi, S. V., & Ghourchian, H. (2015). Colorimetric monitoring of rolling circle amplification for detection of H5N1 influenza virus using metal indicator. Biosensors and Bioelectronics, 72, 121–126. https://doi.org/10.1016/j.bios.2015.04.078
Hamidi, S. V., & Perreault, J. (2019). Simple rolling circle amplification colorimetric assay based on pH for target DNA detection. Talanta, 201, 419–425. https://doi.org/10.1016/j.talanta.2019.04.003
Harcourt, J., Tamin, A., Lu, X., Kamili, S., Sakthivel, S. K., Murray, J., Queen, K., Tao, Y., Paden, C. R., Zhang, J., Li, Y., Uehara, A., Wang, H., Goldsmith, C., Bullock, H. A., Wang, L., Whitaker, B., Lynch, B., Gautam, R., … Thornburg, N. J. (n.d.). Severe Acute Respiratory Syndrome Coronavirus 2 from Patient with Coronavirus Disease, United States—Volume 26, Number 6—June 2020—Emerging Infectious Diseases journal—CDC. https://doi.org/10.3201/eid2606.200516
Hematian, A., Sadeghifard, N., Mohebi, R., Taherikalani, M., Nasrolahi, A., Amraei, M., & Ghafourian, S. (2016). Traditional and Modern Cell Culture in Virus Diagnosis. Osong Public Health and Research Perspectives, 7(2), 77. https://doi.org/10.1016/j.phrp.2015.11.011
Influenza (Seasonal). (n.d.). Retrieved September 21, 2020, from https://www.who.int/news-room/fact-sheets/detail/influenza-(seasonal)
Iwasaki, S., Fujisawa, S., Nakakubo, S., Kamada, K., Yamashita, Y., Fukumoto, T., Sato, K., Oguri, S., Taki, K., Senjo, H., Hayasaka, K., Sugita, J., Konno, S., Nishida, M., & Teshima, T. (2020). Comparison of SARS-CoV-2 detection in nasopharyngeal swab and saliva. MedRxiv, 2020.05.13.20100206. https://doi.org/10.1101/2020.05.13.20100206
Jamal, A. J., Mozafarihashjin, M., Coomes, E., Powis, J., Li, A. X., Paterson, A., Anceva-Sami, S., Barati, S., Crowl, G., Faheem, A., Farooqi, L., Khan, S., Prost, K., Poutanen, S., Taylor, M., Yip, L., Zhong, X. Z., McGeer, A. J., Mubareka, S., … Walmsley, S. (n.d.). Sensitivity of Nasopharyngeal Swabs and Saliva for the Detection of Severe Acute Respiratory Syndrome Coronavirus 2. Clinical Infectious Diseases. https://doi.org/10.1093/cid/ciaa848
Joffroy, B., Uca, Y. O., Prešern, D., Doye, J. P. K., & Schmidt, T. L. (2018). Rolling circle amplification shows a sinusoidal template length-dependent amplification bias. Nucleic Acids Research, 46(2), 538–545. https://doi.org/10.1093/nar/gkx1238
Koczula, K. M., & Gallotta, A. (2016). Lateral flow assays. Essays in Biochemistry, 60(1), 111. https://doi.org/10.1042/EBC20150012
Larsson, C., Koch, J., Nygren, A., Janssen, G., Raap, A. K., Landegren, U., & Nilsson, M. (2004). In situ genotyping individual DNA molecules by target-primed rolling-circle amplification of padlock probes. Nature Methods, 1(3), 227–232. https://doi.org/10.1038/nmeth723
Li, X.-Y., Du, Y.-C., Zhang, Y.-P., & Kong, D.-M. (2017). Dual functional Phi29 DNA polymerase-triggered exponential rolling circle amplification for sequence-specific detection of target DNA embedded in long-stranded genomic DNA. Scientific Reports, 7(1), 6263. https://doi.org/10.1038/s41598-017-06594-1
Lippi, G., Mattiuzzi, C., Bovo, C., & Plebani, M. (2020). Current laboratory diagnostics of coronavirus disease 2019 (COVID-19). Acta Bio Medica Atenei Parmensis, 91(2), 137–145. https://doi.org/10.23750/abm.v91i2.9548
Marty, F. M., Chen, K., & Verrill, K. A. (2020). How to Obtain a Nasopharyngeal Swab Specimen. New England Journal of Medicine, 382(22), e76. https://doi.org/10.1056/NEJMvcm2010260
Nasopharyngeal swab vs. Saliva for COVID-19 diagnosis. (2020, May 21). News-Medical.Net. https://www.news-medical.net/news/20200521/Nasopharyngeal-swab-vs-saliva-for-COVID-19-diagnosis.aspx
Rapid Influenza Diagnostic Tests | CDC. (2019, November 12). https://www.cdc.gov/flu/professionals/diagnosis/clinician_guidance_ridt.htm
RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection | Scientific Reports. (n.d.). Retrieved September 22, 2020, from https://www.nature.com/articles/s41598-018-26132-x
Roberts, J., Bebenek, K., & Kunkel, T. (1988). The accuracy of reverse transcriptase from HIV-1. Science, 242(4882), 1171–1173. https://doi.org/10.1126/science.2460925
Sajid, M., Kawde, A.-N., & Daud, M. (2015). Designs, formats and applications of lateral flow assay: A literature review. Journal of Saudi Chemical Society, 19(6), 689–705. https://doi.org/10.1016/j.jscs.2014.09.001
Tan, S. C., & Yiap, B. C. (2009). DNA, RNA, and Protein Extraction: The Past and The Present. Journal of Biomedicine and Biotechnology, 2009, 1–10. https://doi.org/10.1155/2009/574398
Virus contaminations of cell cultures – A biotechnological view. (n.d.). Retrieved September 21, 2020, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3463984/
WHO Coronavirus Disease (COVID-19) Dashboard. (n.d.). Retrieved September 21, 2020, from https://covid19.who.int