Team:Austin LASA/Description

const r = i => h('a', {href: '#ref_' + i}, i); h(g.Page, {title: 'Description', prev: 'https://2018.igem.org/Team:Austin_LASA/Collaborations', next: 'https://2018.igem.org/Team:Austin_LASA/Design', selector: [2, 0]}, h(g.Section, {title: 'Why HIV Detection in Infants?'}, h('p', null, 'HIV diagnosis of infants in the developing world continues to pose many problems. It is estimated that there are approximately 1500 new cases of HIV infection in children under the age of 15, 90% of which occur in the developing world and contracted HIV at birth [', r(1), ']. Infants often show symptoms of HIV very early on, and in some cases, HIV can rapidly evolve into AIDS [', r(2), ']. As such, it is imperative that infants with HIV are diagnosed as early as possible.'), h('p', null, 'The WHO has stated that earlier diagnosis of HIV in infants and children:'), h('ul', null, h('li', null, 'enables the early identification of who have HIV-infection, as a first step in securing their treatment and care'), h('li', null, 'enables the identification of those who are HIV-exposed but uninfected, facilitating follow-up care and prevention measures that will help to ensure that they remain uninfected'), h('li', null, 'assists in the effective use of essential resources by targeting ART on children who need treatment'), h('li', null, 'improves the psychosocial well-being of families and children, reducing potential stigma, discrimination and psychological distress for HIV-uninfected children and increasing the chances of adoption for orphans'), h('li', null, 'facilitates life-planning for parents and/or children who have HIV [', r(4), ']') ), h('p', null, 'Unfortunately, HIV diagnosis in infants faces more challenges than HIV diagnosis in adults. Typical serological assays result in large false positives and negatives as infants retain maternal HIV antibodies for up to 18 months, initially obtaining them through transplacental fluid in utero or through breastfeeding milk postnatally, and are therefore unreliable for use as a diagnostic method [', r(2), ']. As a result, HIV DNA/RNA PCR tests, p24 core antigen tests, and HIV cultures are the only reliable diagnostic tests for HIV in infants [', r(2), ', ', r(4), ']. PCR tests, although reliable, are frequently unavailable in resource-constrained settings where the proper lab equipment and skilled personnel are limited [', r(5), ']. HIV cultures, similarly to PCR tests, require proper lab equipment and skilled personnel in addition to taking up to two weeks to grow and obtain results [', r(3), ']. p24 core antigen tests also require proper lab equipment and skilled personnel, but are undergoing testing and clinical evaluation for their utility [', r(5), '].') ), h(g.Section, {title: 'How Does Our Project Tie In?'}, h('p', null, 'As mentioned previously, often in order to perform PCR, a clinic or other diagnostic center must send their sample to another location with more sophisticated equipment. Often by the time the results are ready to get back to the patient, the disease has progressed too far or the patient is impossible to contact. This is why our project incorporates an alternative amplification method, Loop Mediated Isothermal Amplification (LAMP), which can be done at the point of care. Performing LAMP does not require a thermal cycler, so it can be done in a water bath that is kept at a constant temperature of 60–65 °C. LAMP requires 4 primers that result in an end product of a multitude of copies attached by loops [', r(8), ']. In our project, we tested primers for LAMP since sometimes primer sets that should theoretically work, do not do so. Furthermore, we examined the specificity and efficiency of the reaction. The amplicons from the LAMP reaction would subsequently be detected using Cas12a, which is discussed in the next paragraph.'), h('p', null, 'Recent research demonstrates CRISPR-associated enzyme Cas12a’s ability to indiscriminately cleave ssDNA following recognition and cleavage of a dsDNA target strand. This property of Cas12a has been utilized for the detection of specific nucleotide sequences [', r(6), ', ', r(7), ']. In these detection assays, Cas12a is assembled with a crRNA including a spacer sequence specific to a duplexed target strand. A fluorophore-quencher pair connected by a short ssDNA sequence is present in the same reaction. Once the Cas12a-crRNA complex is binded to and cleaves a duplexed target strand, Cas12a indiscriminately cleaves any ssDNA or RNA sequence in its vicinity, including the fluorophore-quencher pair. The released fluorophore can then be detected.'), h('p', null, 'We aimed to use this property of Cas12a for the detection of HIV viral DNA in infected human cells. We chose specifically to focus on HIV as:'), h('ol', {type: 'a'}, h('li', null, 'There is a need for the accurate detection of HIV in infants in developing countries'), h('li', null, 'Following diagnosis of HIV in infants, infants can be treated accordingly'), h('li', null, 'HIV’s genome has been very well-documented, as opposed to the genomes of other viruses such as Zika') ) ), h(g.Section, {title: 'What Would a Point-of-Care Diagnostic System Look Like?'}, h('p', null, 'Our project aims to design a point-of-care HIV1 diagnostic system for infants. For actual applicability in the field, our kit would consist of three components:'), h('ol', null, h('li', null, 'Isolation of viral DNA from infected cells'), h('li', null, 'Isothermal amplification of DNA for greater sensitivity'), h('li', null, 'Detection of DNA by a Cas12a assay.') ), h('p', null, 'In our project, we only chose to focus on parts 2 and 3 for reasons stated further below.'), h('p', null, 'A point-of-care diagnostic system would also need to be easily accessible in resource limited settings. Much like with PCR and HIV culture tests, many resource-constrained settings also lack the ability to house specific reagents and proper transportation of enzymes to remote areas is difficult [', r(5), ']. For these reasons, we also aimed to look into the use of lyophilized bacterial reagents, termed “cellular reagents.” Bacteria can be modified to express certain enzymes, grown, and then lyophilized with their produced enzyme. These “cellular reagents” can then be transported dry, stored at room temperature for extended periods of time, and resuspended later at the time of use. Introducing cellular reagents into our HIV-1 diagnostic kit would make it much more accessible to resource-limited settings.'), h('ol', null, h('li', null, 'Isolation of Viral DNA from Infected Cells', h('p', null, 'After consultation with field expert Dr. Leautaud at Rice University, our team was told that in the field samples of nucleotides would most likely be obtained from infected white blood cells. Because loop-mediated isothermal amplification is a fairly robust procedure, isolation of nucleotides can be done fairly simply with detergent or high temperature and not risk messing up the isothermal amplification reaction. Dr. Leautaud further advised us to focus on our isothermal amplification and detection assays, stating that, if it was necessary, we could collaborate with other teams or labs on the matter of nucleotide isolation.'), h('p', null, '(To read more on how human practices impacted the development of our kit, go to our Kit Considerations page ', h('a', {href: 'https://2018.igem.org/Team:Austin_LASA/Kit'}, 'here'), '.)')), h('li', null, 'Isothermal Amplification of Viral DNA for Greater Sensitivity', h('p', null, 'The sensitivity of our Cas12a detection method depends on the amount of viral DNA present for detection to occur. For this reason, an amplification step of the target sequence prior to detection is necessary. However, as PCR is limited in resource-deficient environments, we looked into different methods of isothermal amplification of DNA that would be more accessible.'), h('p', null, 'There are several methods of isothermal amplification. We chose to work with loop-mediated isothermal amplification (LAMP) as our PI and lab mentors have prior experience with LAMP.'), h('p', null, 'LAMP is a reaction that relies on four specific primers. These primers are more difficult to design than the primers for PCR so we used PrimerExplorer to design primers that would amplify various regions of Rev. At the location of point of care, someone would pipette the isolated viral DNA into a tube with rehydrated LAMP cellular regents and then add a mastermix (that was previously stored at a lower temperature). This reaction would subsequently be placed in a water bath at 60–65 °C for 90 minutes, which would allow the viral DNA to be amplified.')), h('li', null, 'Detection of DNA by a Cas12a Assay', h('p', null, 'The final step in our kit would be our Cas12a detection assay. As stated earlier, our detection assay would take advantage of Cas12a’s characteristic to indiscriminately cleave any single-stranded nucleotides in its vicinity following cleavage of a duplexed target strand. We would use our amplified DNA from our LAMP reactions as our target strands and design corresponding crRNA sequences to specifically detect our viral DNA. As mentioned before, we would use a fluorophore-quencher pair to detect Cas12a activity. In order to translate our detection assay to cellular reagents, we would express Cas12a and crRNA in bacteria, then subsequently lyophilize said bacteria to create cellular reagents. In the field, the cellular reagents would be rehydrated and used for a reaction.')) ) ), h(g.Section, {title: 'OUR iGEM PROJECT GOAL'}, h('p', null, 'Our team focused on demonstrating loop-mediated isothermal amplification and subsequent detection of HIV-1 DNA via Cas12a with purified enzymes and cellular reagents.') ), h(g.Section, {title: 'References'}, h('p', null, '[', h('a', {id: 'ref_1'}, '1'), '] Who.int. (2018). WHO | Antiretroviral therapy of HIV infection in infants and children. [online] Available at: ', h('a', {href: 'http://www.who.int/hiv/pub/paediatric/infants/en/'}, 'http://www.who.int/hiv/pub/paediatric/infants/en/'), ' [Accessed 17 Oct. 2018].'), h('p', null, '[', h('a', {id: 'ref_2'}, '2'), '] Krist AH, Crawford-Faucher A. Management of Newborns Exposed to Maternal HIV Infection. American Family Physician. ', h('a', {href: 'https://www.aafp.org/afp/2002/0515/p2049.html'}, 'https://www.aafp.org/afp/2002/0515/p2049.html'), ' Published May 15, 2002. Accessed October 17, 2018.'), h('p', null, '[', h('a', {id: 'ref_3'}, '3'), '] Dr G Vercauteren, Dr Siobhan Crowley. Early detection of HIV infection in infants and children.'), h('p', null, '[', h('a', {id: 'ref_4'}, '4'), '] Diagnosis of HIV Infection in Infants and Children Pediatric ARV. National Institutes of Health. ', h('a', {href: 'https://aidsinfo.nih.gov/guidelines/html/2/pediatric-arv/55/diagnosis-of-hiv-infection-in-infants-and-children'}, 'https://aidsinfo.nih.gov/guidelines/html/2/pediatric-arv/55/diagnosis-of-hiv-infection-in-infants-and-children'), '. Published November 15, 2017. Accessed October 17, 2018.'), h('p', null, '[', h('a', {id: 'ref_5'}, '5'), '] Shafiee H, Wang S, Inci F, et al. Emerging Technologies for Point-of-Care Management of HIV Infection. Annual Review of Medicine. 2015;66(1):387-405. doi:10.1146/annurev-med-092112-143017.'), h('p', null, '[', h('a', {id: 'ref_6'}, '6'), '] Gootenberg JS, Abudayyeh OO, Kellner MJ, Joung J, Collins JJ, Zhang F. Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6. Science. 2018;360(6387):439-444. doi:10.1126/science.aaq0179.'), h('p', null, '[', h('a', {id: 'ref_7'}, '7'), '] Montoya G. Faculty of 1000 evaluation for CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. F1000 - Post-publication peer review of the biomedical literature. 2018. doi:10.3410/f.732670635.793547776.'), h('p', null, '[', h('a', {id: 'ref_8'}, '8'), '] Notomi T. Loop-mediated isothermal amplification of DNA. Nucleic Acids Research. 2000;28(12). doi:10.1093/nar/28.12.e63.'), ) );