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<div class="nonumtoc">__TOC__</div>


==3. Adding COVID-19 and other virus testing to your laboratory==
Does using one method make the most sense, or will your lab turn to multiple methods for virus testing?
===3.1 What methodologies will you use?===
====3.1.1 PCR====
In the previous chapter, the most common testing methodologies for [[COVID-19]] and other [[Coronavirus|coronaviruses]] were discussed in detail. The prevailing method (often called the "gold standard") among them all was real-time reverse-transcription [[polymerase chain reaction]] (rRT-PCR) assays for testing. Broadly speaking, PCR is useful in pharmaceutical, biotechnology, and genetic engineering endeavors, as well as clinical diagnostics. As such, labs in those industries that already have PCR ifrastructure in place have a theoretical step-up over a lab that doesn't.
PCR technology has advanced to the point where it is more efficient and user-friendly than prior, yet "the high cost of the instruments, servicing contracts, and reagents pose major challenges for the market, especially to the price-sensitive academics."<ref name="MWPoly20">{{cite web |url=https://www.marketwatch.com/press-release/polymerase-chain-reaction-market-sector-analysis-report-regional-outlook-competitive-share-forecast---2023-2020-06-23 |title=Polymerase Chain Reaction Market Sector Analysis Report, Regional Outlook & Competitive Share & Forecast - 2023 |author=Kenneth Research |work=MarketWatch |date=23 June 2020 |accessdate=06 August 2020}}</ref> Writing about the thirty-fifth anniversary of PCR in 2018, science writer Alan Dove not only highlighted these cost issues but also the size and energy requirments for running the equipment. "As a result, one of the defining techniques of modern molecular biology has remained stubbornly inaccessible to educators and unusable in many remote locations."<ref name="DovePCR18">{{cite journal |title=PCR: Thirty-five years and counting |journal=Science |author=Dove, A. |volume=360 |issue=6389 |pages=670–672 |year=2018 |doi=10.1126/science.360.6389.673-c}}</ref> Various efforts have been made over the years to bring costs down by modifying how heating and temperature control are performed<ref name="WongARapid15">{{cite journal |title=A Rapid and Low-Cost PCR Thermal Cycler for Low Resource Settings |journal=PLoS One |author=Wong, G.; Wong, I. Chan, K. et al. |volume=10 |issue=7 |at=e0131701 |year=2015 |doi=10.1371/journal.pone.0131701}}</ref><ref name="KuznetsovDIYbio15">{{cite journal |title=DIYbio Things: Open Source Biology Tools as Platforms for Hybrid Knowledge Production and Scientific Participation |journal=Proceedings of the 33rd Annual ACM Conference on Human Factors in Computing Systems |author=Kuznetsov, S.; Doonan, C.; Wilson, N. et al. |pages=4065–68 |year=2015 |doi=10.1145/2702123.2702235}}</ref><ref name="NortonPhila16">{{cite web |url=https://www.bizjournals.com/philadelphia/news/2016/05/11/government-contract-biomeme-hiring-med-tech.html |title=Phila. med tech startup working on multimillion dollar government contract |author=Norton, D. |work=Philadelphia Business Journal |date=11 May 2016 |accessdate=06 August 2020}}</ref><ref name="AnLow20">{{cite journal |title=Low-Cost Battery-Powered and User-Friendly Real-Time Quantitative PCR System for the Detection of Multigene |journal=Micromachines |author=An, J.; Jiang, Y.; Shi, B. et al. |volume=11 |at=435 |year=2020 |doi=10.3390/mi11040435}}</ref>, but many of those system aren't typically optimal during a pandemic when turnaround time is critical.
Amidst the pandemic, additional challenges also exist to those wanting to conduct PCR testing for COVID-19 and other viruses. As was discussed at the end of the previous chapter, supplies of reagents and consumables are not particularly robust mid-pandemic, with shortages being reported since March 2020.<ref name="HerperShortage20">{{cite web |url=https://www.statnews.com/2020/03/10/shortage-crucial-chemicals-us-coronavirus-testing/ |title=Shortage of crucial chemicals creates new obstacle to U.S. coronavirus testing |work=STAT |author=Herper, M.; Branswell, H. |date=10 March 2020 |accessdate=10 April 2020}}</ref><ref name="HaleQiagen20">{{cite web |url=https://www.fiercebiotech.com/medtech/qiagen-aims-to-more-than-quadruple-its-covid-19-reagent-production-6-weeks |title=Qiagen aims to more than quadruple its COVID-19 reagent production in 6 weeks |work=Fierce Biotech |author=Hale, C. |date=18 March 2020 |accessdate=10 April 2020}}</ref><ref name="MehtaMystery20">{{cite web |url=https://www.chemistryworld.com/mystery-surrounds-uk-claim-of-covid-19-test-reagent-shortage/4011457.article |title=Mystery surrounds UK claim of Covid-19 test reagent ‘shortage’ |work=Chemistry World |author=Mehta, A. |date=03 April 2020 |accessdate=10 April 2020}}</ref><ref name="RocheIrish20">{{cite web |url=https://www.irishtimes.com/news/science/irish-scientists-develop-reagent-in-effort-to-ease-covid-19-testing-delays-1.4223897 |title=Irish scientists develop reagent in effort to ease Covid-19 testing delays |work=The Irish Times |author=Roche, B. |date=08 April 2020 |accessdate=10 April 2020}}</ref><ref name="PadmaEfforts20">{{cite web |url=https://www.chemistryworld.com/news/efforts-to-combat-covid-19-in-india-hit-by-imported-reagent-shortages/4011718.article#/ |title=Efforts to combat Covid-19 in India hit by imported reagent shortages |author=Padma, T.V. |work=Chemistry World |date=13 May 2020 |accessdate=19 May 2020}}</ref><ref name="DavidSurvey20">{{cite web |url=https://abcnews.go.com/Health/survey-shows-resources-covid-19-diagnostic-testing-limited/story?id=71341885 |title=Survey shows resources for COVID-19 diagnostic testing still limited months later |author=David, E.; Farber, S.E. |work=ABC News |date=20 June 2020 |accessdate=08 July 2020}}</ref><ref name="JohnsonNCLabs20">{{cite web |url=https://patch.com/north-carolina/charlotte/nc-labs-facing-shortages-covid-19-testing-chemicals |title=NC Labs Facing Shortages In COVID-19 Testing Chemicals |author=Johnson, K. |work=Patch |date=02 July 2020 |accessdate=08 July 2020}}</ref><ref name="MervoshItsLike20">{{cite web |url=https://www.nytimes.com/2020/08/04/us/virus-testing-delays.html |title=‘It’s Like Having No Testing’: Coronavirus Test Results Are Still Delayed |author=Mervosh, S.; Fernandez, M. |work=The New York Times |date=04 August 2020 |accessdate=05 August 2020}}</ref><ref name="CourageCovid20">{{cite web |url=https://www.vox.com/2020/7/31/21336212/covid-19-test-results-delays |title=Should we be testing fewer people to stop the spread of Covid-19? |author=Courage, K.H. |work=Vox |date=31 July 2020 |accessdate=05 August 2020}}</ref> These shortages may eventually work themselves out, but they highlight the need for other varying methods that don't necessarily depend on the same reagents and consumables that are in short supply.
For those labs wishing to adopt PCR testing of viruses—particularly COVID-19—into their workflow while providing reasonable turnaround times, all is not lost. However, careful planning is required. For example, you'll want to keep in mind that some PCR machines require vendor-specific reagents. If you're going to acquire a particular instrument, you'll want to do due diligence by verifying not only the supported reagents but also those reagents' overall availability (real and projected). You'll also want to consider factors such as anticipated workload (tests per day), what your workflow will look like, and how to balance overall investent with the need for reasonable turnaround times.
As of August 2020, an increasing body of research is being produced suggesting ways to improve turnaround times with PCR testing for COVID-19, with many research efforts focusing on cutting out RNA extraction steps entirely. Alcoba-Florez ''et al.'' [[Journal:Fast SARS-CoV-2 detection by RT-qPCR in preheated nasopharyngeal swab samples|propose]] direct heating of the sample-containing nasopharyngeal swab at 70 °C for 10 minutes in place of RNA extraction.<ref name="Alcoba-FlorezFast20">{{cite journal |title=Fast SARS-CoV-2 detection by RT-qPCR in preheated nasopharyngeal swab samples |journal=International Journal of Infectious Diseases |author=Alcoba-Florez, J.; González-Montelongo, R.; Íñigo-Campos, A.; García-Martínezde Artola, D.; Gil-Campesino, H.;<br />The Microbiology Technical Support Team; Ciuffreda, L.; Valenzuela-Fernández, A.; Flores, C. |volume=97 |pages=66–68 |year=2020 |doi=10.1016/j.ijid.2020.05.099}}</ref> Adams ''et al.'' have proposed an "adaptive PCR" method using a non-standard reagent mix that skips RNA extraction and can act "as a contingency for resource‐limited settings around the globe."<ref name="ShapiroStream20">{{cite web |url=https://news.vanderbilt.edu/2020/07/29/streamlined-diagnostic-approach-to-covid-19-can-avoid-potential-testing-logjam/ |title=Streamlined diagnostic approach to COVID-19 can avoid potential testing logjam |author=Shapiro, M. |work=Research News @ Vanderbilt |date=29 July 2020 |accessdate=06 August 2020}}</ref><ref name="AdamsCOVID20">{{cite journal |title=COVID‐19 diagnostics for resource‐limited settings: Evaluation of “unextracted” qRT‐PCR |journal=Journal of Medical Virology |author=Adams, N.M.; Leelawong, M.; Benton, A. et al. |year=2020 |doi=10.1002/jmv.26328}}</ref> Wee ''et al.'' skip RNA extraction and nucleic acid purification by using a single-tube homogeneous reaction method run on a lightweight, portable thermocycler.<ref name="MeharImproving20">{{cite web |url=https://www.techexplorist.com/improving-speed-gold-standard-covid-19-diagnostic-test/34069/ |title=Improving the speed of gold-standard COVID-19 diagnostic test |author=Mehar, P. |work=Tech Explorist |date=27 July 2020 |accessdate=06 August 2020}}</ref><ref name="WeeRapid20">{{cite journal |title=Rapid Direct Nucleic Acid Amplification Test without RNA Extraction for SARS-CoV-2 Using a Portable PCR Thermocycler |journal=Genes |author=Wee, S.K.; Sivalingam, S.P.; Yap, E.P.H. |volume=11 |issue=6 |at=664 |year=2020 |doi=10.3390/genes11060664}}</ref> Other innovations include tweaking reagents and enzymes to work with one step, skipping the reverse transcription step,<ref name="CSIRFaster20">{{cite web |url=https://www.engineeringnews.co.za/article/faster-local-covid-19-test-kits-could-be-ready-by-year-end-2020-07-30/ |title=Faster, local COVID-19 test kits could be ready by year-end |author=Council for Scientific and Industrial Research |work=Engineering News |publisher=Creamer Media |date=30 July 2020 |accessdate=07 August 2020}}</ref> and using saliva-based molecular testing that skips RNA extraction.<ref name="RanoaSaliva20">{{cite journal |title=Saliva-Based Molecular Testing for SARS-CoV-2 that Bypasses RNA Extraction |journal=bioRxiv |author=Ranoa, D.R.E.; Holland, R.L.; Alnaji, F.G. et al. |year=2020 |doi=10.1101/2020.06.18.159434}}</ref>
====3.1.2 Pooled testing====
Another method some labs are taking to speed up turnaround time is using pooled testing. The general concept involves placing two or more test specimens together and testing the pool as one specimen. The most obvious adventage to this is that the process saves on reagents and other supplies, particularly when supply chains are disrupted. This methodology is best used "in situations where disease prevalence is low, since each negative pool test eliminates the need to individually test those specimens and maximizes the number of individuals who can be tested over a given amount of time."<ref name="RohdeCOVID20">{{cite web |url=https://asm.org/Articles/2020/July/COVID-19-Pool-Testing-Is-It-Time-to-Jump-In |title=COVID-19 Pool Testing: Is It Time to Jump In? |author=Rohde, R. |publisher=American Society for Microbiology |date=20 July 2020 |accessdate=06 August 2020}}</ref> However, it's best left to situations where expectations are that less than 10 percent of the population being tested is affected by what's being tested for.<ref name="RohdeCOVID20" /><ref name="MashaPooled20">{{cite web |url=https://asiatimes.com/2020/08/pooled-virus-tests-help-stretched-health-services/ |title=Pooled virus tests help stretched health services |author=Masha, M.; Chau, S. |work=Asia Times |date=04 August 2020 |accessdate=06 August 2020}}</ref><ref name="CitronerHowPool20">{{cite web |url=https://www.healthline.com/health-news/how-pooled-testing-can-help-us-fight-spread-of-covid-19 |title=How Pooled Testing Can Help Us Fight Spread of COVID-19 |author=Citroner, G. |work=Healthline |date=03 August 2020 |accessdate=06 August 2020}}</ref>
The downside of pooled testing comes with the issues of dillution, contamination, and populations with 10 or more percent infected. A target-positive specimen that comingles with other target-free specimens is itself diluted and in some cases may cause issues with the limit of detection for the assay. Additionally, if the pool tests positive, target-free specimens may become contaminated by a target-positive specimen. This may cause issues with any individual specimen assays that get ran. And the workflows involving pooling must be precise, as a technician working with multiple specimens at the same time increases the chance of lab errors.<ref name="RohdeCOVID20" /><ref name="MashaPooled20" /><ref name="CitronerHowPool20" />Finally, at least in the U.S., an Food and Drug Administration (FDA) emergency use authorization (EUA) for a validated pooled testing method is required.<ref name="RohdeCOVID20" /> (Validation of pooled methods may differ in other countries.<ref name="MashaPooled20" />) The U.S. Centers for Disease Control and Prevention (CDC) has published [https://www.cdc.gov/coronavirus/2019-ncov/lab/pooling-procedures.html interim guidance] on pooled testing strategies for [[SARS-CoV-2]].
====3.1.3 Rapid antigen testing====
An antigen is a substance—often a protein but may also be an environmental like a virus—that provokes the immune system to produce an antibody against it.<ref name="MPAntigen">{{cite web |url=https://medlineplus.gov/ency/article/002224.htm |title=Antigen |work=MedlinePlus |publisher=U.S. National Library of Medicine |accessdate=07 August 2020}}</ref> As such, another approach to testing for the presence of a virus in a specimen is to test for the antigen rather than the antibody. An antigen test is useful as a repeated surveillance test, but it has drawbacks as a one-time diagnostic test.<ref name="Anderson5Invest20">{{cite web |url=https://www.wcvb.com/article/5-investigates-concerns-about-current-use-of-rapid-antigen-tests-for-covid-19/33538332 |title=5 Investigates: Concerns about current use of rapid antigen tests for COVID-19 |author=Anderson, K. |work=WCVB 5 ABC |date=06 August 2020 |accessdate=07 August 2020}}</ref><ref name="ServiceRadical2020">{{cite journal |title=Radical shift in COVID-19 testing needed to reopen schools and businesses, researchers say |journal=Science |author=Service, R.F. |year=2020 |doi=10.1126/science.abe1546}}</ref><ref name="KremerUWSys20">{{cite web |url=https://urbanmilwaukee.com/2020/08/07/uw-system-orders-350000-covid-19-tests/ |title=UW System Orders 350,000 COVID-19 Tests |author=Kremer, R. |work=Urban Milwaukee |date=07 August 2020 |accessdate=07 August 2020}}</ref> For COVID-19 and other viral infections, an antigen test has the advantage that specimen collection can typically be done with a simple nasal swab rather than a more invasive [[nasopharyngeal swab]]. Another advantage, on one hand, is that antigen testing is more rapid and convenient because the extraction and amplification steps of PCR are not used. On the other, antigen testing is less sensitive for the same reason: you test only what's there (rather than amplifying the amount for greater sensitivity).<ref name="ServiceRadical2020" /><ref name="GuglielmiTheExp20">{{cite journal |title=The explosion of new coronavirus tests that could help to end the pandemic |journal=Nature |author=Guglielmi, G. |volume=583 |pages=506–09 |year=2020 |doi=10.1038/d41586-020-02140-8}}</ref>
A theory increasingly gaining traction, however, is that "[a] higher frequency of testing makes up for poor sensitivity.”<ref name="ServiceRadical2020" /><ref name="KremerUWSys20" /><ref name="ClarkCOVID20">{{cite web |url=https://www.medpagetoday.com/infectiousdisease/covid19/87930 |title=COVID Antigen Tests: Coming to Case Counts Near You? |author=Clark, C. |work=MedPage Today |date=06 August 2020 |accessdate=07 August 2020}}</ref> Several researchers have shared pre-print and published research suggesting this outcome<ref name="ServiceRadical2020" />:
<blockquote>Larremore and his colleagues have modeled the benefits of more frequent tests, even ones that are less accurate than today’s. Fast tests repeated every three days, with isolation of people who test positive, prevents 88% of viral transmission compared with no tests; a more sensitive test used every two weeks reduced viral transmission by about 40%, they report in a 27 June preprint on medRxiv. Paltiel and his colleagues reached much the same conclusion when they modeled a variety of testing regimes aimed at safely reopening a 5000-student university. In a 31 July paper in JAMA Network Open, they found that, with 10 students infected at the start of the semester, a test that identified only 70% of positive cases, given to every student every two days, could limit the number of infections to 28 by the end of the semester. Screening every seven days allowed greater viral spread, with the model predicting 108 infections.</blockquote>
As such, the utility of antigen testing, despite its lower sensitivity, appears to be surveillance situations where a large group of individuals who are at risk can at regularly scheduled intervals of two to four days be screened. The end result, in theory, would be few people who are target-positive would be missed, positives could be isolated and verified with a more sensitive test, and more target-positive people would be identified and isolated before reaching peak infectivity.<ref name="ServiceRadical2020" /><ref name="CourageCovid20">{{cite web |url=https://www.vox.com/2020/7/31/21336212/covid-19-test-results-delays |title=Should we be testing fewer people to stop the spread of Covid-19? |author=Courage, K.H. |work=Vox |date=31 July 2020 |accessdate=05 August 2020}}</ref> To be clear, it's not a perfect solution, but as Harvard epidemioligist Michael Mina and Boston University economist Laurence Kotlikoff suggest, "[w]e need the best means of detecting and containing the virus, not a perfect test no one can use."<ref name="CourageCovid20" /> A coalition of six U.S. state governors has bought into that concept and agreed to work together with the Rockefeller Foundation, as well as the Quidel Corporation and Becton, Dickinson and Company, which have received FDA EUAs to market antigen tests for SARS-CoV-2.<ref name="ClarkCOVID20" /><ref name="MervoshItsLike20">{{cite web |url=https://www.nytimes.com/2020/08/04/us/virus-testing-delays.html |title=‘It’s Like Having No Testing’: Coronavirus Test Results Are Still Delayed |author=Mervosh, S.; Fernandez, M. |work=The New York Times |date=04 August 2020 |accessdate=05 August 2020}}</ref> However, it's not clear how those six states will best put the tests to use despite 1. their moderate sensitivity (and thus a greater chance of false negatives<ref name="ClarkCOVID20" />) and 2. the question of whether or not the two companies can produce enough test kits for repeat testing in those states.<ref name="ServiceRadical2020" />
====3.1.4 LAMP and CRISPR====
Early on in the pandemic, while PCR was getting most of the attention, reverse transcription loop-mediated isothermal amplification (RT-LAMP), an isothermal [[Nucleic acid test|nucleic acid amplification]] technique that allows for RNA amplification, was also quietly being discussed<ref name="LambRapid20">{{cite journal |title=Rapid Detection of Novel Coronavirus (COVID-19) by Reverse Transcription-Loop-Mediated Isothermal Amplification |journal=medRxiv |author=Lamb, L.E.; Barolone, S.N.; Ward, E. et al. |year=2020 |doi=10.1101/2020.02.19.20025155}}</ref><ref name="Schmid-BurgkLAMP20">{{cite journal |title=LAMP-Seq: Population-Scale COVID-19 Diagnostics Using Combinatorial Barcoding |journal=bioRxiv |author=Schmid-Burgk, J.L.; Li, D.; Feldman, D. et al. |year=2020 |url=https://www.biorxiv.org/content/10.1101/2020.04.06.025635v2.article-info |doi=10.1101/2020.04.06.025635}}</ref>, and it has since gained more attention.<ref name="YuRapid20">{{cite journal |title=Rapid Detection of COVID-19 Coronavirus Using a Reverse Transcriptional Loop-Mediated Isothermal Amplification (RT-LAMP) Diagnostic Platform |journal=Clinical Chemistry |author=Yu, L.; Wu, S.; Hao, X. et al. |volume=66 |issue=7 |pages=975–77 |year=2020 |doi=10.1093/clinchem/hvaa102 |pmid=32315390 |pmc=PMC7188121}}</ref><ref name="ParkDevelop20">{{cite journal |title=Development of Reverse Transcription Loop-Mediated Isothermal Amplification Assays Targeting Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) |journal=Journal of Molecular Diagnostics |author=Park, G.-S.; Ku, K.; Baek, S.-H. et al. |volume=22 |issue=6 |pages=729–35 |year=2020 |doi=10.1016/j.jmoldx.2020.03.006 |pmid=32276051 |pmc=PMC7144851}}</ref><ref name="KellnerARapid20">{{cite journal |title=A rapid, highly sensitive and open-access SARS-CoV-2 detection assay for laboratory and home testing |journal=bioRxiv |author=Kellner, M.J.; Ross, J.J.; Schnabl, J. et al. |year=2020 |doi=10.1101/2020.06.23.166397}}</ref><ref name="ThiAColor20">{{cite journal |title=A colorimetric RT-LAMP assay and LAMP-sequencing for detecting SARS-CoV-2 RNA in clinical samples |journal=Science Translational Medicine |author=Thi, V.L.D.; Herbst, K.; Boerner, K. et al. |at=eabc7075 |year=2020 |doi=10.1126/scitranslmed.abc7075 |pmid=32719001}}</ref><ref name="EsbinOver20">{{cite journal |title=Overcoming the bottleneck to widespread testing: a rapid review of nucleic acid testing approaches for COVID-19 detection |journal=RNA |author=Esbin, M.N.; Whitney, O.N.; Chong, S. et al. |volume=26 |issue=7 |pages=771–83 |year=2020 |doi=10.1261/rna.076232.120 |pmid=32358057 |pmc=PMC7297120}}</ref><ref name="HaleOxford20">{{cite web |url=https://www.fiercebiotech.com/medtech/oxford-researchers-develop-portable-covid-19-test-costing-less-than-25 |title=Oxford researchers develop portable COVID-19 test costing less than $25 |author=Hale, C. |work=Fierce Biotech |date=09 July 2020 |accessdate=07 August 2020}}</ref> The University of Oxford, for example, is in the process of getting a rapid, affordable, clinically-validated RT-LAMP test approved for the European market. Oxford also notes that "[a]n advantage of using LAMP technology is that it uses different reagents to most laboratory-based PCR tests."<ref name="HaleOxford20" /> Thi ''et al.'' have tested a two-color RT-LAMP assay with an N gene primer set and diagnostic validation using LAMP-sequencing, concluding that the pairing of the two "could offer scalable testing that would be difficult to achieve with conventional qRT-PCR based tests."<ref name="ThiAColor20" /> And California-based Color Genomics have set up their own proprietary RT-LAMP system, capable of handling up to 10,000 tests per day.<ref name="SheridanThisCal20">{{cite web |url=https://www.statnews.com/2020/08/06/better-simpler-faster-covid-19-test/ |title=This California company has a better version of a simpler, faster Covid-19 test |author=Sheridan, K. |work=STAT |date=06 August 2020 |accessdate=08 August 2020}}</ref>
In most cases, LAMP-based testing is much simpler than PCR, lacking the requirement of specialized instruments. Despite LAMP generally being thought of as less sensitive than PCR<ref name="GuglielmiTheExp20" /><ref name="SheridanThisCal20" /><ref name="HeidtSaliva20">{{cite web |url=https://www.the-scientist.com/news-opinion/saliva-tests-how-they-work-and-what-they-bring-to-covid-19-67720 |title=Saliva Tests: How They Work and What They Bring to COVID-19 |author=Heidt, A. |work=The Scientist |date=09 July 2020 |accessdate=08 August 2020}}</ref>, the recent explosion of research into RT-LAMP methods for testing for the presence of SARS-CoV-2 seems to gradually indicate that "under optimized conditions," RT-LAMP methods may actually be able to rival the sensitivity and specificity of many RT-PCR COVID-19 test.<ref name="EsbinOver20" /> Esbin ''et al.'' add<ref name="EsbinOver20" />:
<blockquote>These methods allow for faster amplification, less specialized equipment, and easy readout. LAMP methods also benefit from the ability to multiplex targets in a single reaction and can be combined with other isothermal methods, like &#91;[[recombinase polymerase amplification]]&#93; in the RAMP technique, to increase test accuracy even more. These techniques may be particularly useful for rapid, point-of-care diagnoses or for remote clinical testing without the need for laboratory equipment.</blockquote>
CRISPR methods are also being used in conjunction with RT-LAMP.<ref name="GuglielmiTheExp20" /><ref name="EsbinOver20" /><ref name="BroughtonCRISPR20">{{cite journal |title=CRISPR–Cas12-based detection of SARS-CoV-2 |journal=Nature Biotechnology |author=Broughton, J.P.; Deng, X.; Yu, G. et al. |volume=38 |pages=870–74 |year=2020 |doi=10.1038/s41587-020-0513-4 |pmid=32300245}}</ref> RT-LAMP creates complementary double-stranded DNA (cDNA) from specimen RNA and then copies (amplifies) it. Then CRISPR methods are used to detect a predefined coronavirus sequence (from a cleaved molecular marker) in the resulting amplified specimen. Though as of August 2020 approved assays using CRISPR-based detection of SARS-CoV-2 are limited to a handful of companies<ref name="GuglielmiTheExp20" /><ref name="GDHCRISPR20">{{cite web |url=https://www.medicaldevice-network.com/comment/crispr-biotechnology-disrupt-covid-19-testing-market/ |title=CRISPR biotechnology set to disrupt Covid-19 testing market |author=GlobalData Healthcare |work=Verdict Medical Devices |date=14 July 2020}}</ref>, the technology has some promise as an alternative testing method. It has the additional advantage of being readily couples with lateral flow assay technology to be deployed in the point-of-care setting.<ref name="EsbinOver20" /><ref name="GDHCRISPR20" />
====3.1.5 Point-of-care and other alternative testing====
Examples include<ref name="Leichman10Ways20">{{cite web |url=https://www.israel21c.org/how-israeli-scientists-are-improving-corona-testing/ |title=10 ways Israeli scientists are improving corona testing |author=Leichman, A.K. |work=Isael21c |date=27 July 2020 |accessdate=11 August 2020}}</ref>:
* a rapid breath test to detect volatile organic chemicals from the lungs;
* an affordable, hand-held spectral imaging device to detect virus in blood or saliva in seconds;
* an ultrahigh frequency spectroscopic scanning device to see virus particules resonating;
* a method that combines optical devices and magnetic particles to detect virus RNA;
* an RNA extraction protocol that uses magnetic bears;
* the addtional use of an [[artificial intelligence]] (AI) application to better scrutenize test results; and
* the miniaturization of PCR technology to make it more portable and user-friendly.
POLs:
* https://hitconsultant.net/2020/08/03/talis-nih-contract-for-point-of-care-covid-19-testing/
* https://www.nih.gov/news-events/news-releases/nih-delivering-new-covid-19-testing-technologies-meet-us-demand
* <ref name="HeidtSaliva20" /> (Saliva)
===3.2 What equipment and supplies will you need?===
====Instruments====
[[File:Eppendorf Mastercycler Pro S.jpg|right|400px|thumb|Eppendorf Mastercycler Pro S, a thermal cycler for PCR and other applications]] Thermal cyclers (a.k.a. PCR machines) - standard PCR systems, RT PCR -  advantages of digital PCR systems such as precision, sensitivity, accuracy, reproducibility, direct quantification and multiplexing, and speed of the analysis systems, and digital PCR systems "The market is witnessing an emerging trend of digital and droplet digital PCR technology, which is sensitive and accurate than the traditional method."
https://www.thermofisher.com/search/browse/category/us/en/602552/PCR+Machines+%28Thermal+Cyclers%29+%26+Accessories
https://blog.biomeme.com/how-do-you-test-for-covid-19
====Reagents====
template DNA, PCR primers and probes, dNTPs, PCR buffers, enzymes, and master mixes
====Consumables====
PCR tubes, plates, and other accessories
https://www.sigmaaldrich.com/labware/labware-products.html?TablePage=9577275
====Software and services====
====Vendors====
Major players operating in the global PCR market are Bio-Rad Laboratories, Inc., QIAGEN N.V., F. Hoffmann-La Roche AG, Thermo Fisher Scientific, Inc. Becton, Dickinson and Company, Abbott, Siemens Healthcare GmbH (Siemens AG), bioMérieux SA, Danaher Corporation, and Agilent Technologies. Merck KGaA, Promega
==References==
{{Reflist|colwidth=30em}}

Latest revision as of 13:14, 6 September 2021