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| <div class="nonumtoc">__TOC__</div>
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| ==3. Adding COVID-19 and other virus testing to your laboratory==
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| Does using one method make the most sense, or will your lab turn to multiple methods for virus testing?
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| ===3.1 What methodologies will you use?===
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| ====3.1.1 PCR====
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| 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.
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| 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.
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| 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.
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| 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.
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| 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>
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| ====3.1.2 Pooled testing====
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| 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>
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| 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 Emergency Use Authorization 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]].
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| ====3.1.3 Rapid antigen testing====
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| 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. On one hand, antigen testing is more rapid and convenient because the extraction and amplification steps of PCR are not use, but 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">{{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="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>
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| A theory increasingly gaining traction, however, is that "[a] higher frequency of testing makes up for poor sensitivity.”<ref name="ServiceRadical2020" />
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| * <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>
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| * <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>
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| * Also see Nature article below
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| ====3.1.4 LAMP and CRISPR====
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| Alternatives such as LAMP and CRISPR:
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| * https://www.fiercebiotech.com/medtech/oxford-researchers-develop-portable-covid-19-test-costing-less-than-25 (LAMP)
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| * <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> (LAMP, sequencing, CRISPR, antigen)
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| * https://thejewishvoice.com/2020/08/10-ways-israeli-scientists-are-improving-corona-testing-part-1/ (Rapid saliva, breath, AI, spectrometry, ultrahigh frequency radiation)
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| ====3.1.5 Point-of-care testing====
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| POLs:
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| * https://hitconsultant.net/2020/08/03/talis-nih-contract-for-point-of-care-covid-19-testing/
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| * https://www.nih.gov/news-events/news-releases/nih-delivering-new-covid-19-testing-technologies-meet-us-demand
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| ===3.2 What equipment and supplies will you need?===
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| ====Instruments====
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| [[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."
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| https://www.thermofisher.com/search/browse/category/us/en/602552/PCR+Machines+%28Thermal+Cyclers%29+%26+Accessories
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| https://blog.biomeme.com/how-do-you-test-for-covid-19
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| ====Reagents====
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| template DNA, PCR primers and probes, dNTPs, PCR buffers, enzymes, and master mixes
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| ====Consumables====
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| PCR tubes, plates, and other accessories
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| https://www.sigmaaldrich.com/labware/labware-products.html?TablePage=9577275
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| ====Software and services====
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| ====Vendors====
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| 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
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| ==References==
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| {{Reflist|colwidth=30em}}
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