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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.  
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>
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>




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Methods for speeding up PCR (and alternatives such as LAMP and CRISPR):
Methods for speeding up PCR (and alternatives such as LAMP and CRISPR):


* https://news.vanderbilt.edu/2020/07/29/streamlined-diagnostic-approach-to-covid-19-can-avoid-potential-testing-logjam/
 
* https://www.techexplorist.com/improving-speed-gold-standard-covid-19-diagnostic-test/34069/
* https://www.sciencemag.org/news/2020/08/radical-shift-testing-strategy-needed-reopen-schools-and-businesses-researchers-say
* https://www.sciencemag.org/news/2020/08/radical-shift-testing-strategy-needed-reopen-schools-and-businesses-researchers-say
* https://asiatimes.com/2020/08/pooled-virus-tests-help-stretched-health-services/
* https://asiatimes.com/2020/08/pooled-virus-tests-help-stretched-health-services/

Revision as of 18:21, 6 August 2020

3. Adding COVID-19 and other virus testing to your laboratory

3.1 Prepping based on methodology

In the previous chapter, the most common testing methodologies for COVID-19 and other 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."[1] 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."[2] Various efforts have been made over the years to bring costs down by modifying how heating and temperature control are performed[3][4][5][6], 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.[7][8][9][10][11][12][13][14][15] 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. propose direct heating of the sample-containing nasopharyngeal swab at 70 °C for 10 minutes in place of RNA extraction.[16] 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."[17][18] Wee et al. skip RNA extraction and nucleic acid purification by using a single-tube homogeneous reaction method run on a lightweight, portable thermocycler.[19][20]



The explosion of new coronavirus tests that could help to end the pandemic https://www.nature.com/articles/d41586-020-02140-8

Methods for speeding up PCR (and alternatives such as LAMP and CRISPR):


Alternative testing:

Alternative reagents:

POLs:


3.2 The equipment

Instruments

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

3.3 The 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

  1. ↑ Kenneth Research (23 June 2020). "Polymerase Chain Reaction Market Sector Analysis Report, Regional Outlook & Competitive Share & Forecast - 2023". MarketWatch. https://www.marketwatch.com/press-release/polymerase-chain-reaction-market-sector-analysis-report-regional-outlook-competitive-share-forecast---2023-2020-06-23. Retrieved 06 August 2020. 
  2. ↑ Dove, A. (2018). "PCR: Thirty-five years and counting". Science 360 (6389): 670–672. doi:10.1126/science.360.6389.673-c. 
  3. ↑ Wong, G.; Wong, I. Chan, K. et al. (2015). "A Rapid and Low-Cost PCR Thermal Cycler for Low Resource Settings". PLoS One 10 (7): e0131701. doi:10.1371/journal.pone.0131701. 
  4. ↑ Kuznetsov, S.; Doonan, C.; Wilson, N. et al. (2015). "DIYbio Things: Open Source Biology Tools as Platforms for Hybrid Knowledge Production and Scientific Participation". Proceedings of the 33rd Annual ACM Conference on Human Factors in Computing Systems: 4065–68. doi:10.1145/2702123.2702235. 
  5. ↑ Norton, D. (11 May 2016). "Phila. med tech startup working on multimillion dollar government contract". Philadelphia Business Journal. https://www.bizjournals.com/philadelphia/news/2016/05/11/government-contract-biomeme-hiring-med-tech.html. Retrieved 06 August 2020. 
  6. ↑ An, J.; Jiang, Y.; Shi, B. et al. (2020). "Low-Cost Battery-Powered and User-Friendly Real-Time Quantitative PCR System for the Detection of Multigene". Micromachines 11: 435. doi:10.3390/mi11040435. 
  7. ↑ Herper, M.; Branswell, H. (10 March 2020). "Shortage of crucial chemicals creates new obstacle to U.S. coronavirus testing". STAT. https://www.statnews.com/2020/03/10/shortage-crucial-chemicals-us-coronavirus-testing/. Retrieved 10 April 2020. 
  8. ↑ Hale, C. (18 March 2020). "Qiagen aims to more than quadruple its COVID-19 reagent production in 6 weeks". Fierce Biotech. https://www.fiercebiotech.com/medtech/qiagen-aims-to-more-than-quadruple-its-covid-19-reagent-production-6-weeks. Retrieved 10 April 2020. 
  9. ↑ Mehta, A. (3 April 2020). "Mystery surrounds UK claim of Covid-19 test reagent ‘shortage’". Chemistry World. https://www.chemistryworld.com/mystery-surrounds-uk-claim-of-covid-19-test-reagent-shortage/4011457.article. Retrieved 10 April 2020. 
  10. ↑ Roche, B. (8 April 2020). "Irish scientists develop reagent in effort to ease Covid-19 testing delays". The Irish Times. https://www.irishtimes.com/news/science/irish-scientists-develop-reagent-in-effort-to-ease-covid-19-testing-delays-1.4223897. Retrieved 10 April 2020. 
  11. ↑ Padma, T.V. (13 May 2020). "Efforts to combat Covid-19 in India hit by imported reagent shortages". Chemistry World. https://www.chemistryworld.com/news/efforts-to-combat-covid-19-in-india-hit-by-imported-reagent-shortages/4011718.article#/. Retrieved 19 May 2020. 
  12. ↑ David, E.; Farber, S.E. (20 June 2020). "Survey shows resources for COVID-19 diagnostic testing still limited months later". ABC News. https://abcnews.go.com/Health/survey-shows-resources-covid-19-diagnostic-testing-limited/story?id=71341885. Retrieved 08 July 2020. 
  13. ↑ Johnson, K. (2 July 2020). "NC Labs Facing Shortages In COVID-19 Testing Chemicals". Patch. https://patch.com/north-carolina/charlotte/nc-labs-facing-shortages-covid-19-testing-chemicals. Retrieved 08 July 2020. 
  14. ↑ 14.0 14.1 Mervosh, S.; Fernandez, M. (4 August 2020). "‘It’s Like Having No Testing’: Coronavirus Test Results Are Still Delayed". The New York Times. https://www.nytimes.com/2020/08/04/us/virus-testing-delays.html. Retrieved 05 August 2020. 
  15. ↑ 15.0 15.1 Courage, K.H. (31 July 2020). "Should we be testing fewer people to stop the spread of Covid-19?". Vox. https://www.vox.com/2020/7/31/21336212/covid-19-test-results-delays. Retrieved 05 August 2020. 
  16. ↑ Alcoba-Florez, J.; González-Montelongo, R.; Íñigo-Campos, A.; García-Martínezde Artola, D.; Gil-Campesino, H.;
    The Microbiology Technical Support Team; Ciuffreda, L.; Valenzuela-Fernández, A.; Flores, C. (2020). "Fast SARS-CoV-2 detection by RT-qPCR in preheated nasopharyngeal swab samples". International Journal of Infectious Diseases 97: 66–68. doi:10.1016/j.ijid.2020.05.099.     
  17. ↑ Shapiro, M. (29 July 2020). "Streamlined diagnostic approach to COVID-19 can avoid potential testing logjam". Research News @ Vanderbilt. https://news.vanderbilt.edu/2020/07/29/streamlined-diagnostic-approach-to-covid-19-can-avoid-potential-testing-logjam/. Retrieved 06 August 2020. 
  18. ↑ Adams, N.M.; Leelawong, M.; Benton, A. et al. (2020). "COVID‐19 diagnostics for resource‐limited settings: Evaluation of “unextracted” qRT‐PCR". Journal of Medical Virology. doi:10.1002/jmv.26328. 
  19. ↑ Mehar, P. (27 July 2020). "Improving the speed of gold-standard COVID-19 diagnostic test". Tech Explorist. https://www.techexplorist.com/improving-speed-gold-standard-covid-19-diagnostic-test/34069/. Retrieved 06 August 2020. 
  20. ↑ Wee, S.K.; Sivalingam, S.P.; Yap, E.P.H. (2020). "Rapid Direct Nucleic Acid Amplification Test without RNA Extraction for SARS-CoV-2 Using a Portable PCR Thermocycler". Genes 11 (6): 664. doi:10.3390/genes11060664. 
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