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As the pandemic has progressed, you may have heard talk of a "delta" variant of SARS-CoV-2, which is reportedly more contagious and virulent than the initial strain that kicked off the pandemic.<ref name="CDCDelta21">{{cite web |url=https://www.cdc.gov/coronavirus/2019-ncov/variants/delta-variant.html |title=Delta Variant: What We Know About the Science |author=Centers for Disease Control and Prevention |publisher=Centers for Disease Control and Prevention |date=26 August 2021 |accessdate=18 September 2021}}</ref> One or more variants of a virus are expected as time progresses, and some of those variants can cause significantly more problems than the source virus. As such, analytical testing of the virus over time is vital to public health.
'''PCR considerations'''


The purpose of variant testing can be described in two ways, one for public health reasons and another for clinical care reasons. On the public health side, analysis of SARS-CoV-2 variants provides an unbiased, population-level view "of the specific viral strains in circulation and monitors changes in the viral genome over time."<ref name="BuchanSARS21">{{cite web |url=https://www.amp.org/AMP/assets/File/clinical-practice/COVID/AMP_RC_VariantTestingforSARSCOV2_4_28_21.pdf |format=PDF |title=SARS-CoV-2 Variant Testing |work=Rapid Communication |author=Buchan, B.W.; Wolk, D.M.; Yao, J.D. |publisher=Association for Molecular Pathology |date=28 April 2021 |accessdate=18 September 2021}}</ref> With enough public health laboratories conducting this type of analysis—typically whole-genome sequencing (WGS) using [[next-generation sequencing]] (NGS) techniques—a clearer picture of how an outbreak spreads is gained, as well as what variants are taking hold and further threatening human populations (even those that are vaccinated). This information is typically shared through the public health system for surveillance and reporting purposes, though the affected patients themselves may never see the data.<ref name="BuchanSARS21" />
Whether adding PCR to your existing laboratory, modifying existing PCR workflows, or starting from scratch, preventing contamination is a top priority. As PCR can effectively amplify even the tiniest of quantities of DNA and RNA, the risk of amplifying a contaminant and ruining the validity of an assay is very real.<ref name="MifflinSetting03">{{cite book |url=http://www.biosupplynet.com/pdf/01_pcr_primer_p.5_14.pdf |format=PDF |chapter=Chapter 1: Setting Up a PCR Laboratory |title=PCR Primer |author=Mifflin, T.E. |editor=Dieffenbach, C.; Dveksler, G. |edition=2nd |publisher=Cold Spring Harbor Laboratory Press |pages=5–14 |year=2003 |isbn=9780879696542 |accessdate=13 August 2020}}</ref><ref name="RochePCR06">{{cite book |url=https://www.gene-quantification.de/ras-pcr-application-manual-3rd-ed.pdf |format=PDF |chapter=Chapter 2: General Guidelines |title=PCR Applications Manual |editor=Degen, H.-J.; Deufel, A.; Eisel, D. et al. |edition=3rd |publisher=Roche Diagnostics GmbH |pages=19–38 |year=2006 |accessdate=13 August 2020}}</ref><ref name="AhmedSetting14">{{cite book |url=http://grcpk.com/wp-content/uploads/2014/10/12.-Setting-up-PCR-Lab.pdf |format=PDF |chapter=Chapter 12: Setting-up a PCR Lab |title=Manual of PCR |author=Ahmed, S. |publisher=Genetics Resource Centre |year=2014 |accessdate=13 August 2020}}</ref><ref name="RedigTheDevil14">{{cite web |url=https://bitesizebio.com/19880/the-devil-is-in-the-details-how-to-setup-a-pcr-laboratory/ |title=The Devil is in the Details: How to Setup a PCR Laboratory |author=Redig, J. |work=BiteSizeBio |date=01 August 2014 |accessdate=13 August 2020}}</ref><ref name="BioChekTheBasics18">{{cite web |url=https://www.biochek.com/wp-content/uploads/2018/05/BioChek-E-book-The-basics-of-PCR.pdf |format=PDF |title=The basics of PCR: Detecting viruses and bacteria red-handed |publisher=BioChek BV |date=May 2018 |accessdate=13 August 2020}}</ref><ref name="DasMitig18">{{cite journal |title=Mitigating PCR /Amplicon Contamination in a High Risk High Burden Mycobacterial Reference Laboratory in a Resource Limited Setting |journal=Mycobacterial Diseases |author=Das, P.K.; Ganguly, S.B.; Mandal, B. |volume=8 |issue=2 |at=261 |year=2018 |doi=10.4172/2161-1068.1000261}}</ref><ref name="WHODos18">{{cite web |url=https://www.who.int/teams/global-malaria-programme/case-management/diagnosis/nucleic-acid-amplification-based-diagnostics/dos-and-don-ts-for-molecular-testing |title=Dos and Don'ts for molecular testing |author=World Health Organization |publisher=World Health Organization |date=31 January 2018 |accessdate=08 September 2021}}</ref> Contamination typically comes from non-amplified environmental substances such as aerosols, and from carryover contamination of amplicons from earlier PCR cycles. As such, not only do best-practice processes and procedures (P&P) need to be followed (e.g., unidirectional workflow, thorough cleaning procedures, proper preparation and disposal), but also where to place PCR-related equipment must be carefully considered.<ref name="MifflinSetting03" /><ref name="RochePCR06" /><ref name="RedigTheDevil14" /><ref name="DasMitig18" />  


On the clinical care side, analysis of SARS-CoV-2 variants provides further insights into improving COVID-19 patient outcomes. Buchan ''et al.'' identify three potential insights that clinicians may gain, noting that variant testing allows the clinician<ref name="BuchanSARS21" />:
When possible, separate rooms for sample preparation, PCR setup, and post-PCR activities, each with their own airflow control, are encouraged.<ref name="MifflinSetting03" /><ref name="RochePCR06" /><ref name="BioChekTheBasics18" /><ref name="DasMitig18" /><ref name="WHODos18" /> However, the laboratory attempting to add PCR to an already small clinical diagnostic lab may not have the luxury of having multiple rooms. In that case, a single-room setup may suffice, if the workflow areas remain demarcated or physically partitioned. Additionally, a single-room setup must also have stricter P&P and design controls to offset the space constraints. For example, the sample preparation area of the room should have a laminar flow hood with UV light that is regularly cleaned, and post-PCR analysis may need to occur later in the day after cleanup from prior steps.<ref name="MifflinSetting03" /><ref name="AhmedSetting14" /><ref name="WHODos18" /> Of course, always maintaining unidirectional workflow—regardless of number of rooms—is also critical to minimizing contamination. For example, technicians shouldn't be transporting amplified materials into the DNA extraction area.


* to distinguish between an existing, persistent infection caused by one viral strain vs. re-infection by a different viral strain;
Although dated, Roche Diagnostics' 2006 ''[https://www.gene-quantification.de/ras-pcr-application-manual-3rd-ed.pdf PCR Applications Manual]''<ref name="RochePCR06" /> provides a detailed breakdown of setting up the laboratory for PCR. [https://www.longdom.org/open-access/mitigating-pcr-amplicon-contamination-in-a-high-risk-high-burden-mycobacterial-reference-laboratory-in-a-resource-limited-setting-2161-1068-1000261.pdf Das ''et al.'']<ref name="DasMitig18" />  and [https://bitesizebio.com/19880/the-devil-is-in-the-details-how-to-setup-a-pcr-laboratory/ Dr. Jennifer Redig]<ref name="RedigTheDevil14" /> provide additional valuable insight. The World Health Organization (WHO) also provides [https://www.who.int/teams/global-malaria-programme/case-management/diagnosis/nucleic-acid-amplification-based-diagnostics/dos-and-don-ts-for-molecular-testing guidance] for setting up molecular testing in the lab.<ref name="WHODos18" />
* to determine whether a patient not responding to a treatment is affected by a specific viral spike protein (S) gene mutation that is "potentially resistant or less susceptible to neutralizing antibodies or monoclonal antibodies"; and
* to detect in the serum or plasma of a patient post-vaccination "viral S gene substitutions in specific variants that are potentially resistant or less susceptible" to the antibodies the vaccine generates.


If, for example, a patient is diagnosed with a variant that is tied to heightened disease severity, the clinician can opt for additional treatments early on to counteract the variant's effects on the patient. This testing is done in a hospital or reference lab by WGS or by targeting a portion of the genome (e.g., a spike protein) or a specific mutation (using RT-PCR). However, according to Buchan ''et al.'', the contributions a mutation makes to a "variant's attributes is not entirely understood, and there is no definitive evidence that directly links a given mutation to poor outcomes, significantly reduced efficacy of SARS-CoV-2 therapies, or vaccine coverage."<ref name="BuchanSARS21" />
'''Isothermal amplification considerations'''


That said, and leaving the public health element to the side, if you are a laboratory conducting clinical analyses of SARS-CoV-2 specimens, the likelihood of including viral sequencing and sequence analysis for variant testing may be low for your facility. Such testing is a multi-step process requiring a non-trivial set of resources, often available to large commercial diagnostic laboratories.<ref name="BuchanSARS21" /><ref name="WilliamsEnhanc21">{{cite web |url=https://health.mo.gov/emergencies/ert/alertsadvisories/pdf/update21921.pdf |format=PDF |title=Enhancing Public Health Surveillance for Variant SARSCoV-2 Viruses in Missouri |author=Williams, R.W. |publisher=Missouri Department of Health & Senior Services |date=19 February 2021 |accessdate=18 September 2021}}</ref> The CDC represents one possible workflow for genomic sequencing as such<ref name="CDCRole21">{{cite web |url=https://www.cdc.gov/coronavirus/2019-ncov/variants/cdc-role-surveillance.html |title=CDC’s Role in Tracking Variants |author=Centers for Disease Control and Prevention |publisher=Centers for Disease Control and Prevention |date=08 September 2021 |accessdate=18 September 2021}}</ref>:
Similarly, because DNA and RNR amplification is involved, contamination concerns exist with isothermal amplification techniques. Multiple pipetting steps and repeated freezing and thawing of reagents can still lead to cross-contamination<ref name="DiegoProgress19">{{cite journal |title=Progress in loop-mediated isothermal amplification assay for detection of Schistosoma mansoni DNA: Towards a ready-to-use test |journal=Scientific Reports |author=Diego, J. G.-B.; Fernández-Soto, P.; Crego-Vicente, B. et al. |volume=9 |at=14744 |year=2019 |doi=10.1038/s41598-019-51342-2 |pmid=31611563 |pmc=PMC6791938}}</ref>, as does opening the reaction chamber after reaction is completed.<ref name="MartzyChall19">{{cite journal |title=Challenges and perspectives in the application of isothermal DNA amplification methods for food and water analysis |journal=Analytical and Bioanalytical Chemistry |author=Martzy, R.; Kolm, C.; Krska, R. et al. |volume=411 |pages=1695–1702 |year=2019 |doi=10.1007/s00216-018-1553-1 |pmid=30617408 |pmc=PMC6453865}}</ref> However, the advent of microfluidics and lateral flow technologies in isothermal amplification processes has seen the development of "fully enclosed microstructured devices into which performing the isothermal amplification reduces the risk of sample contamination and allows integration and portable device realization."<ref name="ZanoliIsotherm13">{{cite journal |title=Isothermal Amplification Methods for the Detection of Nucleic Acids in Microfluidic Devices |journal=Biosensors |author=Zanoli, L.M.; Spoto, G. |volume=3 |issue=1 |pages=18–43 |year=2013 |doi=10.3390/bios3010018 |pmid=25587397 |pmc=PMC4263587}}</ref><ref name="RoskosSimple13">{{cite journal |title=Simple System for Isothermal DNA Amplification Coupled to Lateral Flow Detection |journal=PLoS One |author=Roskos, K.; Hickerson, A.I.; Lu, H.-W. et al. |volume=8 |issue=7 |at=e69355 |year=2013 |doi=10.1371/journal.pone.0069355 |pmid=23922706 |pmc=PMC3724848}}</ref> Even more cutting-edge techniques to reduce contamination such as the CUT-LAMP technique of Bao ''et al.''<ref name="BaoCUT20">{{cite journal |title=CUT-LAMP: Contamination-Free Loop-Mediated Isothermal Amplification Based on the CRISPR/Cas9 Cleavage |journal=ACS Sensors |author=Bao, Y.; Jiang, Y.; Xiong, E. et al. |volume=5 |issue=4 |pages=1082–91 |year=2020 |doi=10.1021/acssensors.0c00034 |pmid=32242409}}</ref> or the dUTP/UDG system for COVID-19 RT-LAMP reactions of Kellner ''et al.''<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> hold further promise in making isothermal amplification processes in the laboratory easier to manage. That said, labs running isothermal amplification processes such as LAMP requiring analysis with agarose gel electrophoresis or a method requiring the opening of reaction vessels will preferably have a secondary area set up for analysis steps so as to minimize the chances of contamination.<ref name="NEBLoop14">{{cite web |url=https://www.neb.com/protocols/2014/06/17/loop-mediated-isothermal-amplification-lamp |title=Loop-mediated Isothermal Amplification (LAMP) |publisher=New England BioLabs |date=17 June 2014 |accessdate=14 August 2020}}</ref><ref name="Fernández-SotoDevelop14">{{cite journal |title=Development of a Highly Sensitive Loop-Mediated Isothermal Amplification (LAMP) Method for the Detection of ''Loa loa'' |journal=PLoS One |author=Fernández-Soto, P.; Mvoulouga, P.O.; Akue, J.P. et al. |volume=9 |issue=4 |at=e94664 |year=2014 |doi=10.1371/journal.pone.0094664 |pmid=24722638 |pmc=PMC3983228}}</ref>
 
# A specimen containing the SARS-CoV-2 virus is received by the lab and promptly entered into the [[laboratory information system]] (LIS).
# The RNA of the SARS-CoV-2 virus is extracted from the sample and then converted to complimentary DNA. It is then enriched and loaded into the appropriate NGS instrument.
# The instrument runs the sequencing and raw data is collected, with the lab maintaining quality control steps. The raw data is turned into actionable sequence data.
# The sequence data is verified for suitability, with a resequencing occurring if found to be inadequate. Otherwise, the data is then analyzed and interpreted.
# The final approved sequencing results are reported to the appropriate state, local, tribal, or territorial public health department.<ref name="CDCGuidanceSeq21">{{cite web |url=https://www.cdc.gov/coronavirus/2019-ncov/lab/resources/reporting-sequencing-guidance.html |title=Guidance for Reporting SARS-CoV-2 Sequencing Result |author=Centers for Disease Control and Prevention |publisher=Centers for Disease Control and Prevention |date=23 June 2021 |accessdate=19 September 2021}}</ref>
 
If your diagnostic lab has or is planning on adding sequencing tools to supplement clinical diagnostics, it may make sense to consider adding variant testing to your available options. But in reality, this sort of testing may largely be left to large institutions, such as the University of Rochester Vaccine Treatment Evaluation Unit or the Yale School of Public Health.<ref name="DupuyCOVID21">{{cite web |url=https://apnews.com/article/fact-checking-549965482111 |title=COVID-19 variants tested through genome sequencing |author=Dupuy, B. |work=Reuters Fact-Checking |date=28 July 2021 |accessdate=18 September 2021}}</ref>


==References==
==References==
{{Reflist|colwidth=30em}}
{{Reflist|colwidth=30em}}

Revision as of 19:34, 3 February 2022

PCR considerations

Whether adding PCR to your existing laboratory, modifying existing PCR workflows, or starting from scratch, preventing contamination is a top priority. As PCR can effectively amplify even the tiniest of quantities of DNA and RNA, the risk of amplifying a contaminant and ruining the validity of an assay is very real.[1][2][3][4][5][6][7] Contamination typically comes from non-amplified environmental substances such as aerosols, and from carryover contamination of amplicons from earlier PCR cycles. As such, not only do best-practice processes and procedures (P&P) need to be followed (e.g., unidirectional workflow, thorough cleaning procedures, proper preparation and disposal), but also where to place PCR-related equipment must be carefully considered.[1][2][4][6]

When possible, separate rooms for sample preparation, PCR setup, and post-PCR activities, each with their own airflow control, are encouraged.[1][2][5][6][7] However, the laboratory attempting to add PCR to an already small clinical diagnostic lab may not have the luxury of having multiple rooms. In that case, a single-room setup may suffice, if the workflow areas remain demarcated or physically partitioned. Additionally, a single-room setup must also have stricter P&P and design controls to offset the space constraints. For example, the sample preparation area of the room should have a laminar flow hood with UV light that is regularly cleaned, and post-PCR analysis may need to occur later in the day after cleanup from prior steps.[1][3][7] Of course, always maintaining unidirectional workflow—regardless of number of rooms—is also critical to minimizing contamination. For example, technicians shouldn't be transporting amplified materials into the DNA extraction area.

Although dated, Roche Diagnostics' 2006 PCR Applications Manual[2] provides a detailed breakdown of setting up the laboratory for PCR. Das et al.[6] and Dr. Jennifer Redig[4] provide additional valuable insight. The World Health Organization (WHO) also provides guidance for setting up molecular testing in the lab.[7]

Isothermal amplification considerations

Similarly, because DNA and RNR amplification is involved, contamination concerns exist with isothermal amplification techniques. Multiple pipetting steps and repeated freezing and thawing of reagents can still lead to cross-contamination[8], as does opening the reaction chamber after reaction is completed.[9] However, the advent of microfluidics and lateral flow technologies in isothermal amplification processes has seen the development of "fully enclosed microstructured devices into which performing the isothermal amplification reduces the risk of sample contamination and allows integration and portable device realization."[10][11] Even more cutting-edge techniques to reduce contamination such as the CUT-LAMP technique of Bao et al.[12] or the dUTP/UDG system for COVID-19 RT-LAMP reactions of Kellner et al.[13] hold further promise in making isothermal amplification processes in the laboratory easier to manage. That said, labs running isothermal amplification processes such as LAMP requiring analysis with agarose gel electrophoresis or a method requiring the opening of reaction vessels will preferably have a secondary area set up for analysis steps so as to minimize the chances of contamination.[14][15]

References

  1. ↑ 1.0 1.1 1.2 1.3 Mifflin, T.E. (2003). "Chapter 1: Setting Up a PCR Laboratory". In Dieffenbach, C.; Dveksler, G. (PDF). PCR Primer (2nd ed.). Cold Spring Harbor Laboratory Press. pp. 5–14. ISBN 9780879696542. http://www.biosupplynet.com/pdf/01_pcr_primer_p.5_14.pdf. Retrieved 13 August 2020. 
  2. ↑ 2.0 2.1 2.2 2.3 Degen, H.-J.; Deufel, A.; Eisel, D. et al., ed. (2006). "Chapter 2: General Guidelines" (PDF). PCR Applications Manual (3rd ed.). Roche Diagnostics GmbH. pp. 19–38. https://www.gene-quantification.de/ras-pcr-application-manual-3rd-ed.pdf. Retrieved 13 August 2020. 
  3. ↑ 3.0 3.1 Ahmed, S. (2014). "Chapter 12: Setting-up a PCR Lab" (PDF). Manual of PCR. Genetics Resource Centre. http://grcpk.com/wp-content/uploads/2014/10/12.-Setting-up-PCR-Lab.pdf. Retrieved 13 August 2020. 
  4. ↑ 4.0 4.1 4.2 Redig, J. (1 August 2014). "The Devil is in the Details: How to Setup a PCR Laboratory". BiteSizeBio. https://bitesizebio.com/19880/the-devil-is-in-the-details-how-to-setup-a-pcr-laboratory/. Retrieved 13 August 2020. 
  5. ↑ 5.0 5.1 "The basics of PCR: Detecting viruses and bacteria red-handed" (PDF). BioChek BV. May 2018. https://www.biochek.com/wp-content/uploads/2018/05/BioChek-E-book-The-basics-of-PCR.pdf. Retrieved 13 August 2020. 
  6. ↑ 6.0 6.1 6.2 6.3 Das, P.K.; Ganguly, S.B.; Mandal, B. (2018). "Mitigating PCR /Amplicon Contamination in a High Risk High Burden Mycobacterial Reference Laboratory in a Resource Limited Setting". Mycobacterial Diseases 8 (2): 261. doi:10.4172/2161-1068.1000261. 
  7. ↑ 7.0 7.1 7.2 7.3 World Health Organization (31 January 2018). "Dos and Don'ts for molecular testing". World Health Organization. https://www.who.int/teams/global-malaria-programme/case-management/diagnosis/nucleic-acid-amplification-based-diagnostics/dos-and-don-ts-for-molecular-testing. Retrieved 08 September 2021. 
  8. ↑ Diego, J. G.-B.; Fernández-Soto, P.; Crego-Vicente, B. et al. (2019). "Progress in loop-mediated isothermal amplification assay for detection of Schistosoma mansoni DNA: Towards a ready-to-use test". Scientific Reports 9: 14744. doi:10.1038/s41598-019-51342-2. PMC PMC6791938. PMID 31611563. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6791938. 
  9. ↑ Martzy, R.; Kolm, C.; Krska, R. et al. (2019). "Challenges and perspectives in the application of isothermal DNA amplification methods for food and water analysis". Analytical and Bioanalytical Chemistry 411: 1695–1702. doi:10.1007/s00216-018-1553-1. PMC PMC6453865. PMID 30617408. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6453865. 
  10. ↑ Zanoli, L.M.; Spoto, G. (2013). "Isothermal Amplification Methods for the Detection of Nucleic Acids in Microfluidic Devices". Biosensors 3 (1): 18–43. doi:10.3390/bios3010018. PMC PMC4263587. PMID 25587397. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4263587. 
  11. ↑ Roskos, K.; Hickerson, A.I.; Lu, H.-W. et al. (2013). "Simple System for Isothermal DNA Amplification Coupled to Lateral Flow Detection". PLoS One 8 (7): e69355. doi:10.1371/journal.pone.0069355. PMC PMC3724848. PMID 23922706. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3724848. 
  12. ↑ Bao, Y.; Jiang, Y.; Xiong, E. et al. (2020). "CUT-LAMP: Contamination-Free Loop-Mediated Isothermal Amplification Based on the CRISPR/Cas9 Cleavage". ACS Sensors 5 (4): 1082–91. doi:10.1021/acssensors.0c00034. PMID 32242409. 
  13. ↑ Kellner, M.J.; Ross, J.J.; Schnabl, J. et al. (2020). "A rapid, highly sensitive and open-access SARS-CoV-2 detection assay for laboratory and home testing". bioRxiv. doi:10.1101/2020.06.23.166397. 
  14. ↑ "Loop-mediated Isothermal Amplification (LAMP)". New England BioLabs. 17 June 2014. https://www.neb.com/protocols/2014/06/17/loop-mediated-isothermal-amplification-lamp. Retrieved 14 August 2020. 
  15. ↑ Fernández-Soto, P.; Mvoulouga, P.O.; Akue, J.P. et al. (2014). "Development of a Highly Sensitive Loop-Mediated Isothermal Amplification (LAMP) Method for the Detection of Loa loa". PLoS One 9 (4): e94664. doi:10.1371/journal.pone.0094664. PMC PMC3983228. PMID 24722638. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3983228. 
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