Microfluidic Nano-Scale qPCR Enables Ultra-Sensitive and Quantitative Detection of SARS-CoV-2
A major challenge in controlling the COVID-19 pandemic is the high false-negative rate of the commonly used RT-PCR methods for SARS-CoV-2 detection in clinical samples. Accurate detection is particularly challenging in samples with low viral loads that are below the limit of detection (LoD) of stand...
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| Veröffentlicht in: | Processes 2020-11, Vol.8 (11), p.1425 |
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| creator | Xie, Xin Gjorgjieva, Tamara Attieh, Zaynoun Dieng, Mame Massar Arnoux, Marc Khair, Mostafa Moussa, Yasmine Al Jallaf, Fatima Rahiman, Nabil Jackson, Christopher A. Messery, Lobna El Pamplona, Khristine Victoria, Zyrone Zafar, Mohammed Ali, Raghib Piano, Fabio Gunsalus, Kristin C. Idaghdour, Youssef |
| description | A major challenge in controlling the COVID-19 pandemic is the high false-negative rate of the commonly used RT-PCR methods for SARS-CoV-2 detection in clinical samples. Accurate detection is particularly challenging in samples with low viral loads that are below the limit of detection (LoD) of standard one- or two-step RT-PCR methods. In this study, we implemented a three-step approach for SARS-CoV-2 detection and quantification that employs reverse transcription, targeted cDNA preamplification, and nano-scale qPCR based on a commercially available microfluidic chip. Using SARS-CoV-2 synthetic RNA and plasmid controls, we demonstrate that the addition of a preamplification step enhances the LoD of this microfluidic RT-qPCR by 1000-fold, enabling detection below 1 copy/µL. We applied this method to analyze 182 clinical NP swab samples previously diagnosed using a standard RT-qPCR protocol (91 positive, 91 negative) and demonstrate reproducible and quantitative detection of SARS-CoV-2 over five orders of magnitude ( |
| doi_str_mv | 10.3390/pr8111425 |
| format | Article |
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Accurate detection is particularly challenging in samples with low viral loads that are below the limit of detection (LoD) of standard one- or two-step RT-PCR methods. In this study, we implemented a three-step approach for SARS-CoV-2 detection and quantification that employs reverse transcription, targeted cDNA preamplification, and nano-scale qPCR based on a commercially available microfluidic chip. Using SARS-CoV-2 synthetic RNA and plasmid controls, we demonstrate that the addition of a preamplification step enhances the LoD of this microfluidic RT-qPCR by 1000-fold, enabling detection below 1 copy/µL. We applied this method to analyze 182 clinical NP swab samples previously diagnosed using a standard RT-qPCR protocol (91 positive, 91 negative) and demonstrate reproducible and quantitative detection of SARS-CoV-2 over five orders of magnitude (<1 to 106 viral copies/µL). Crucially, we detect SARS-CoV-2 with relatively low viral load estimates (<1 to 40 viral copies/µL) in 17 samples with negative clinical diagnosis, indicating a potential false-negative rate of 18.7% by clinical diagnostic procedures. In summary, this three-step nano-scale RT-qPCR method can robustly detect SARS-CoV-2 in samples with relatively low viral loads (<1 viral copy/µL) and has the potential to reduce the false-negative rate of standard RT-PCR-based diagnostic tests for SARS-CoV-2 and other viral infections.</description><identifier>ISSN: 2227-9717</identifier><identifier>EISSN: 2227-9717</identifier><identifier>DOI: 10.3390/pr8111425</identifier><language>eng</language><ispartof>Processes, 2020-11, Vol.8 (11), p.1425</ispartof><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c330t-e41b753dd75eaae4f96cadbce7f41bf474f4eece3639aec44389308586b9e8b73</citedby><cites>FETCH-LOGICAL-c330t-e41b753dd75eaae4f96cadbce7f41bf474f4eece3639aec44389308586b9e8b73</cites><orcidid>0000-0002-2514-3580 ; 0000-0002-2768-9376 ; 0000-0002-6784-4860</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,780,784,27924,27925</link.rule.ids></links><search><creatorcontrib>Xie, Xin</creatorcontrib><creatorcontrib>Gjorgjieva, Tamara</creatorcontrib><creatorcontrib>Attieh, Zaynoun</creatorcontrib><creatorcontrib>Dieng, Mame Massar</creatorcontrib><creatorcontrib>Arnoux, Marc</creatorcontrib><creatorcontrib>Khair, Mostafa</creatorcontrib><creatorcontrib>Moussa, Yasmine</creatorcontrib><creatorcontrib>Al Jallaf, Fatima</creatorcontrib><creatorcontrib>Rahiman, Nabil</creatorcontrib><creatorcontrib>Jackson, Christopher A.</creatorcontrib><creatorcontrib>Messery, Lobna El</creatorcontrib><creatorcontrib>Pamplona, Khristine</creatorcontrib><creatorcontrib>Victoria, Zyrone</creatorcontrib><creatorcontrib>Zafar, Mohammed</creatorcontrib><creatorcontrib>Ali, Raghib</creatorcontrib><creatorcontrib>Piano, Fabio</creatorcontrib><creatorcontrib>Gunsalus, Kristin C.</creatorcontrib><creatorcontrib>Idaghdour, Youssef</creatorcontrib><title>Microfluidic Nano-Scale qPCR Enables Ultra-Sensitive and Quantitative Detection of SARS-CoV-2</title><title>Processes</title><description>A major challenge in controlling the COVID-19 pandemic is the high false-negative rate of the commonly used RT-PCR methods for SARS-CoV-2 detection in clinical samples. Accurate detection is particularly challenging in samples with low viral loads that are below the limit of detection (LoD) of standard one- or two-step RT-PCR methods. In this study, we implemented a three-step approach for SARS-CoV-2 detection and quantification that employs reverse transcription, targeted cDNA preamplification, and nano-scale qPCR based on a commercially available microfluidic chip. Using SARS-CoV-2 synthetic RNA and plasmid controls, we demonstrate that the addition of a preamplification step enhances the LoD of this microfluidic RT-qPCR by 1000-fold, enabling detection below 1 copy/µL. We applied this method to analyze 182 clinical NP swab samples previously diagnosed using a standard RT-qPCR protocol (91 positive, 91 negative) and demonstrate reproducible and quantitative detection of SARS-CoV-2 over five orders of magnitude (<1 to 106 viral copies/µL). Crucially, we detect SARS-CoV-2 with relatively low viral load estimates (<1 to 40 viral copies/µL) in 17 samples with negative clinical diagnosis, indicating a potential false-negative rate of 18.7% by clinical diagnostic procedures. In summary, this three-step nano-scale RT-qPCR method can robustly detect SARS-CoV-2 in samples with relatively low viral loads (<1 viral copy/µL) and has the potential to reduce the false-negative rate of standard RT-PCR-based diagnostic tests for SARS-CoV-2 and other viral infections.</description><issn>2227-9717</issn><issn>2227-9717</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2020</creationdate><recordtype>article</recordtype><recordid>eNpNUEtLxDAYDKLgsu7Bf5Crh2jSpE1zXOr6gPW1db1J-Zp-gUhN1yYr-O9dH4hzmWEGBmYIORb8VErDzzZjKYRQWb5HJlmWaWa00Pv_9CGZxfjCdzBClnkxIc833o6D67e-85beQhhYbaFH-nZfregiQNtjpOs-jcBqDNEn_44UQkcfthCST_BtnGNCm_wQ6OBoPV_VrBqeWHZEDhz0EWe_PCXri8VjdcWWd5fX1XzJrJQ8MVSi1bnsOp0jACpnCgtda1G7XeKUVk4hWpSFNIBWKVkaycu8LFqDZavllJz89O62xDiiazajf4XxoxG8-bqm-btGfgJgj1aR</recordid><startdate>20201109</startdate><enddate>20201109</enddate><creator>Xie, Xin</creator><creator>Gjorgjieva, Tamara</creator><creator>Attieh, Zaynoun</creator><creator>Dieng, Mame Massar</creator><creator>Arnoux, Marc</creator><creator>Khair, Mostafa</creator><creator>Moussa, Yasmine</creator><creator>Al Jallaf, Fatima</creator><creator>Rahiman, Nabil</creator><creator>Jackson, Christopher A.</creator><creator>Messery, Lobna El</creator><creator>Pamplona, Khristine</creator><creator>Victoria, Zyrone</creator><creator>Zafar, Mohammed</creator><creator>Ali, Raghib</creator><creator>Piano, Fabio</creator><creator>Gunsalus, Kristin C.</creator><creator>Idaghdour, Youssef</creator><scope>AAYXX</scope><scope>CITATION</scope><orcidid>https://orcid.org/0000-0002-2514-3580</orcidid><orcidid>https://orcid.org/0000-0002-2768-9376</orcidid><orcidid>https://orcid.org/0000-0002-6784-4860</orcidid></search><sort><creationdate>20201109</creationdate><title>Microfluidic Nano-Scale qPCR Enables Ultra-Sensitive and Quantitative Detection of SARS-CoV-2</title><author>Xie, Xin ; Gjorgjieva, Tamara ; Attieh, Zaynoun ; Dieng, Mame Massar ; Arnoux, Marc ; Khair, Mostafa ; Moussa, Yasmine ; Al Jallaf, Fatima ; Rahiman, Nabil ; Jackson, Christopher A. ; Messery, Lobna El ; Pamplona, Khristine ; Victoria, Zyrone ; Zafar, Mohammed ; Ali, Raghib ; Piano, Fabio ; Gunsalus, Kristin C. ; Idaghdour, Youssef</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c330t-e41b753dd75eaae4f96cadbce7f41bf474f4eece3639aec44389308586b9e8b73</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2020</creationdate><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Xie, Xin</creatorcontrib><creatorcontrib>Gjorgjieva, Tamara</creatorcontrib><creatorcontrib>Attieh, Zaynoun</creatorcontrib><creatorcontrib>Dieng, Mame Massar</creatorcontrib><creatorcontrib>Arnoux, Marc</creatorcontrib><creatorcontrib>Khair, Mostafa</creatorcontrib><creatorcontrib>Moussa, Yasmine</creatorcontrib><creatorcontrib>Al Jallaf, Fatima</creatorcontrib><creatorcontrib>Rahiman, Nabil</creatorcontrib><creatorcontrib>Jackson, Christopher A.</creatorcontrib><creatorcontrib>Messery, Lobna El</creatorcontrib><creatorcontrib>Pamplona, Khristine</creatorcontrib><creatorcontrib>Victoria, Zyrone</creatorcontrib><creatorcontrib>Zafar, Mohammed</creatorcontrib><creatorcontrib>Ali, Raghib</creatorcontrib><creatorcontrib>Piano, Fabio</creatorcontrib><creatorcontrib>Gunsalus, Kristin C.</creatorcontrib><creatorcontrib>Idaghdour, Youssef</creatorcontrib><collection>CrossRef</collection><jtitle>Processes</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Xie, Xin</au><au>Gjorgjieva, Tamara</au><au>Attieh, Zaynoun</au><au>Dieng, Mame Massar</au><au>Arnoux, Marc</au><au>Khair, Mostafa</au><au>Moussa, Yasmine</au><au>Al Jallaf, Fatima</au><au>Rahiman, Nabil</au><au>Jackson, Christopher A.</au><au>Messery, Lobna El</au><au>Pamplona, Khristine</au><au>Victoria, Zyrone</au><au>Zafar, Mohammed</au><au>Ali, Raghib</au><au>Piano, Fabio</au><au>Gunsalus, Kristin C.</au><au>Idaghdour, Youssef</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Microfluidic Nano-Scale qPCR Enables Ultra-Sensitive and Quantitative Detection of SARS-CoV-2</atitle><jtitle>Processes</jtitle><date>2020-11-09</date><risdate>2020</risdate><volume>8</volume><issue>11</issue><spage>1425</spage><pages>1425-</pages><issn>2227-9717</issn><eissn>2227-9717</eissn><abstract>A major challenge in controlling the COVID-19 pandemic is the high false-negative rate of the commonly used RT-PCR methods for SARS-CoV-2 detection in clinical samples. 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