Sensitivity assessment of droplet digital PCR for SARS-CoV-2 detection

Falzone, Luca; Musso, Nicolò; Gattuso, Giuseppe; Bongiorno, Dafne; Palermo, Concetta; Scalia, Guido; Libra, Massimo; Stefani, Stefania

doi:10.3892/ijmm.2020.4673

Cited by 199 publications

(205 citation statements)

References 29 publications

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“…As of today, few studies reported the use of ddPCR for a more sensitive SARS-CoV-2 detection compared to RT-PCR 12 , 14 – 16 . To our knowledge, this is the first report of direct quantitation of SARS-CoV-2 RNA performed on a consistent number of clinical samples and comparing two different nasopharyngeal swabs.…”

Section: Discussionmentioning

confidence: 99%

Assessment of the direct quantitation of SARS-CoV-2 by droplet digital PCR

Deiana

Mori

Piubelli

et al. 2020

Sci Rep

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Droplet digital PCR (ddPCR) is a sensitive and reproducible technology widely used for quantitation of several viruses. The aim of this study was to evaluate the 2019-nCoV CDC ddPCR Triplex Probe Assay (BioRad) performance, comparing the direct quantitation of SARS-CoV-2 on nasopharyngeal swab with the procedure applied to the extracted RNA. Moreover, two widely used swab types were compared (UTM 3 mL and ESwab 1 mL, COPAN). A total of 50 nasopharyngeal swabs (n = 25 UTM 3 mL and n = 25 ESwab 1 mL) from SARS-CoV-2 patients, collected during the pandemic at IRCCS Sacro Cuore Don Calabria Hospital (Veneto Region, North-East Italy), were used for our purpose. After heat inactivation, an aliquot of swab medium was used for the direct quantitation. Then, we compared the direct method with the quantitation performed on the RNA purified from nasopharyngeal swab by automated extraction. We observed that the direct approach achieved generally equal RNA copies compared to the extracted RNA. The results with the direct quantitation were more accurate on ESwab with a sensitivity of 93.33% [95% CI, 68.05 to 99.83] and specificity of 100.00% for both N1 and N2. On the other hand, on UTM we observed a higher rate of discordant results for N1 and N2. The human internal amplification control (RPP30) showed 100% of both sensitivity and specificity independent of swabs and approaches. In conclusion, we described a direct quantitation of SARS-CoV-2 in nasopharyngeal swab. Our approach resulted in an efficient quantitation, without automated RNA extraction and purification. However, special care needs to be taken on the potential bias due to the conservation of samples and to the heating treatment, as we used thawed and heat inactivated material. Further studies on a larger cohort of samples are warranted to evaluate the clinical value of this direct approach.

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Section: Discussionmentioning

confidence: 99%

Assessment of the direct quantitation of SARS-CoV-2 by droplet digital PCR

Deiana

Mori

Piubelli

et al. 2020

Sci Rep

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“…Another approach is to detect the nucleocapsid (N) gene and to use an open reading frame 1a/b (ORF1b) gene or E gene assay as a confirmatory test [3]. In addition, to improve assay sensitivity, other studies have been focusing on the detection of N, E, or ORF1b using droplet digital polymerase chain reaction (ddPCR) [4][5][6][7].…”

Section: Introductionmentioning

confidence: 99%

Sensitive detection and quantification of SARS-CoV-2 by multiplex droplet digital RT-PCR

Kock

Baselmans

Scharnhorst

et al. 2020

Eur J Clin Microbiol Infect Dis

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The purpose of this study is to develop a one-step droplet digital RT-PCR (RT-ddPCR) multiplex assay that allows for sensitive quantification of SARS-CoV-2 RNA with respect to human-derived RNA and could be used for screening and monitoring of Covid-19 patients. A one-step RT-ddPCR multiplex assay was developed for simultaneous detection of SARS-CoV-2 E, RdRp and N viral RNA, and human Rpp30 DNA and GUSB mRNA, for internal nucleic acid (NA) extraction and RT-PCR control. Dilution series of viral RNA transcripts were prepared in water and total NA extract of Covid-19-negative patients. As reference assay, an E-GUSB duplex RT-PCR was used. GUSB mRNA detection was used to set validity criteria to assure viral RNA and RT-PCR assay quality and to enable quantification of SARS-CoV-2 RNA. In a background of at least 100 GUSB mRNA copies, 5 copies of viral RNA are reliably detectable and 10 copies viral RNA copies are reliably quantifiable. It was found that assay sensitivity of the RT-ddPCR was not affected by the total NA background while assay sensitivity of the gold standard RT-PCR assay is drastically decreased when SARS-CoV-2 copies were detected in a background of total NA extract compared with water. The present study describes a robust and sensitive one-step ddRT-PCR multiplex assay for reliable quantification of SARS-CoV-2 RNA. By determining the fractional abundance of viral RNA with respect to a human housekeeping gene, viral loads from different samples can be compared, what could be used to investigate the infectiveness and to monitor Covid-19 patients.

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“…While reverse-transcriptase RT-PCR is still the gold standard, the ndings in the present study indicate that the assay sensitivity of the RT-PCR assay is reduced due to background NA from the patient sample. By contrast, the sensitivity of the RT-ddPCR multiplex assay was not affected by background NA, and is more sensitive than the gold standard reverse-transcriptase RT-PCR [4][5][6][7] in the clinical setting.…”

Section: Discussionmentioning

confidence: 89%

“…Another approach is to detect the nucleocapsid (N) gene, and to use an open reading frame 1a/b (ORF1b) gene or E gene assay as a con rmatory test [3]. In addition, to improve assay sensitivity, other studies have been focusing on the detection of N, E or ORF1b using droplet digital polymerase chain reaction (ddPCR) [4][5][6][7].…”

Section: Introductionmentioning

confidence: 99%

Sensitive Detection and Quantification of SARS-CoV-2 by Multiplex Droplet Digital RT-PCR

Kock

Baselmans

Scharnhorst

et al. 2020

Preprint

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Purpose. We aimed to develop a one-step droplet digital RT-PCR (RT-ddPCR) multiplex assay that allows for sensitive detection of SARS-CoV-2 RNA with respect to human derived RNA and could be used for triage and monitoring of Covid-19 patients. Methods. A one step RT-ddPCR multiplex assay was developed for simultaneous detection of SARS-CoV-2 E, RdRp and N viral RNA, and human Rpp30 DNA and GUSB mRNA, for internal nucleic acid (NA) extraction and RT-PCR control. Dilution series of viral RNA transcripts were prepared in water and total NA extract of Covid-19 negative patients. As reference assay, an E-GUSB duplex RT-PCR was used. Results. Assay sensitivity of the RT-PCR assay drastically decreased when SARS-CoV-2 copies were detected in a background of total NA extract compared to water, while the sensitivity of the RT-ddPCR was not affected by the total NA background. GUSB mRNA detection was used to set validity criteria to assure viral RNA and RT-PCR assay quality. In a background of at least 100 GUSB mRNA copies, 5 copies of viral RNA are reliably detectable and 10 copies viral RNA copies are reliably quantifiable. Conclusion. The present study describes a robust and sensitive one-step RT-ddPCR multiplex assay for reliable detection and quantification of SARS-CoV-2 RNA. By determining the fractional abundance of viral RNA with respect to a human housekeeping gene, viral loads from different samples can be compared, what could be used to investigate the infectiveness and to monitor Covid-19 patients.

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Sensitivity assessment of droplet digital PCR for SARS-CoV-2 detection

Cited by 199 publications

References 29 publications

Assessment of the direct quantitation of SARS-CoV-2 by droplet digital PCR

Assessment of the direct quantitation of SARS-CoV-2 by droplet digital PCR

Sensitive detection and quantification of SARS-CoV-2 by multiplex droplet digital RT-PCR

Sensitive Detection and Quantification of SARS-CoV-2 by Multiplex Droplet Digital RT-PCR

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