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Sensitivity of different RT-qPCR solutions for SARS-CoV-2 detection.

Author(s): Alcoba-Florez J, Gil-Campesino H, Artola DG, González-Montelongo R, Valenzuela-Fernández A, Ciuffreda L, Flores C

Publication: Int J Infect Dis, 2020, Vol. 99, Page 190-192

PubMed ID: 32745627 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to compare detection of SARS-CoV-2 using different targets and six different real-time PCR based assays. The potential to heat the specimen instead of extracting RNA was also evaluated using the most sensitive assay. Specimens from 98 patients with SARS-CoV-2 were collected in viral transport media (VTM). No other details of specimen collection were provided. RNA was extracted from VTM using the MagNA Pure Compact Nucleic Acid Isolation Kit I or the STARMag Viral DNA/RNA 200C Kit. SARS-CoV-2 virus was detected using the real-time PCR based TaqMan Fast Virus 1-Step Master Mix combined with validated primer-probe sets for the E, N, and RdRp genes; the LightMix1 Modular SARS-CoV (COVID19) Assay with primers for the E, N, and RdRp genes; the SARS-COV-2 R-GENE Kit with primers for the N and RdRp genes; the TaqPath COVID-19 CE-IVD RT-PCR Kit with primers for ORF1ab, S, and N genes; the Genesig Real-Time PCR COVID-19 Kit with primers for the RdRp gene; and the Real Accurate Quadruplex corona-plus PCR Kit with primers for the N gene. False negatives were defined as no detection after 45 cycles. To investigate the need for RNA extraction, specimens were heated for 10 min to 70°C and SARS-CoV-2 was detected directly using the LightMix1 Modular SARS-CoV (COVID19) Assay with primers for the E gene.

   Summary of Findings:

   The fewest false negative results were obtained using the LightMix1 Modular SARS-CoV (COVID19) Assay with the E-gene primers (2 false negatives, sensitivity 97.9%), followed by the TaqMan Fast Virus 1-Step Master Mix also with primers for the E-gene (4 false negatives, 95.9% sensitivity). The highest number of false negative results were found using the SARS-COV-2 R-GENE Kit with primers for RdRp (39 false negatives, sensitivity 60.2%), E (34 false negatives, sensitivity 66.3%), and N (33 false negatives, sensitivity 65.3%) genes or the TaqPath COVID-19 CE-IVD RT-PCR Kit and primers for ORF1ab (34 false negatives, sensitivity 65.3%). Combining E and RdRp genes in the TaqMan Fast Virus 1-Step Master Mix Kit increased sensitivity to be equivalent to that using the LightMix1 Modular SARS-CoV (COVID-9) Assay with primers for the E-gene. Use of primers for the E gene in combination with the N or RdRp genes slightly improved the sensitivity of the SARS-COV-2 R-GENE Kit (71.4% and 69.4%, respectively), but use of multiple genes did not improve the sensitivity of any other kits. Importantly, the most sensitive method, the LightMix1 Modular SARS-CoV (COVID19) assay with the E-gene primers, had a sensitivity of 72.5% when specimens were heated for 10 min to 70°C instead of RNA extraction.

   Biospecimens

   • Cell - Nasal cavity

   Preservative Types

   • None (Fresh)

   Diagnoses:

   • Pneumonia/Respiratory Infection

   Platform:

   Analyte	Technology Platform
   RNA	Real-time qRT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Real-time qRT-PCR Specific	Technology platform	TaqMan Fast Virus 1-Step Master Mix LightMix1 Modular SARS-CoV assay SARS-COV-2 R-GENE assay TaqPath COVID-19 CE-IVD RT-PCR Kit Genesig Real-Time PCR COVID-19 kit Real Accurate Quadruplex corona-plus PCR Kit
   Real-time qRT-PCR Specific	Targeted nucleic acid	E-gene N-gene RdRp-gene ORF1ab gene S-gene
   Analyte Extraction and Purification	Analyte isolation method	MagNA Pure Compact Nucleic Acid Isolation Kit I or the STARMag Viral DNA/RNA 200C kits Heat to 70°C

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