Extraction-free SARS-CoV-2 detection by rapid RT-qPCR universal for all primary respiratory materials

Lübke, Nadine; Senff, Tina; Scherger, Sara; Hauka, Sandra; Andrée, Marcel; Adams, Ortwin; Timm, Jörg; Walker, Andreas

doi:10.1016/j.jcv.2020.104579

Cited by 80 publications

(76 citation statements)

References 7 publications

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“…For samples with Ct < 30, observed sensitivities were similar in all three assays. A previous study assessing PrimeDirect among 91 samples showed higher sensitivity rate than in our study ( Lübke et al, 2020 ). This study showed an overall sensitivity of 84.9% rising to 95.8% among samples with Ct <35 while it is 55.1% and 91.9% among samples with Ct <30 in our study ( Lübke et al, 2020 ).…”

Section: Discussioncontrasting

confidence: 85%

Evaluation of three extraction-free SARS-CoV-2 RT-PCR assays: A feasible alternative approach with low technical requirements

Visseaux

Collin

Houhou‐Fidouh

et al. 2021

Journal of Virological Methods

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The worldwide demand for SARS-CoV-2 RT-PCR testing resulted in a shortage of diagnostic kits. RNA extraction step constitutes a major bottleneck to perform diagnostic. The aim of this study was to assess performances of different extraction-free SARS-CoV-2 RT-PCR assays compared to a reference RT-PCR assay. The panel of evaluation consisted of 94 samples: 69 positive and 25 negative for SARS-CoV-2 by reference RT-PCR. Three extraction-free RT-PCR assays were assessed: (i) PrimeDirect® Probe RT-qPCR Mix (Takara), (ii) PrimeScript®RT-PCR (Takara), and (iii) SARS-CoV-2 SANSURE®BIOTECH Novel Coronavirus (Sansure). The overall sensitivity of PrimeDirect, PrimeScript and Sansure assays was 55.1%, 69.6% and 69.6%, respectively. The sensitivity increased among samples with Ct < 30: 91.9% (n = 34/37), 89.2% (n = 33/37) and 94.6% (n = 35/37) for PrimeDirect, PrimeScript and Sansure assays, respectively. The specificity was 88%, 100% and 100% for PrimeDirect, PrimeScript and Sansure assays, respectively. In the present study, we showed a good sensitivity of extraction-free PCR assays, especially for high viral loads (Ct < 30), except PrimeDirect that displayed imperfect sensitivity and specificity. Despite a lower sensitivity for low viral loads, extraction-free reagents can provide a valuable option, cheaper, easier and less reagent consuming for SARS-CoV-2 diagnostic, especially in laboratory with lower experience and equipment for molecular assays.

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Section: Discussioncontrasting

confidence: 85%

Evaluation of three extraction-free SARS-CoV-2 RT-PCR assays: A feasible alternative approach with low technical requirements

Visseaux

Collin

Houhou‐Fidouh

et al. 2021

Journal of Virological Methods

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“…To date, several protocols that effectively inactivate SARS-CoV-2 in clinical and some research settings have been described, including use of heat to ≥95 °C as a part of diagnostic polymerase chain reaction (quantitative PCR) assays performed in clinical specimens [ 7 , 8 , 9 , 10 ], use of heat to 56 °C for 30 min. in serum samples [ 10 ], use of alcohol-based hand sanitizers [ 11 ], use of ultraviolet (UV) light to disinfect surface contamination [ 12 ] use of lysis buffers containing sodium dodecyl sulfate (SDS) or Triton-X100 in cell culture supernatant or nasopharyngeal samples [ 13 , 14 , 15 , 16 ], and use of heat to 80 °C, Trizol ® , detergents and UV energy in stocks of SARS-CoV-2 harvested from infected Vero E6 cells [ 15 ].…”

Section: Introductionmentioning

confidence: 99%

Inactivation of Material from SARS-CoV-2-Infected Primary Airway Epithelial Cell Cultures

Barrow¹,

Rich²,

Vanderwall³

et al. 2021

MPs

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Given that the airway epithelium is the initial site of infection, study of primary human airway epithelial cells (AEC) infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) will be crucial to improved understanding of viral entry factors and innate immune responses to the virus. Centers for Disease Control and Prevention (CDC) guidance recommends work with live SARS-CoV-2 in cell culture be conducted in a Biosafety Level 3 (BSL-3) laboratory. To facilitate downstream assays of materials from experiments there is a need for validated protocols for SARS-CoV-2 inactivation to facilitate safe transfer of material out of a BSL-3 laboratory. We propagated stocks of SARS-CoV-2, then evaluated the effectiveness of heat (65 °C) or ultraviolet (UV) light inactivation. We infected differentiated human primary AECs with SARS-CoV-2, then tested protocols designed to inactivate SARS-CoV-2 in supernatant, protein isolate, RNA, and cells fixed for immunohistochemistry by exposing Vero E6 cells to materials isolated/treated using these protocols. Heating to 65 °C for 10 min or exposing to UV light fully inactivated SARS-CoV-2. Furthermore, we found in SARS-CoV-2-infected primary AEC cultures that treatment of supernatant with UV light, isolation of RNA with Trizol®, isolation of protein using a protocol including sodium dodecyl sulfate (SDS) 0.1% and Triton X100 1%, and fixation of AECs using 10% formalin and Triton X100 1%, each fully inactivated SARS-CoV-2.

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“…Fortunately, several studies have already looked into SARS-CoV-2 sample preparation to expedite and simplify the process ( Marais et al, 2020 ; Rabe and Cepko 2020 ; Wozniak et al, 2020 ). In addition, some studies have also examined sample preparation-free assay by utilizing RT-PCR ( Lübke et al, 2020 ) and RT-LAMP ( Wei et al, 2020 ). Future works should focus on developing a fully streamlined process (raw sample-in-answer-out) by seamlessly combining the sample preparation with the CRISPR assay.…”

Section: Summary and Future Perspectivementioning

confidence: 99%

CRISPR-based detection of SARS-CoV-2: A review from sample to result

Nouri

Tang

Dong

et al. 2021

Biosensors and Bioelectronics

114

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Extraction-free SARS-CoV-2 detection by rapid RT-qPCR universal for all primary respiratory materials

Abstract: Highlights Rapid SARS-CoV-2 detection without RNA extraction. Universal direct RT-qPCR protocol suitable for all respiratory materials. Significant correlation of Ct values between direct and RNA RT-qPCR. High SARS-CoV-2 detection rate by direct RT-qPCR of 95.8 % for Ct values <35.

Cited by 80 publications

References 7 publications

Evaluation of three extraction-free SARS-CoV-2 RT-PCR assays: A feasible alternative approach with low technical requirements

Evaluation of three extraction-free SARS-CoV-2 RT-PCR assays: A feasible alternative approach with low technical requirements

Inactivation of Material from SARS-CoV-2-Infected Primary Airway Epithelial Cell Cultures

CRISPR-based detection of SARS-CoV-2: A review from sample to result

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