Rapid, convenient and efficient kit-independent detection of SARS-CoV-2 RNA

Michel, Detlef; Danzer, Karin M; Groß, Rüdiger; Conzelmann, Carina; Müller, Janis A.; Freischmidt, Axel; Weishaupt, Jochen H.; Heller, Sandra; Münch, Jan; Michel, Manuela; Stamminger, Thomas; Kleger, Alexander; Otto, Markus

doi:10.1016/j.jviromet.2020.113965

Cited by 12 publications

(20 citation statements)

References 7 publications

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“…Here, we combined heat inactivation with a pretreatment with PK, protease routinely used in nucleic acid preparations and thus widely available in most clinical laboratories. We mentioned previous works using PK to treat nasopharyngeal swab samples; the protease is also starting to be used in the analysis of saliva samples for the detection of SARS-CoV-2 in extraction-free RT-qPCR determinations [21,22,30,31].…”

Section: Discussionmentioning

confidence: 99%

Extraction-free protocol combining proteinase K and heat inactivation for detection of SARS-CoV-2 by RT-qPCR

et al. 2021

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Real-time reverse transcription PCR (RT-qPCR) is the gold-standard technique for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection in nasopharyngeal swabs specimens. The analysis by RT-qPCR usually requires a previous extraction step to obtain the purified viral RNA. Unfortunately, RNA extraction constitutes a bottleneck for early detection in many countries since it is expensive, time-consuming and depends on the availability of commercial kits. Here, we describe an extraction-free protocol for SARS-CoV-2 detection by RT-qPCR from nasopharyngeal swab clinical samples in saline solution. The method includes a treatment with proteinase K followed by heat inactivation (PK+HID method). We demonstrate that PK+HID improves the RT-qPCR performance in comparison to the heat-inactivation procedure. Moreover, we show that this extraction-free protocol can be combined with a variety of multiplexing RT-qPCR kits. The method combined with a multiplexing detection kit targeting N and ORF1ab viral genes showed a sensitivity of 0.99 and a specificity of 0.99 from the analysis of 106 positive and 106 negative clinical samples. In conclusion, PK+HID is a robust, fast and inexpensive procedure for extraction-free RT-qPCR determinations of SARS-CoV-2. The National Administration of Drugs, Foods and Medical Devices of Argentina has recently authorized the use of this method.

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Section: Discussionmentioning

confidence: 99%

Extraction-free protocol combining proteinase K and heat inactivation for detection of SARS-CoV-2 by RT-qPCR

et al. 2021

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“…Cycling conditions were: 45°C for 15 min, 95°C for 10 min followed by 45 cycles of 95°C for 15 s and 60°C for 30 s. The E-Gene primer and probe sequences were taken from Corman et al (Corman et al, 2020). The SARS-CoV-2 specific ORF1ab assay was designed based on the 72 sequences that were available at the time from Michel et al (Michel et al, 2020). KoMa and human cell cmyc were detected to serve as quality and internal amplification (KoMa) control for potential inhibitors of RT-PCR and the respective sampling technique (Kirchner et al, 2010).…”

Section: Laboratory Analysesmentioning

confidence: 99%

Self-collected oral, nasal and saliva samples yield sensitivity comparable to professionally collected oro-nasopharyngeal swabs in SARS-CoV-2 diagnosis among symptomatic outpatients

Gertler

Krause

Loon

et al. 2021

International Journal of Infectious Diseases

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“…The main disadvantage of extraction-free RT-PCR is reduction in sensitivity, which ranges between 81.3 and 94.6%. 50 , 51 , 52 …”

Section: Innovations In Molecular Assaysmentioning

confidence: 99%

Use of emerging testing technologies and approaches for SARS-CoV-2: review of literature and global experience in an Australian context

et al. 2021

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Emerging testing technologies for detection of SARS-CoV-2 include those that are rapid and can be used at point-of-care (POC), and those facilitating high throughput laboratory-based testing. Tests designed to be performed at POC (such as antigen tests and molecular assays) have the potential to expedite isolation of infectious patients and their contacts, but most are less sensitive than standard-of-care reverse transcription polymerase chain reaction (RT-PCR). Data on clinical performance of the majority of emerging assays is limited with most evaluations performed on contrived or stored laboratory samples. Further evaluations of these assays are required, particularly when performed at POC on symptomatic and asymptomatic patients and at various time-points after symptom onset. A few studies have so far shown several of these assays have high specificity. However, large prospective evaluations are needed to confirm specificity, particularly before the assays are implemented in low prevalence settings or asymptomatic populations. High throughput laboratory-based testing includes the use of new sample types (e.g., saliva to increase acceptability) or innovative uses of existing technology (e.g., sample pooling). Information detailing population-wide testing strategies for SARS-COV-2 is largely missing from peer-reviewed literature. Logistics and supply chains are key considerations in any plan to ‘scale up’ testing in the Australian context. The strategic use of novel assays will help strike the balance between achieving adequate test numbers without overwhelming laboratory capacity. To protect testing of high-risk populations, the aims of testing with respect to the phase of the pandemic must be considered.

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Rapid, convenient and efficient kit-independent detection of SARS-CoV-2 RNA

Cited by 12 publications

References 7 publications

Extraction-free protocol combining proteinase K and heat inactivation for detection of SARS-CoV-2 by RT-qPCR

Extraction-free protocol combining proteinase K and heat inactivation for detection of SARS-CoV-2 by RT-qPCR

Self-collected oral, nasal and saliva samples yield sensitivity comparable to professionally collected oro-nasopharyngeal swabs in SARS-CoV-2 diagnosis among symptomatic outpatients

Use of emerging testing technologies and approaches for SARS-CoV-2: review of literature and global experience in an Australian context

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