Use of a simplified sample processing step without RNA extraction for direct SARS-CoV-2 RT-PCR detection

Barza, Ruby; Patel, Parul; Sabatini, Linda M.; Singh, Kamaljit

doi:10.1016/j.jcv.2020.104587

Cited by 28 publications

(32 citation statements)

References 4 publications

(5 reference statements)

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“…In Argentina, a trained lab technician processes approximately 24 samples every 1.5 hours. Additionally, the skyrocketing increment on the clinical use of RT-qPCR due to the COVID-19 pandemic led to a worldwide shortage of RNA purification reagents [4,5] thus, many research groups around the world have concentrated their efforts on either simplifying or eliminating the extraction step [6][7][8][9][10][11][12][13][14][15][16].…”

Section: Introductionmentioning

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: Introductionmentioning

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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“…Lübke et al 3 showed with their protocol without extraction using the PrimeDirect TM Probe RT‐qPCR Mix (TaKara) that the SARS‐CoV‐2 detection was 82.4% with 16/91 negative samples characterized by low viral loads (Ct > 35). Barza et al, with their method using the ChromaCode HDPCR TM SARS‐CoV‐2 Research Use Only correctly identified 94% of the samples (81/86) with the same observations concerning the negative returned samples (Ct > 35) 8 . The extraction‐free strategy was also investigated with three other commercial kits showing 51% (37/73) positive detection with SARS‐CoV‐2 RdRp plus EAV control (Roche), 62% (48/77) with real‐time fluorescent RT‐qPCR kit for detecting 2019‐nCoV (BGI), and 45% (41/74) for Detection kit for 2019‐nCoV RNA (Sun Yat‐sen University) 9 .…”

Section: Discussionmentioning

confidence: 73%

“…Various studies have already focused on extraction‐free protocols 3–10 . Lübke et al 3 showed with their protocol without extraction using the PrimeDirect TM Probe RT‐qPCR Mix (TaKara) that the SARS‐CoV‐2 detection was 82.4% with 16/91 negative samples characterized by low viral loads (Ct > 35).…”

Section: Discussionmentioning

confidence: 99%

“…The production capacity can be limited either by shortages of reagents, disposables, instrument saturation, or lack of qualified staff. To save reagents and/or increase testing capacities, various strategies have been developed such as sample pooling, parallel acquisition of new molecular biology techniques, and/or extraction‐free SARS ‐CoV‐2 detection 2–11 …”

Section: Introductionmentioning

confidence: 99%

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Development and implementation of a RT‐qPCR extraction‐free protocol for the detection of SARS‐CoV‐2 and impact on the turn‐around‐time

Blairon

Piteüs

Beukinga

et al. 2021

Journal of Medical Virology

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The occurrence of the COVID‐19 second‐wave outbreak in Europe has pushed laboratories performing molecular SARS‐CoV‐2 tests to increase their throughput and decrease the result rendering time. In this evaluation, we tested for the first time a new, extraction‐free, protocol with the Allplex SARS‐CoV‐2 Assay RT‐qPCR kit on a Nimbus platform. Ninety‐one samples, of which 71 previously tested positive with RT‐qPCR with extraction were immediately analyzed without extraction, using only a dilution and thermal shock protocol. The positive and negative percentage agreements were respectively 97.2% (95% confidence interval [CI]: 0.90–0.99) and 95.0% (95% CI: 0.76–0.99). The two false negatives observed were very weakly positive with the comparison method. Moderate variations in Ct of the targeted genes were observed (median ± 95% CI): E gene, +2.49 ± 0.44; N gene, +0.98 ± 0.54; RdRP/S genes, +2.64 ± 0.48. On the other hand, the number of tests performed within 24 h raised from 86.4% to 97.8%, the turn‐around time decreased from 19:18 to 09:03 (p < .0001), and the number of tests that can be performed per day doubled since this technique was introduced routinely in our laboratory.

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“…Additionally, the skyrocketing increment on the clinical use of RT-qPCR due to the COVID-19 pandemic led to a worldwide shortage of RNA purification reagents [4,5] thus, many research groups around the world have concentrated their efforts on either simplifying or eliminating the extraction step [6][7][8][9][10][11][12][13][14][15][16].…”

Section: Introductionmentioning

confidence: 99%

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

Genoud

Stortz

Waisman

et al. 2020

Preprint

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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.

show abstract

Use of a simplified sample processing step without RNA extraction for direct SARS-CoV-2 RT-PCR detection

Cited by 28 publications

References 4 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

Development and implementation of a RT‐qPCR extraction‐free protocol for the detection of SARS‐CoV‐2 and impact on the turn‐around‐time

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

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