SARS-CoV-2 detection by direct rRT-PCR without RNA extraction

Mérindol, Natacha; Pépin, Geneviève; Marchand, Caroline; Rheault, Marylène; Peterson, Christine; Poirier, André; Houle, Claudia; Germain, Hugo; Danylo, Alexis

doi:10.1016/j.jcv.2020.104423

Cited by 100 publications

(96 citation statements)

References 10 publications

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“…Hence the RT-nPCR test was able to detect presence of all three samples in a pool of 1:5. Direct testing of samples by RT-qPCR without RNA isolation has been described in recent reports (Bruce et al, 2020;Merindol et al, 2020;Smyrlaki et al, 2020). We performed direct testing of heat inactivated positive samples by the RT-nPCR test.…”

Section: Pooled and Direct Testingmentioning

confidence: 99%

An Inexpensive RT-PCR Endpoint Diagnostic Assay for SARS-CoV-2 Using Nested PCR: Direct Assessment of Detection Efficiency of RT-qPCR Tests and Suitability for Surveillance

Davda

Frank

Prakash

et al. 2020

Preprint

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With a view to extending testing capabilities for the ongoing SARS-CoV-2 pandemic we have developed a test that lowers cost and does not require real time quantitative reverse transcription polymerase chain reaction (RT-qPCR). We developed a reverse transcription nested PCR endpoint assay (RT-nPCR) and showed that RT-nPCR has comparable performance to the standard RT-qPCR test. In the course of comparing the results of both tests, we found that the standard RT-qPCR test can have low detection efficiency (less than 50%) in a real testing scenario which may be only partly explained by low viral representation in many samples. This finding points to the importance of directly monitoring detection efficiency in test environments. We also suggest measures that would improve detection efficiency.

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Section: Pooled and Direct Testingmentioning

confidence: 99%

An Inexpensive RT-PCR Endpoint Diagnostic Assay for SARS-CoV-2 Using Nested PCR: Direct Assessment of Detection Efficiency of RT-qPCR Tests and Suitability for Surveillance

Davda

Frank

Prakash

et al. 2020

Preprint

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“…[13][14][15] While we are unaware of direct SARS-CoV-2 detection from saliva that bypasses RNA isolation/purification, there are several reports of detection from swab/VTM that bypasses RNA isolation/purification (Figure 1C). [16][17][18][19][20][21][22][23] With the ultimate goal of providing convenient, scalable, and costeffective molecular diagnostic testing for >10,000 individuals per day using a single COVID-19 testing center, here we report the discovery of a sensitive saliva-based detection method for SARS-CoV-2 that bypasses RNA isolation/purification (Figure 1D). This SARS-CoV-2 testing process and workflow is convenient, simple, rapid, and inexpensive, and can be readily adopted by any testing facility currently using RT-qPCR.…”

mentioning

confidence: 99%

Saliva-Based Molecular Testing for SARS-CoV-2 that Bypasses RNA Extraction

Rañoa

Holland

Alnaji

et al. 2020

Preprint

124

178

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Convenient, repeatable, large-scale molecular testing for SARS-CoV-2 would be a key weapon to help control the COVID-19 pandemic. Unfortunately, standard SARS-CoV-2 testing protocols are invasive and rely on numerous items that can be subject to supply chain bottlenecks, and as such are not suitable for frequent repeat testing. Specifically, personal protective equipment (PPE), nasopharyngeal (NP) swabs, the associated viral transport media (VTM), and kits for RNA isolation and purification have all been in short supply at various times during the COVID-19 pandemic. Moreover, SARS-CoV-2 is spread through droplets and aerosols transmitted through person-to-person contact, and thus saliva may be a relevant medium for diagnosing SARS-CoV-2 infection status. Here we describe a saliva-based testing method that bypasses the need for RNA isolation/purification. In experiments with inactivated SARS-CoV-2 virus spiked into saliva, this method has a limit of detection of 500-1000 viral particles per mL, rivalling the standard NP swab method, and initial studies also show excellent performance with 100 clinical samples. This saliva-based process is operationally simple, utilizes readily available materials, and can be easily implemented by existing testing sites, thus allowing for high-throughput, rapid, and repeat testing of large populations. Graphical Abstract3 BackgroundThe slow roll-out and inconsistent availability of diagnostic testing for SARS-CoV-2 has hobbled efforts to control the COVID-19 pandemic in many countries. Testing protocols based on the use of nasopharyngeal (NP) swabs as the collection agent, placed in a tube containing viral transport media (VTM), followed by RNA isolation/purification and subsequent analysis by RT-qPCR is currently the most common method ( Figure 1A). 1,2 While some variant of this process has been implemented worldwide, there are multiple challenges with this workflow. Sample collection using NP swabs requires healthcare workers wearing personal protective equipment (PPE) to collect samples, the swabs can be uncomfortable for the patients during collection, and the swabs and the associated VTM have been in short supply at many times and in most locations. In addition, RNA isolation/purification is another significant bottleneck, both in the time and labor required for this process, and in the availability of the equipment and reagents. All of these components also add to the cost of the testing process.There is emerging consensus that widespread, frequently repeated testing is necessary for a safer return to activities that are important for society. Given the data suggesting that SARS-CoV-2 can be spread by pre-symptomatic/asymptomatic carriers, 3-6 localized outbreaks could be dramatically reduced or prevented if individuals shedding SARS-CoV-2 could be readily identified and isolated. For example, imagine a testing bubble placed over a group that desires face-to-face interaction -employees of a company, members of a sports team, extended family networks, etc. If all members of...

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“…Whilst there has been a global shortage of necessary reagents, 19 an increasing number of novel extractions methods have been described in the literature. [20][21][22][23] Furthermore, nested PCR is also less amenable to automation and requires equipment for casting agarose gels and amplicon visualization using UV light. The poor performance of the nested PCR for detecting RNA control 1 is hypothesized to be due to the design of the transcript standard.…”

Section: Discussionmentioning

confidence: 99%

Evaluation of Different PCR Assay Formats for Sensitive and Specific Detection of SARS-CoV-2 RNA

Ratcliff

Nguyen

Andersson

et al. 2020

Preprint

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ABSTRACTAccurate identification of individuals infected with SARS-CoV-2 is crucial for efforts to control the ongoing COVID-19 pandemic. Polymerase chain reaction (PCR)-based assays are the gold standard for detecting viral RNA in patient samples and are used extensively in clinical settings. Most currently used quantitative PCR (RT-qPCRs) rely upon real-time detection of PCR product using specialized laboratory equipment. To enable the application of PCR in resource-poor or non-specialist laboratories, we have developed and evaluated a nested PCR method for SARS-CoV-2 RNA using simple agarose gel electrophoresis for product detection. Using clinical samples tested by conventional qPCR methods and RNA transcripts of defined RNA copy number, the nested PCR based on the RdRP gene demonstrated high sensitivity and specificity for SARS-CoV-2 RNA detection in clinical samples, but showed variable and transcript length-dependent sensitivity for RNA transcripts. Samples and transcripts were further evaluated in an additional N protein real-time quantitative PCR assay. As determined by 50% endpoint detection, the sensitivities of three RT-qPCRs and nested PCR methods varied substantially depending on the transcript target with no method approaching single copy detection. Overall, these findings highlight the need for assay validation and optimization and demonstrate the inability to precisely compare viral quantification from different PCR methodologies without calibration.

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SARS-CoV-2 detection by direct rRT-PCR without RNA extraction

Cited by 100 publications

References 10 publications

An Inexpensive RT-PCR Endpoint Diagnostic Assay for SARS-CoV-2 Using Nested PCR: Direct Assessment of Detection Efficiency of RT-qPCR Tests and Suitability for Surveillance

An Inexpensive RT-PCR Endpoint Diagnostic Assay for SARS-CoV-2 Using Nested PCR: Direct Assessment of Detection Efficiency of RT-qPCR Tests and Suitability for Surveillance

Saliva-Based Molecular Testing for SARS-CoV-2 that Bypasses RNA Extraction

Evaluation of Different PCR Assay Formats for Sensitive and Specific Detection of SARS-CoV-2 RNA

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