Overcoming the bottleneck to widespread testing: a rapid review of nucleic acid testing approaches for COVID-19 detection

Esbin, Meagan; Whitney, Oscar N.; Chong, Shasha; Maurer, Anna; Darzacq, Xavier; Tjian, Robert

doi:10.1261/rna.076232.120

Cited by 473 publications

(512 citation statements)

References 60 publications

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“…Currently, this is normally done by showing the presence of SARS-CoV-2 RNA in a frontal nose swab using a real-time RT-PCR (rRT-PCR) test 3 . Generally, these SARS-CoV-2 rRT-PCR test panels have several targets in one or more genes of the virus, complemented with several positive controls and negative controls for checking the correct execution of the whole procedure, including RNA extraction and RNA reverse-transcription efficacy (RT) 4 . Early 2020, the Division of Viral Disease of the Centers for Disease Control and Prevention (CDC, Atlanta, USA) has put a rRT-PCR diagnostic panel together for the detection of the 2019-Novel Coronavirus (https://www.fda.gov/media/134922/download).…”

Section: Introductionmentioning

confidence: 99%

Overhauling a faulty control in the CDC-recommended SARS-CoV-2 RT-PCR test panel

Dekker

Ensink

Leeuwen

et al. 2020

Preprint

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To battle the COVID-19 pandemic, widespread testing for the presence of the SARS-CoV-2 virus is worldwide being employed by specific real-time RT-PCR (rRT-PCR) of viral RNA. The CDC has issued a recommended panel of PCR-based test sets that entail several primer/probe sets that target the SARS-CoV-2 N-gene, but also one that targets the human RNase P gene (h-RP) as a positive control for RNA extraction and/or reverse-transcription (RT) efficacy.We discovered that the CDC-recommended h-RP primer/probe set has a faulty design, because both PCR primers are located in the same exon, which allows for unwanted PCR-amplification of background genomic DNA (gDNA). By removing RNA from nose-swab samples by an RNase treatment, we showed that the presence of gDNA in samples resulted in false-positive signals for the h-RP test control. This is rather serious, because it could lead to false-negative test outcomes, since the CDC interpretation of an absent SARS-CoV-2 rRT-PCR signal plus a positive h-RP rRT-PCR signal is interpreted as "2019-nCoV not detected", whereas a false-positive h-RP rRT-PCR signal resulting from amplification of gDNA should be interpreted as "Invalid Result" and the procedure should be repeated.In order to overhaul the faulty h-RP rRT-PCR primer/probe set with minimal modification, we designed and tested several new h-RP reverse primers. Replacement of the CDC-recommended PCR reverse primer with our selected exon-exon junction reverse primer corrected the problem of false-positive results with this important SARS-CoV-2 RT-PCR test control and thus eliminated the problem of potential false-negative COVID-19 diagnoses.

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Section: Introductionmentioning

confidence: 99%

Overhauling a faulty control in the CDC-recommended SARS-CoV-2 RT-PCR test panel

Dekker

Ensink

Leeuwen

et al. 2020

Preprint

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“…Not least among the latter has been a dramatic increase in sample testing load (1). Efforts to meet the demand have included increased use of automated instrumentation, multiplexing of molecular detection assays, streamlined testing protocols, as well as increasingly varied acceptable sample types, collection devices and transport media (2)(3)(4). Accommodating the testing workload and reagent shortage during the symptomatic pandemic wave was a significant undertaking (1).…”

Section: Introductionmentioning

confidence: 99%

Assessment of sample pooling for clinical SARS-CoV-2 testing

Griesemer

Slyke

George

2020

Preprint

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Accommodating large increases in sample workloads has presented one of the biggest challenges to clinical laboratories during the COVID-19 pandemic. Despite the implementation of new automated detection systems, and previous efficiencies such as barcoding, electronic data transfer and extensive robotics, throughput capacities have struggled to meet the demand. Sample pooling has been suggested as an additional strategy to further address this need. The greatest concern with this approach in a clinical setting is the potential for reduced sensitivity, particularly the risk of false negative results when weak positive samples are pooled. To investigate this possibility, detection rates in pooled samples were evaluated, with extensive assessment of pools containing weak positive specimens. Additionally, the frequency of occurrence of weak positive samples across ten weeks of the pandemic were reviewed. Weak positive specimens were detected in all five-sample pools but failed to be detected in four of the 24 nine-sample pools tested. Weak positive samples comprised an average 16.5% of the positive specimens tested during the pandemic thus far, slightly increasing in frequency during later weeks. Other aspects of the testing process should be considered, however, such as accessioning and reporting, which are not streamlined and may be complicated by pooling procedures. Therefore, the impact on the entire laboratory process needs to be carefully assessed prior to implementing such a strategy.

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“…Quantitative reverse-transcription (qRT)-PCR has become a critical tool for detecting SARS-CoV-2, by amplifying virus-derived RNA, due to its increased sensitivity and fast processing time (Esbin et al 2020) . However, this has been limited by the requirement for labor-intensive RNA extractions in order to enrich viral RNA.…”

Section: Introductionmentioning

confidence: 99%

SYBR green one-step qRT-PCR for the detection of SARS-CoV-2 RNA in saliva

Ganguly

Rottet

Yee

et al. 2020

Preprint

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We describe our efforts at developing a one-step quantitative reverse-transcription (qRT)-PCR protocol to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA directly from saliva samples, without RNA purification. We find that both heat and the presence of saliva impairs the ability to detect synthetic SARS-CoV-2 RNA. Buffer composition (for saliva dilution) was also crucial to effective PCR detection. Using the SG2 primer pair, designed by Sigma-Aldrich, we were able to detect the equivalent of 1.7×10 6 viral copies per mL of saliva after heat inactivation; approximately equivalent to the median viral load in symptomatic patients. This would make our assay potentially useful for rapid detection of high-shedding infected individuals. We also provide a comparison of the PCR efficiency and specificity, which varied considerably, across 9 reported primer pairs for SARS-CoV-2 detection. Primer pairs SG2 and CCDC-N showed highest specificity and PCR efficiency. Finally, we provide an alternate primer pair to use as a positive control for human RNA detection in SARS-CoV-2 assays, as we found that the widely used US CDC primers (targeting human RPP30 ) do not span an exon-exon junction and therefore does not provide an adequate control for the reverse transcription reaction.

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Overcoming the bottleneck to widespread testing: a rapid review of nucleic acid testing approaches for COVID-19 detection

Cited by 473 publications

References 60 publications

Overhauling a faulty control in the CDC-recommended SARS-CoV-2 RT-PCR test panel

Overhauling a faulty control in the CDC-recommended SARS-CoV-2 RT-PCR test panel

Assessment of sample pooling for clinical SARS-CoV-2 testing

SYBR green one-step qRT-PCR for the detection of SARS-CoV-2 RNA in saliva

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