Detection of SARS-CoV-2 RNA residue on object surfaces in nucleic acid testing laboratory using droplet digital PCR

Jun, LV; Jin, Yang; Xue, Juan; Zhu, Ping; Liu, Lanfang; Li, Shan

doi:10.1016/j.scitotenv.2020.140370

Cited by 57 publications

(54 citation statements)

References 15 publications

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“…In our flowchart ( Figure 8A), we also highlight this as a possibility even though we did not explore it. However, majority of the research done on SARS-CoV-2 using ddPCR have used samples that have undergone RNA extraction [6][7][8][9][10][11][12][13]. This is similar to our work.…”

Section: Discussionsupporting

confidence: 69%

“…This poses a challenge to prospective researchers and clinicians who want to develop in-house multiplex assays for SARS-CoV-2 detection, diagnosis, and/or research. In this study, we used already ddPCR tested [6][7][8][9]11] China CDC SARS-CoV-2 primers [16] and additional in-house primers to show steps on how to develop simplex, duplex, triplex probe mix, and fourplex assays that can reliably detect and also quantify SARS-CoV-2 from clinical and research samples.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Developing multiplex ddPCR assays for SARS-CoV-2 detection based on probe mix and amplitude based multiplexing

Nyaruaba

Mwaliko

et al. 2020

Preprint

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Multiplexing has been highlighted to save on costs, increase sample throughput, and maximize on the number of targets that can be sensitively detected within a small sample. With the ongoing SARS-CoV-2 pandemic, different articles have been published highlighting the superiority of droplet digital PCR (ddPCR) over the gold reverse transcription PCR (RT-PCR) in SARS-CoV-2 detection. However, few studies have been reported on developing multiplex ddPCR assays for SARS-CoV-2 detection and their performance. In this study, we developed simplex (1 target), duplex (2 targets), triplex probe mix (3 targets), and fourplex (4 targets) assays based on a two color ddPCR system for SARS-CoV-2 detection. Results showed that the fourplex assay had the similar limits of detection and accuracy to the lower multiplex assays. Analyzing 94 clinical isolates demonstrated that the ddPCR triplex probe mix assay had better sensitivity than the RT-qPCR assay. Additionally, the ddPCR multiplex assay showed that remdesivir could inhibit the growth of SARS-CoV-2 in vitro while another testing drug couldn't. Conclusively, our research shows that developing multiplex ddPCR assays is possible by combing probe mix and amplitude based multiplexing, which will help in developing multiplexed ddPCR assays for different SARS-CoV-2 applications.

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Section: Discussionsupporting

confidence: 69%

Section: Introductionmentioning

confidence: 99%

Developing multiplex ddPCR assays for SARS-CoV-2 detection based on probe mix and amplitude based multiplexing

Nyaruaba

Mwaliko

et al. 2020

Preprint

View full text Add to dashboard Cite

show abstract

“…The copyright holder for this preprint this version posted August 25, 2020. ; https://doi.org/10.1101/2020.08.22.20179754 doi: medRxiv preprint was carefully surveilled and preventive measures were strictly enforced Di Maria, 2020;Van Doremalen, et al, 2020;Chia et al, 2020;Guo, et al, 2020;Ong et al, 2020;Lv et al, 2020;Razzini et al, 2020;Foladori et al, 2020;Wei et al, 2020). From our findings, it seems that the environmental spread of SARS-CoV-2 was not widely diffused, with the only exception of the surfaces near a hospitalized infected patient.…”

Section: Sars-cov-2 In Hospitals and Public Placesmentioning

confidence: 67%

Monitoring COVID-19 transmission risks by RT-PCR tracing of droplets in hospital and living environments

Piana¹,

Colucci

Valeriani³

et al. 2020

Preprint

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SARS-CoV-2 environmental contamination occurs through droplets and biological fluids released in the surroundings from patients or asymptomatic carriers. Surfaces and objects contaminated by saliva or nose secretions represent a risk for indirect transmission of COVID-19. We assayed surfaces from hospital and living spaces to identify the presence of viral RNA and the spread of fomites in the environment. Anthropic contamination by droplets and biological fluids was monitored by detecting the microbiota signature using multiplex RT-PCR on selected species and massive sequencing on 16S-amplicons. A total of 92 samples (flocked swab) were collected from critical areas during the pandemic, including indoor (3 hospitals and 3 public buildings) and outdoor surfaces exposed to anthropic contamination (handles and handrails, playgrounds). Traces of biological fluids were frequently detected in spaces open to the public and on objects that are touched with the hands (>80%). However, viral RNA was not detected in hospital wards or other indoor and outdoor surfaces either in the air system of a COVID-hospital, but only in the surroundings of an infected patient, in consistent association with droplets traces and fomites. Handled objects accumulated the highest level of multiple contaminations by saliva, nose secretions and faecal traces, further supporting the priority role of handwashing in prevention. In conclusion, anthropic contamination by droplets and biological fluids is widespread in spaces open to the public and can be traced by RT-PCR. Monitoring fomites can support evaluation of indirect transmission risks for Coronavirus or other flu-like viruses in the environment.

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“…This may support the suggestion by Yu et.al to use ddPCR in quantifying antiviral efficacy against SARS-CoV-2 [ 16 ]. Only two non-diagnostic research applications of ddPCR against SARS-CoV-2 exist [ 11 , 13 ]. We believe that multiplex assays can also help in testing how different targets respond when performing research like gene expression, copy number variations, and rare mutation detections.…”

Section: Discussionmentioning

confidence: 99%

Developing multiplex ddPCR assays for SARS-CoV-2 detection based on probe mix and amplitude based multiplexing

Nyaruaba

Mwaliko

et al. 2020

Expert Review of Molecular Diagnostics

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Introduction: With the ongoing SARS-CoV-2 pandemic, different articles have been published highlighting the superiority of droplet digital PCR (ddPCR) over the gold-standard reverse transcription PCR (RT-PCR) in SARS-CoV-2 detection. However, few studies have been reported on developing multiplex ddPCR assays for SARS-CoV-2 detection and their performance. This study shows steps on how to develop different ddPCR SAR-CoV-2 assays including higher order multiplex assays for SARS-CoV-2 detection and antiviral screening. Methods: Using multiple primer/probe sets, we developed, optimized, and analyzed the performance of simplex (1 target), duplex (2 targets), triplex probe mix (3 targets), and quadruplex (4 targets) SARS-CoV-2 ddPCR assays based on a two-color ddPCR detection system. Results: Results showed that the quadruplex assay had similar limits of detection and accuracy to the lower multiplex assays. Analyzing 94 clinical samples demonstrated that the ddPCR triplex probe mix assay had better sensitivity than the RT-qPCR assay. Additionally, the ddPCR multiplex assay showed that remdesivir could inhibit the growth of SARS-CoV-2 in vitro while another testing drug could not. Conclusion:Our research shows that developing multiplex ddPCR assays is possible by combing probe mix and amplitude-based multiplexing, which will help in developing multiplexed ddPCR assays for different SARS-CoV-2 applications.

show abstract

Detection of SARS-CoV-2 RNA residue on object surfaces in nucleic acid testing laboratory using droplet digital PCR

Cited by 57 publications

References 15 publications

Developing multiplex ddPCR assays for SARS-CoV-2 detection based on probe mix and amplitude based multiplexing

Developing multiplex ddPCR assays for SARS-CoV-2 detection based on probe mix and amplitude based multiplexing

Monitoring COVID-19 transmission risks by RT-PCR tracing of droplets in hospital and living environments

Developing multiplex ddPCR assays for SARS-CoV-2 detection based on probe mix and amplitude based multiplexing

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