scholarly journals Assessment of Specimen Pooling to Conserve SARS CoV-2 Testing Resources

2020 ◽  
Author(s):  
Baha Abdalhamid ◽  
Christopher R. Bilder ◽  
Emily L. McCutchen ◽  
Steven H. Hinrichs ◽  
Scott A. Koepsell ◽  
...  
Keyword(s):  
Incidence Rate ◽  
Group Testing ◽  
Viral Rna ◽  
Optimal Parameters ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Web Based ◽  
Negative Patient ◽  
Key Points ◽  
Unselected Group

AbstractObjectivesTo establish the optimal parameters for group testing of pooled specimens for the detection of SARS-CoV-2.MethodsThe most efficient pool size was determined to be 5 specimens using a web-based application. From this analysis, 25 experimental pools were created using 50 microliter from one SARS-CoV-2 positive nasopharyngeal specimen mixed with 4 negative patient specimens (50 microliter each) for a total volume of 250 microliter l. Viral RNA was subsequently extracted from each pool and tested using the CDC SARS-CoV-2 RT-PCR assay. Positive pools were consequently split into individual specimens and tested by extraction and PCR. This method was also tested on an unselected group of 60 nasopharyngeal specimens grouped into 12-pools.ResultsAll 25 pools were positive with Cycle threshold (Ct) values within 0 and 5.03 Ct of the original individual specimens. The analysis of 60 specimens determined that two pools were positive followed by identification of two individual specimens among the 60 tested. This testing was accomplished while using 22 extractions/PCR tests, a savings of 38 reactions.ConclusionsWhen the incidence rate of SARS-CoV-2 infection is 10% or less, group testing will result in the saving of reagents and personnel time with an overall increase in testing capability of at least 69%.Key PointsSARS CoV-2, COVID-19, Group testing

scholarly journals Assessment of Specimen Pooling to Conserve SARS CoV-2 Testing Resources

2020 ◽  
Vol 153 (6) ◽  
pp. 715-718 ◽  
Author(s):  
Baha Abdalhamid ◽  
Christopher R Bilder ◽  
Emily L McCutchen ◽  
Steven H Hinrichs ◽  
Scott A Koepsell ◽  
...  
Keyword(s):  
Incidence Rate ◽  
Group Testing ◽  
Viral Rna ◽  
Optimal Parameters ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Web Based ◽  
Negative Patient ◽  
Cycle Threshold ◽  
Unselected Group

Abstract Objectives To establish the optimal parameters for group testing of pooled specimens for the detection of SARS-CoV-2. Methods The most efficient pool size was determined to be five specimens using a web-based application. From this analysis, 25 experimental pools were created using 50 µL from one SARS-CoV-2 positive nasopharyngeal specimen mixed with 4 negative patient specimens (50 µL each) for a total volume of 250 µL. Viral RNA was subsequently extracted from each pool and tested using the CDC SARS-CoV-2 RT-PCR assay. Positive pools were consequently split into individual specimens and tested by extraction and PCR. This method was also tested on an unselected group of 60 nasopharyngeal specimens grouped into 12 pools. Results All 25 pools were positive with cycle threshold (Ct) values within 0 and 5.03 Ct of the original individual specimens. The analysis of 60 specimens determined that 2 pools were positive followed by identification of 2 individual specimens among the 60 tested. This testing was accomplished while using 22 extractions/PCR tests, a savings of 38 reactions. Conclusions When the incidence rate of SARS-CoV-2 infection is 10% or less, group testing will result in the saving of reagents and personnel time with an overall increase in testing capability of at least 69%.

scholarly journals Assessing Viral Shedding and Infectivity of Asymptomatic or Mildly Symptomatic Patients with COVID-19 in a Later Phase

2020 ◽  
Vol 9 (9) ◽  
pp. 2924 ◽  
Author(s):  
Yujin Sohn ◽  
Su Jin Jeong ◽  
Won Suk Chung ◽  
Jong Hoon Hyun ◽  
Yae Jee Baek ◽  
...  
Keyword(s):  
Viral Rna ◽  
Community Treatment ◽  
Transmission Risk ◽  
Treatment Center ◽  
Public Health Issue ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Viral Shedding ◽  
Viral Culture ◽  
Later Phase

Background: The coronavirus disease (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a major global public health issue. SARS-CoV-2 infection is confirmed by the detection of viral RNA using reverse transcription polymerase chain reaction (RT-PCR). Prolonged viral shedding has been reported in patients with SARS-CoV-2 infection, but the presence of viral RNA does not always correlate with infectivity. Therefore, the present study aimed to confirm the presence of viable virus in asymptomatic or mildly symptomatic patients in the later phase of the disease, more than two weeks after diagnosis. Method: Asymptomatic or mildly symptomatic COVID-19 patients who had been diagnosed with the disease at least two weeks previously and admitted to a community treatment center (CTC) from 15 March to 10 April 2020 were enrolled in this study. Nasopharyngeal and salivary swab specimens were collected from each patient. Using these specimens, RT-PCR assay and viral culture were performed. Result: In total, 48 patients were enrolled in this study. There were no significant differences in baseline characteristics between the asymptomatic and mildly symptomatic patient groups. RT-PCR assay and viral culture of SARS-CoV-2 were performed using nasopharyngeal and salivary swabs. The results of RT-PCR performed using salivary swab specimens, in terms of cycle threshold (Ct) values, were similar to those of RT-PCR using nasopharyngeal swab specimens. In addition, no viable virus could be cultured from swab specimens collected from the late-phase COVID-19 patients with prolonged viral RNA shedding. Conclusions: In conclusion, our study suggests that even if viral shedding is sustained in asymptomatic or mildly symptomatic patients with later phase of COVID-19, it can be expected that the transmission risk of the virus is low. In addition, saliva can be used as a reliable specimen for the diagnosis of SARS-CoV-2 infection.

Detection of Colorado Tick Fever viral RNA in acute human serum samples by a quantitative real-time RT-PCR assay

2007 ◽  
Vol 140 (1-2) ◽  
pp. 43-48 ◽  
Author(s):  
Amy J. Lambert ◽  
Olga Kosoy ◽  
Jason O. Velez ◽  
Brandy J. Russell ◽  
Robert S. Lanciotti
Keyword(s):  
Human Serum ◽  
Real Time ◽  
Viral Rna ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Serum Samples ◽  
Human Serum Samples

scholarly journals Sensitive Detection and Quantification of SARS-CoV-2 by Multiplex Droplet Digital RT-PCR

2020 ◽  
Author(s):  
Remco de Kock ◽  
Mieke Baselmans ◽  
Volkher Scharnhorst ◽  
Birgit Deiman
Keyword(s):  
Simultaneous Detection ◽  
Housekeeping Gene ◽  
Multiplex Assay ◽  
Viral Rna ◽  
Sensitive Detection ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Assay Sensitivity ◽  
One Step ◽  
Detection And Quantification

Abstract Purpose. We aimed to develop a one-step droplet digital RT-PCR (RT-ddPCR) multiplex assay that allows for sensitive detection of SARS-CoV-2 RNA with respect to human derived RNA and could be used for triage and monitoring of Covid-19 patients. Methods. A one step RT-ddPCR multiplex assay was developed for simultaneous detection of SARS-CoV-2 E, RdRp and N viral RNA, and human Rpp30 DNA and GUSB mRNA, for internal nucleic acid (NA) extraction and RT-PCR control. Dilution series of viral RNA transcripts were prepared in water and total NA extract of Covid-19 negative patients. As reference assay, an E-GUSB duplex RT-PCR was used. Results. Assay sensitivity of the RT-PCR assay drastically decreased when SARS-CoV-2 copies were detected in a background of total NA extract compared to water, while the sensitivity of the RT-ddPCR was not affected by the total NA background. GUSB mRNA detection was used to set validity criteria to assure viral RNA and RT-PCR assay quality. In a background of at least 100 GUSB mRNA copies, 5 copies of viral RNA are reliably detectable and 10 copies viral RNA copies are reliably quantifiable. Conclusion. The present study describes a robust and sensitive one-step RT-ddPCR multiplex assay for reliable detection and quantification of SARS-CoV-2 RNA. By determining the fractional abundance of viral RNA with respect to a human housekeeping gene, viral loads from different samples can be compared, what could be used to investigate the infectiveness and to monitor Covid-19 patients.

scholarly journals SARS-CoV-2 RNA detection in nasal mid-turbinate swabs

2020 ◽  
Author(s):  
Laura Dioni ◽  
Benedetta Albetti ◽  
Federica Rota ◽  
Valentina Bollati
Keyword(s):  
Rna Extraction ◽  
Positive Control ◽  
Viral Rna ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Rna Detection ◽  
Nasal Swabs ◽  
Internal Positive Control ◽  
Polymerase Chain ◽  
Genomic Regions

Abstract In this protocol, we describe a method to investigate the presence of SARS-CoV-2 RNA in nasal swabs, using a commercially available high-throughput Real-Time Polymerase Chain Reaction (RT-PCR) assay (TaqPath™ Covid-19 kit, ThermoFisher Scientific) in a 384-Well Plate. After Viral RNA extraction with QIAamp Viral RNA Mini kit, reverse transcription and cDNA amplification, all samples are assessed for the presence of three specific SARS-CoV-2 viral genomic regions and an internal positive control (IPC), in one Multiplex RT-PCR reaction.

scholarly journals Sensitive detection and quantification of SARS-CoV-2 by multiplex droplet digital RT-PCR

2020 ◽  
Author(s):  
Remco de Kock ◽  
Mieke Baselmans ◽  
Volkher Scharnhorst ◽  
Birgit Deiman
Keyword(s):  
Simultaneous Detection ◽  
Housekeeping Gene ◽  
Multiplex Assay ◽  
Viral Rna ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Assay Sensitivity ◽  
Viral Loads ◽  
Pcr Multiplex ◽  
One Step

Abstract The purpose of this study is to develop a one-step droplet digital RT-PCR (RT-ddPCR) multiplex assay that allows for sensitive quantification of SARS-CoV-2 RNA with respect to human-derived RNA and could be used for screening and monitoring of Covid-19 patients. A one-step RT-ddPCR multiplex assay was developed for simultaneous detection of SARS-CoV-2 E, RdRp and N viral RNA, and human Rpp30 DNA and GUSB mRNA, for internal nucleic acid (NA) extraction and RT-PCR control. Dilution series of viral RNA transcripts were prepared in water and total NA extract of Covid-19-negative patients. As reference assay, an E-GUSB duplex RT-PCR was used. GUSB mRNA detection was used to set validity criteria to assure viral RNA and RT-PCR assay quality and to enable quantification of SARS-CoV-2 RNA. In a background of at least 100 GUSB mRNA copies, 5 copies of viral RNA are reliably detectable and 10 copies viral RNA copies are reliably quantifiable. It was found that assay sensitivity of the RT-ddPCR was not affected by the total NA background while assay sensitivity of the gold standard RT-PCR assay is drastically decreased when SARS-CoV-2 copies were detected in a background of total NA extract compared with water. The present study describes a robust and sensitive one-step ddRT-PCR multiplex assay for reliable quantification of SARS-CoV-2 RNA. By determining the fractional abundance of viral RNA with respect to a human housekeeping gene, viral loads from different samples can be compared, what could be used to investigate the infectiveness and to monitor Covid-19 patients.

1089: Prognostic Value of Molecular Staging of Bladder Cancer with RT -PCR Assay for KRT20: Results at 5 Year-Follow-up

2007 ◽  
Vol 177 (4S) ◽  
pp. 360-360
Author(s):  
Ana Agud ◽  
Maria J. Ribal ◽  
Lourdes Mengual ◽  
Mercedes Marin-Aguilera ◽  
Laura Izquierdo ◽  
...  
Keyword(s):  
Bladder Cancer ◽  
Prognostic Value ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Molecular Staging

Polymerase chain reaction amplification and typing of rotavirus in environmental samples

1995 ◽  
Vol 31 (5-6) ◽  
pp. 371-374 ◽  
Author(s):  
R. Gajardo ◽  
R. M. Pintó ◽  
A. Bosch
Keyword(s):  
Polymerase Chain Reaction ◽  
Environmental Samples ◽  
Polymerase Chain Reaction Amplification ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Group A ◽  
Reaction Amplification ◽  
Stool Specimens ◽  
Polymerase Chain

A reverse transcription polymerase chain reaction (RT-PCR) assay is described that has been developed for the detection and serotyping of group A rotavirus in stool specimens and concentrated and non-concentrated sewage specimens.

Scoring System for the Diagnosis of COVID-19

2020 ◽  
Vol 13 (1) ◽  
pp. 413-414 ◽  
Author(s):  
Mohamed Farouk Allam
Keyword(s):  
Scoring System ◽  
Clinical Symptoms ◽  
False Negative ◽  
Nasopharyngeal Swab ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Negative Results ◽  
False Negative Results ◽  
Symptoms And Signs

Due to the international spread of COVID-19, the difficulty of collecting nasopharyngeal swab specimen from all suspected patients, the costs of RT-PCR and CT, and the false negative results of RT-PCR assay in 41% of COVID-19 patients, a scoring system is needed to classify the suspected patients in order to determine the need for follow-up, home isolation, quarantine or the conduction of further investigations. A scoring system is proposed as a diagnostic tool for suspected patients. It includes Epidemiological Evidence of Exposure, Clinical Symptoms and Signs, and Investigations (if available). This scoring system is simple, could be calculated in a few minutes, and incorporates the main possible data/findings of any patient.

Effective optimization of SARS-CoV-2 laboratory testing variables in an era of supply chain constraints

2020 ◽  
Vol 15 (15) ◽  
pp. 1483-1487
Author(s):  
Nikhil S Sahajpal ◽  
Ashis K Mondal ◽  
Allan Njau ◽  
Sudha Ananth ◽  
Kimya Jones ◽  
...  
Keyword(s):  
Supply Chain ◽  
Rna Extraction ◽  
Nasopharyngeal Swab ◽  
Sample Collection ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Short Supply ◽  
Viral Transport ◽  
3D Printed ◽  
Bridging Studies

RT-PCR-based assays for the detection of SARS-CoV-2 have played an essential role in the current COVID-19 pandemic. However, the sample collection and test reagents are in short supply, primarily due to supply chain issues. Thus, to eliminate testing constraints, we have optimized three key process variables: RNA extraction and RT-PCR reactions, different sample types and media to facilitate SARS-CoV-2 testing. By performing various validation and bridging studies, we have shown that various sample types such as nasopharyngeal swab, bronchioalveolar lavage and saliva, collected using conventional nasopharyngeal swabs, ESwab or 3D-printed swabs and, preserved in viral transport media, universal transport media, 0.9% sodium chloride or Amies media are compatible with RT-PCR assay for COVID-19. Besides, the reduction of PCR reagents by up to fourfold also produces reliable results.

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