scholarly journals Self-collection and pooling of samples as resources-saving strategies for RT-PCR-based SARS-CoV-2 surveillance, the example of travelers in French Polynesia

PLoS ONE ◽  
2021 ◽  
Vol 16 (9) ◽  
pp. e0256877
Author(s):  
Maite Aubry ◽  
Iotefa Teiti ◽  
Anita Teissier ◽  
Vaea Richard ◽  
Teheipuaura Mariteragi-Helle ◽  
...  
Keyword(s):  
Large Scale ◽  
Rna Extraction ◽  
French Polynesia ◽  
Rt Pcr ◽  
Spike Gene ◽  
First Case ◽  
Main Challenge ◽  
Positive Individual ◽  
Human Material ◽  
Alpha Variant

In French Polynesia, the first case of SARS-CoV-2 infection was detected on March 10th, 2020, in a resident returning from France. Between March 28th and July 14th, international air traffic was interrupted and local transmission of SARS-CoV-2 was brought under control, with only 62 cases recorded. The main challenge for reopening the air border without requiring travelers to quarantine on arrival was to limit the risk of re-introducing SARS-CoV-2. Specific measures were implemented, including the obligation for all travelers to have a negative RT-PCR test for SARS-CoV-2 carried out within 3 days before departure, and to perform another RT-PCR testing 4 days after arrival. Because of limitation in available medical staff, travelers were provided a kit allowing self-collection of oral and nasal swabs. In addition to increase our testing capacity, self-collected samples from up to 10 travelers were pooled before RNA extraction and RT-PCR testing. When a pool tested positive, RNA extraction and RT-PCR were performed on each individual sample. We report here the results of COVID-19 surveillance (COV-CHECK PORINETIA) conducted between July 15th, 2020, and February 15th, 2021, in travelers using self-collection and pooling approaches. We tested 5,982 pools comprising 59,490 individual samples, and detected 273 (0.46%) travelers positive for SARS-CoV-2. A mean difference of 1.17 Ct (CI 95% 0.93–1.41) was found between positive individual samples and pools (N = 50), probably related to the volume of samples used for RNA extraction (200 μL versus 50 μL, respectively). Retrospective testing of positive samples self-collected from October 20th, 2020, using variants-specific amplification kit and spike gene sequencing, found at least 6 residents infected by the Alpha variant. Self-collection and pooling approaches allowed large-scale screening for SARS-CoV-2 using less human, material and financial resources. Moreover, this strategy allowed detecting the introduction of SARS-CoV-2 variants of concern in French Polynesia.

scholarly journals Self-collection and pooling of samples as resources-saving strategies for RT-PCR-based SARS-CoV-2 surveillance, the example of travelers in French Polynesia

2021 ◽  
Author(s):  
Maite Aubry ◽  
Iotefa Teiti ◽  
Anita Teissier ◽  
Vaea Richard ◽  
Teheipuaura Mariteragi-Helle ◽  
...  
Keyword(s):  
Large Scale ◽  
Rna Extraction ◽  
French Polynesia ◽  
Rt Pcr ◽  
Spike Gene ◽  
First Case ◽  
Main Challenge ◽  
Positive Individual ◽  
Human Material ◽  
Specific Amplification

In French Polynesia, the first case of SARS–CoV–2 infection was detected on March 10th, 2020, in a resident returning from France. Between March 28th and July 14th, international air traffic was interrupted and local transmission of SARS–CoV–2 was brought under control, with only 62 cases recorded. The main challenge for reopening the air border without requiring travelers to quarantine on arrival was to limit the risk of re–introducing SARS–COV–2. Specific measures were implemented, including the obligation for all travelers to have a negative RT–PCR test for SARS–CoV–2 carried out within 3 days before departure, and to perform another RT–PCR testing 4 days after arrival. Because of limitation in available medical staff, travelers were provided a kit allowing self–collection of oral and nasal swabs. In addition to increase our testing capacity, self–collected samples from up to 10 travelers were pooled before RNA extraction and RT–PCR testing. When a pool tested positive, RNA extraction and RT–PCR were performed on each individual sample. We report here the results of COVID–19 surveillance (COV–CHECK PORINETIA) conducted between July 15th, 2020, and February 15th, 2021, in travelers using self–collection and pooling approaches. We tested 5,982 pools comprising 59,490 individual samples, and detected 273 (0.46%) travelers positive for SARS–CoV–2. A mean difference of 1.17 Ct (CI 95% 0.93 – 1.41) was found between positive individual samples and pools (N=50), probably related to the volume of samples used for RNA extraction (200 µL versus 50 µL, respectively). Retrospective testing of positive samples self–collected from October 20th, 2020, using variants–specific amplification kit and spike gene sequencing, found at least 6 residents infected by the B1.1.7 UK variant. Self–collection and pooling approaches allowed large–scale screening for SARS–CoV–2 using less human, material and financial resources. Moreover, this strategy allowed detecting the introduction of SARS–CoV–2 variants in French Polynesia.

scholarly journals Pooling of samples to optimize SARS-CoV-2 diagnosis by RT-qPCR: comparative analysis of two protocols.

2020 ◽  
Author(s):  
Fabiana Volpato ◽  
Daiana Lima-Morales ◽  
Priscila Lamb Wink ◽  
Julia Willig ◽  
Fernanda de-Paris ◽  
...  
Keyword(s):  
Large Scale ◽  
Diagnostic Yield ◽  
Rna Extraction ◽  
Clinical Samples ◽  
The Novel ◽  
Positive Individual ◽  
Sample Pooling ◽  
Novel Coronavirus ◽  
The Individual ◽  
Pooled Samples

RT-qPCR for SARS-CoV-2 is the main diagnostic test used to identify the novel coronavirus. Several countries have used large scale SARS-CoV-2 RT-qPCR testing as one of the important strategies for combating the pandemic. In order to process the massive needs for coronavirus testing, the usual throughput of routine clinical laboratories has reached and often surpassed its limits and new approaches to cope with this challenge must be developed. This study has aimed to evaluate the use pool of samples as a strategy to optimize the diagnostic of SARS-CoV-2 by RT-qPCR in a general population. A total of 220 naso/orofaryngeal swab samples were collected and tested using two different protocols of sample pooling. In the first protocol (Protocol A); 10 clinical samples were pooled before RNA extraction. The second protocol (Protocol B) consisted of pooling the already extracted RNAs from 10 individual samples. Results from Protocol A were identical (100% agreement) with the individual results. However, for results from Protocol B, reduced agreement (91%) was observed in relation to results obtained by individual testing. Inconsistencies observed were related to RT-qPCR results with higher Cycle Thresholds (Ct > 32.73). Furthermore, in pools containing more than one positive individual, the Ct of the pool was equivalent to the lowest Ct among the individual results. These results provide additional evidence in favor of the clinical use of pooled samples for SARS-CoV-2 diagnosis by RT-qPCR and suggest that pooling of samples before RNA extraction is preferrable in terms of diagnostic yield.

scholarly journals Massive Multiplexing Can Deliver a $1 Test for COVID-19

2020 ◽  
Author(s):  
Paul DN Hebert ◽  
Sean WJ Prosser ◽  
Natalia V Ivanova ◽  
Evgeny V Zakharov ◽  
Sujeevan Ratnasingham
Keyword(s):  
Large Scale ◽  
Low Cost ◽  
Rna Extraction ◽  
Respiratory Syndrome Virus ◽  
Major Advance ◽  
Rt Pcr ◽  
Screening Programs ◽  
Global Pandemic ◽  
Gene Coding ◽  
Large Scale Screening

ABSTRACTThe severe acute respiratory syndrome virus, SARS-CoV-2 (hereafter COVID-19), rapidly achieved global pandemic status, provoking large-scale screening programs in many nations. Their activation makes it imperative to identify methods that can deliver a diagnostic result at low cost. This paper describes an approach which employs sequence variation in the gene coding for its envelope protein as the basis for a scalable, inexpensive test for COVID-19. It achieves this by coupling a simple RNA extraction protocol with low-volume RT-PCR, followed by E-Gel screening and sequencing on high-throughput platforms to analyze 10,000 samples in a run. Slight modifications to the protocol could support screening programs for other known viruses and for viral discovery. Just as the $1,000 genome is transforming medicine, a $1 diagnostic test for viral and bacterial pathogens would represent a major advance for public health.

scholarly journals Large-scale implementation of pooled RNA extraction and RT-PCR for SARS-CoV-2 detection

2020 ◽  
Vol 26 (9) ◽  
pp. 1248-1253 ◽  
Author(s):  
R. Ben-Ami ◽  
A. Klochendler ◽  
M. Seidel ◽  
T. Sido ◽  
O. Gurel-Gurevich ◽  
...  
Keyword(s):  
Large Scale ◽  
Rna Extraction ◽  
Rt Pcr

scholarly journals A Streamlined Approach to Rapidly Detect SARS-CoV-2 Infection Avoiding RNA Extraction: Workflow Validation

2020 ◽  
Vol 2020 ◽  
pp. 1-5
Author(s):  
Catia Mio ◽  
Adriana Cifù ◽  
Stefania Marzinotto ◽  
Natascha Bergamin ◽  
Chiara Caldana ◽  
...  
Keyword(s):  
Large Scale ◽  
Rna Extraction ◽  
Cost Effective ◽  
Extraction Methods ◽  
Rapid Identification ◽  
Proteinase K ◽  
Rt Pcr ◽  
Isolation Method ◽  
Pcr Assay ◽  
Sensitivity Specificity

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has rapidly spread worldwide from the beginning of 2020. The presence of viral RNA in samples by nucleic acid (NA) molecular analysis is the only method available to diagnose COVID-19 disease and to assess patients’ viral load. Since the demand for laboratory reagents has increased, there has been a worldwide shortage of RNA extraction kits. We, therefore, developed a fast and cost-effective viral genome isolation method that, combined with quantitative RT-PCR assay, detects SARS-CoV-2 RNA in patient samples. The method relies on the addition of Proteinase K followed by a controlled heat-shock incubation and, then, E gene evaluation by RT-qPCR. It was validated for sensitivity, specificity, linearity, reproducibility, and precision. It detects as low as 10 viral copies/sample, is rapid, and has been characterized in 60 COVID-19-infected patients. Compared to automated extraction methods, our pretreatment guarantees the same positivity rate with the advantage of shortening the time of the analysis and reducing its cost. This is a rapid workflow meant to aid the healthcare system in the rapid identification of infected patients, such as during a pathogen-related outbreak. For its intrinsic characteristics, this workflow is suitable for large-scale screenings.

scholarly journals Lessons from applied large-scale pooling of 133,816 SARS-CoV-2 RT-PCR tests

2021 ◽  
Vol 13 (589) ◽  
pp. eabf2823 ◽  
Author(s):  
Netta Barak ◽  
Roni Ben-Ami ◽  
Tal Sido ◽  
Amir Perri ◽  
Aviad Shtoyer ◽  
...  
Keyword(s):  
Large Scale ◽  
Rna Extraction ◽  
Group Testing ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Open Questions ◽  
Assay Performance ◽  
Theoretical Predictions ◽  
Polymerase Chain ◽  
The Impact

Pooling multiple swab samples before RNA extraction and real-time reverse transcription polymerase chain reaction (RT-PCR) analysis has been proposed as a strategy to reduce costs and increase throughput of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) tests. However, reports on practical large-scale group testing for SARS-CoV-2 have been scant. Key open questions concern reduced sensitivity due to sample dilution, the rate of false positives, the actual efficiency (number of tests saved by pooling), and the impact of infection rate in the population on assay performance. Here, we report an analysis of 133,816 samples collected between April and September 2020 and tested by Dorfman pooling for the presence of SARS-CoV-2. We spared 76% of RNA extraction and RT-PCR tests, despite the frequently changing prevalence (0.5 to 6%). We observed pooling efficiency and sensitivity that exceeded theoretical predictions, which resulted from the nonrandom distribution of positive samples in pools. Overall, our findings support the use of pooling for efficient large-scale SARS-CoV-2 testing.

scholarly journals Presence and distribution of oilseed pumpkin viruses and molecular detection of Zucchini yellow mosaic virus

2009 ◽  
Vol 24 (2) ◽  
pp. 85-94 ◽  
Author(s):  
Ana Vucurovic ◽  
Aleksandra Bulajic ◽  
Ivana Djekic ◽  
Danijela Ristic ◽  
Janos Berenji ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Large Scale ◽  
Molecular Detection ◽  
Rna Extraction ◽  
Zucchini Yellow Mosaic Virus ◽  
Ringspot Virus ◽  
Yellow Mosaic Virus ◽  
Economic Losses ◽  
Watermelon Mosaic Virus ◽  
Rt Pcr

Over the past decade, intensive spread of virus infections of oilseed pumpkin has resulted in significant economic losses in pumpkin crop production, which is currently expanding in our country. In 2007 and 2008, a survey for the presence and distribution of oilseed pumpkin viruses was carried out in order to identify viruses responsible for epidemics and incidences of very destructive symptoms on cucurbit leaves and fruits. Monitoring and collecting samples of oil pumpkin, as well as other species such as winter and butternut squash and buffalo and bottle gourd with viral infection symptoms, was conducted in several localities of Vojvodina Province. The collected plant samples were tested by DAS-ELISA using polyclonal antisera specific for the detection of six most economically harmful pumpkin viruses: Cucumber mosaic virus (CMV), Zucchini yellow mosaic virus (ZYMV), Watermelon mosaic virus (WMW), Squash mosaic virus (SqMV), Papaya ringspot virus (PRSV) and Tobacco ringspot virus (TRSV) that are included in A1 quarantine list of harmful organisms in Serbia. Identification of viruses in the collected samples indicated the presence of three viruses, ZYMV, WMV and CMV, in individual and mixed infections. Frequency of the identified viruses varied depending on locality and year of investigations. In 2007, WMV was the most frequent virus (94.2%), while ZYMV was prevalent (98.04%) in 2008. High frequency of ZYMV determined in both years of investigation indicated the need for its rapid and reliable molecular detection. During this investigation, a protocol for ZYMV detection was developed and optimized using specific primers CPfwd/Cprev and commercial kits for total RNA extraction, as well as for RT-PCR. In RT-PCR reaction using these primers, a DNA fragment of approximately 1100 bp, which included coat protein gene, was amplified in the samples of infected pumkin leaves. Although serological methods are still useful for large-scale testing of a great number of samples, this protocol, due to its high sensitivity and specificity, is an important improvement in rapid diagnosis of diseases caused by this virus. In addition, the protocol provides a basis for further characterization of ZYMV isolates originating from Serbia.

scholarly journals Use of alternative RNA storage and extraction reagents and development of a hybrid PCR-based method for SARS-CoV-2 detection

2020 ◽  
Author(s):  
Julie Yang ◽  
Elias Salfati ◽  
Damian Kao ◽  
Yuliana Mihaylova
Keyword(s):  
Sanger Sequencing ◽  
Large Scale ◽  
Solid Phase ◽  
Rna Extraction ◽  
Positive Control ◽  
Sample Collection ◽  
Animal Testing ◽  
Rt Pcr ◽  
Detection Assay ◽  
Rna Stabilization

AbstractThe COVID-19 pandemic has presented multiple healthcare challenges, one of which is adequately meeting the need for large-scale diagnostic testing. The most commonly used assays for detection of SARS-CoV-2, including those recommended by the Center for Disease Control and Prevention (CDC), rely on a consistent set of core reagents. This has put a serious strain on the reagent supply chain, resulting in insufficient testing. It has also led to restricted animal testing, even though there are now multiple reports of animals, particularly cats, ferrets and minks, contracting the disease. We aimed to address the diagnostic bottleneck by developing a PCR-based SARS-CoV-2 detection assay for cats (and, potentially, other animals) which avoids the use of most common reagents, such as collection kits optimized for RNA stabilization, RNA isolation kits and TaqMan-based RT-PCR reagents. We demonstrated that an inexpensive solid-phase reversible immobilization (SPRI) method can be used for RNA extraction from feline samples collected with DNAGenotek’s ORAcollect RNA OR-100 and PERFORMAgene DNA PG-100 sample collection kits, optimized for RNA or DNA stabilization, respectively. We developed a dual method SARS-CoV-2 detection assay relying on SYBR RT-PCR and Sanger sequencing, using the same set of custom synthesized oligo primers. We validated our test’s specificity with a commercially available SARS-CoV-2 plasmid positive control, as well as two in-house positive control RNA samples. Our assay’s sensitivity was determined to be 10 viral copies per reaction. Our results suggest that a simple SPRI-dependent RNA extraction protocol and certain sample collection kits not specifically optimized for RNA stabilization could potentially be used in cases where reagent shortages are hindering adequate COVID-19 testing. These ‘alternative’ reagents could be used in combination with our COVID-19 testing method, which relies on inexpensive and readily available SYBR RT-PCR and non-fluorescent PCR reagents. Depending on the detection goals and the laboratory setup available, the SYBR RT-PCR method and the Sanger sequencing based method can be used alone or in conjunction, for improved accuracy. Although the test is intended for animal use, it is, in theory, possible to use it with human samples, especially those with higher viral loads.

scholarly journals Proliferation of SARS-CoV-2 B.1.1.7 Variant in Pakistan-A Short Surveillance Account

2021 ◽  
Vol 9 ◽  
Author(s):  
Massab Umair ◽  
Muhammad Salman ◽  
Zaira Rehman ◽  
Nazish Badar ◽  
Qasim Ali ◽  
...  
Keyword(s):  
Large Scale ◽  
Low Frequency ◽  
Control Measures ◽  
Initial Screening ◽  
Rt Pcr ◽  
Gene Target ◽  
Pakistani Population ◽  
Spike Gene ◽  
Thermofisher Scientific ◽  
The United Kingdom

The emergence of a more transmissible variant of SARS-CoV-2 (B1. 1.7) in the United Kingdom (UK) during late 2020 has raised major public health concerns. Several mutations have been reported in the genome of the B.1.1.7 variant including the N501Y and 69-70deletion in the Spike region that has implications on virus transmissibility and diagnostics. Although the B.1.1.7 variant has been reported by several countries, only three cases have been reported in Pakistan through whole-genome sequencing. Therefore, the objective of the study was to investigate the circulation of B.1.1.7 variant of concern (VOC) in Pakistani population. We used a two-step strategy for the detection of B.1.1.7 with initial screening through TaqPathTM COVID-19 CE-IVD RT-PCR kit (ThermoFisher Scientific, Waltham, US) followed by partial spike (S) gene sequencing of a subset of samples having the spike gene target failure (SGTF). From January 01, 2021, to February 21, 2021, a total of 2,650 samples were tested for SARS-CoV-2 and 70.4% (n = 1,867) showed amplification of all the 3 genes (ORF, N, and S). Notably, 29.6% (n=783) samples have been SGTF that represented numbers from all the four provinces and suggest a rather low frequency during the first 3 weeks of January (n = 10, n = 13, and n = 1, respectively). However, the numbers have started to increase in the last week of January, 2021. During February, 726 (93%) cases of SGTF were reported with a peak (n = 345) found during the 3rd week. Based on the partial sequencing of SGTF samples 93.5% (n = 29/31) showed the characteristic N501Y, A570D, P681H, and T716I mutations found in the B.1.1.7 variant. In conclusion, our findings showed an upsurge of B.1.1.7 cases in Pakistan during February, 2021 affecting 15 districts and warranting large scale genomic surveillance, strengthening of laboratory network and implementation of appropriate control measures in the country.

scholarly journals Diagnostic Performance of Pooled RT-PCR Testing for SARS-CoV-2 Detection

2021 ◽  
Author(s):  
Diadem Ricarte ◽  
Aubrey Gador ◽  
Leomill Mendiola ◽  
Ian Christian Gonzales
Keyword(s):  
Predictive Value ◽  
Diagnostic Performance ◽  
Medical Center ◽  
Rna Extraction ◽  
N Gene ◽  
Rt Pcr ◽  
Positive Individual ◽  
Containment Measures ◽  
Pooled Samples ◽  
Pooled Sample

ABSTRACTBackgroundWith the high number of COVID-19 cases, a need to optimize testing strategy must be regarded to obtain timely diagnosis for early containment measures. With this, several studies have employed pooled RT-PCR testing for SARS-CoV-2 as this could potentially conserve laboratory resources while has the capacity to test several individuals. However, this was recommended to firstly validate the method as different laboratory reagents and equipment vary with its diagnostic performance.ObjectiveThe aim of this study was to determine the diagnostic performance of pooled SARS-CoV-2 nasopharyngeal/oropharyngeal swabbed samples using RT-PCR technique.MethodsA records review of two-staged pooled RT-PCR testing data from August 10, 26, 30 and September 5, 2020 was utilized from Northern Mindanao Medical Center COVID-19 Satellite Laboratory (formerly CHDNM TB Regional Center). For the first stage, using known samples, a total of 30 pools were made for each of the pooling size, 5- and 10-pooled, on both pooling phase, pre- and post-RNA extraction. One positive individual was used to represent each of the Cycle threshold values given (<24, 25-28, 29-32, 33-36, and 37-40) while the rest of the samples were negative. For the second stage, 54 pools of five from 270 random unknown samples were used to validate the results. Target gene performance of N gene and RdRp was also determined.Key ResultsResults show that 5-pooled sample has higher sensitivity (SN), specificity (SP), positive predictive value (PPV), and negative predictive value (NPV) of 100% (95% confidence interval (CI) 88.97-100), 66.95% (95% CI, 60.75-72.6), 28.18% (95% CI, 20.62-37.22), and 100% (95% CI, 97.66-100) compared to 10-pooled sample that has 87.1% (95% CI, 71.15-94.87), 56.9% (50.57-63.02), 20.77% (95% CI, 14.68-28.53) and 97.14% (95% CI, 92.88-98.88). Further, these Ct values were only from the N gene, emphasizing its higher diagnostic performance as well to detect SARS-CoV-2 compared to RdRp as only a few samples were detected, thus, no analysis was made.ConclusionThis study found out that 5-pooled sample has better diagnostic performance compared to 10-pooled samples. Specifically, all positive individual samples were detected in 5-pooled samples in pre-RNA extraction phase which these results are evident and consistent on both known and unknown samples. N gene was found out to detect more SARS-CoV-2 samples compared to RdRp.

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