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

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.

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

Disease Markers ◽

10.1155/2020/8869424 ◽

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.

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

Science Translational Medicine ◽

10.1126/scitranslmed.abf2823 ◽

2021 ◽

Vol 13 (589) ◽

pp. eabf2823 ◽

Cited By ~ 1

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.

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

Pesticidi i fitomedicina ◽

10.2298/pif0902085v ◽

2009 ◽

Vol 24 (2) ◽

pp. 85-94 ◽

Cited By ~ 7

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.

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

10.1101/2020.11.21.20236216 ◽

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.

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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

10.1101/2021.06.17.21254195 ◽

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.

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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 ◽

10.1371/journal.pone.0256877 ◽

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.

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

10.1101/2020.10.16.20213405 ◽

2020 ◽

Cited By ~ 3

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 ◽

Report Analysis ◽

The Impact

AbstractPooling multiple swab samples prior to RNA extraction and RT-PCR analysis was proposed as a strategy to reduce costs and increase throughput of 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 analysis of 133,816 samples collected at April-September 2020, tested by pooling for the presence of SARS-CoV-2. We spared 76% of RNA extraction and RT-PCR tests, despite the reality of frequently changing prevalence rate (0.5%-6%). Surprisingly, we observed pooling efficiency and sensitivity that exceed theoretical predictions, which resulted from non-random distribution of positive samples in pools. Overall, the findings strongly support the use of pooling for efficient large high throughput SARS-CoV-2 testing.

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

Future Microbiology ◽

10.2217/fmb-2020-0094 ◽

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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Evaluation of an Automated High-Throughput Liquid-Based RNA Extraction Platform on Pooled Nasopharyngeal or Saliva Specimens for SARS-CoV-2 RT-PCR

Viruses ◽

10.3390/v13040615 ◽

2021 ◽

Vol 13 (4) ◽

pp. 615

Author(s):

Allen Wing-Ho Chu ◽

Cyril Chik-Yan Yip ◽

Wan-Mui Chan ◽

Anthony Chin-Ki Ng ◽

Dream Lok-Sze Chan ◽

...

Keyword(s):

Nucleic Acid ◽

High Throughput ◽

Rna Extraction ◽

Nasopharyngeal Swab ◽

Acid Extraction ◽

Rt Pcr ◽

Nucleic Acid Extraction ◽

Suitable Alternative ◽

Pcr Inhibitors ◽

Pcr Targeting

SARS-CoV-2 RT-PCR with pooled specimens has been implemented during the COVID-19 pandemic as a cost- and manpower-saving strategy for large-scale testing. However, there is a paucity of data on the efficiency of different nucleic acid extraction platforms on pooled specimens. This study compared a novel automated high-throughput liquid-based RNA extraction (LRE) platform (PHASIFYTM) with a widely used magnetic bead-based total nucleic acid extraction (MBTE) platform (NucliSENS® easyMAG®). A total of 60 pools of nasopharyngeal swab and 60 pools of posterior oropharyngeal saliva specimens, each consisting of 1 SARS-CoV-2 positive and 9 SARS-CoV-2 negative specimens, were included for the comparison. Real-time RT-PCR targeting the SARS-CoV-2 RdRp/Hel gene was performed, and GAPDH RT-PCR was used to detect RT-PCR inhibitors. No significant differences were observed in the Ct values and overall RT-PCR positive rates between LRE and MBTE platforms (92.5% (111/120] vs 90% (108/120]), but there was a slightly higher positive rate for LRE (88.3% (53/60]) than MBTE (81.7% (49/60]) among pooled saliva. The automated LRE method is comparable to a standard MBTE method for the detection of SAR-CoV-2 in pooled specimens, providing a suitable alternative automated extraction platform. Furthermore, LRE may be better suited for pooled saliva specimens due to more efficient removal of RT-PCR inhibitors.

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