scholarly journals Clinical validation of innovative, low cost, kit-free, RNA processing protocol for RT-PCR based COVID-19 testing.

2020 ◽  
Author(s):  
Nikhil Shri Sahajpal ◽  
Ashis K Mondal ◽  
Allan Njau ◽  
Sudha Ananth ◽  
Arvind Kothandaraman ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Performance Metrics ◽  
Limit Of Detection ◽  
Rna Extraction ◽  
Limited Resource ◽  
Clinical Samples ◽  
Pcr Analysis ◽  
Nasopharyngeal Swab ◽  
Rt Pcr ◽  
Pcr Assay

The current gold-standard molecular diagnosis for COVID-19 is based on a multi-step assay involving RNA-extraction and RT-PCR analysis for the detection of SARS-CoV-2. RNA-extraction step has been a major rate-limiting step in implementing high-throughput screening for COVID-19 during this pandemic. Moreover, clinical laboratories are facing several challenges that include cost, reagents, instrumentation, turn-around time, trained personnel, and supply-chain constraints to efficiently implement and sustain testing. Cognizant of these limitations, we evaluated the extraction-free methods described in the literature and have developed an innovative, simplified and easy protocol employing limited reagents to extract RNA for subsequent RT-PCR analysis. Nasopharyngeal-swab samples were subjected to the following individual conditions: 65°C for 15 minutes; 80°C for 5 minutes; 90°C for 5 minutes or 80°C for 1 minute, and processed for direct RT-PCR. These groups were also compared with a supplemental protocol adding isopropanol-ethanol-water elution steps followed by RT-PCR assay. The direct RT-PCR assay did not detect SARS-CoV-2 within the various temperature incubation only groups, whereas, the 90°C for 5 minutes-isopropanol-ethanol-water method was found to be comparable to the FDA-EUA method. Evaluation of the performance metrics for 100 clinical samples demonstrated a sensitivity of 94.2% and a specificity of 100%. The limit of detection was ascertained to be ~40 copies/ml by absolute-quantification. The protocol presented for this assay employs limited reagents and yields results with high sensitivity. Additionally, it presents a simplified methodology that would be easier to implement in laboratories in limited resource countries in order to meet the high current COVID-19 testing needs.

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.

scholarly journals Direct diagnostic testing of SARS-CoV-2 without the need for prior RNA extraction

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Shan Wei ◽  
Esther Kohl ◽  
Alexandre Djandji ◽  
Stephanie Morgan ◽  
Susan Whittier ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Point Of Care ◽  
Diagnostic Testing ◽  
Rna Extraction ◽  
Cost Effective ◽  
Clinical Samples ◽  
Nasopharyngeal Swab ◽  
Percentage Agreement ◽  
Testing Method ◽  
Viral Transport

AbstractThe COVID-19 pandemic has resulted in an urgent need for a rapid, point of care diagnostic testing that could be rapidly scaled on a worldwide level. We developed and tested a highly sensitive and robust assay based on reverse transcription loop mediated isothermal amplification (RT-LAMP) that uses readily available reagents and a simple heat block using contrived spike-in and actual clinical samples. RT-LAMP testing on RNA-spiked samples showed a limit of detection (LoD) of 2.5 copies/μl of viral transport media. RT-LAMP testing directly on clinical nasopharyngeal swab samples in viral transport media had an 85% positive percentage agreement (PPA) (17/20), and 100% negative percentage agreement (NPV) and delivered results in 30 min. Our optimized RT-LAMP based testing method is a scalable system that is sufficiently sensitive and robust to test for SARS-CoV-2 directly on clinical nasopharyngeal swab samples in viral transport media in 30 min at the point of care without the need for specialized or proprietary equipment or reagents. This cost-effective and efficient one-step testing method can be readily available for COVID-19 testing world-wide, especially in resource poor settings.

scholarly journals Rapid isothermal amplification and portable detection system for SARS-CoV-2

2020 ◽  
Vol 117 (37) ◽  
pp. 22727-22735 ◽  
Author(s):  
Anurup Ganguli ◽  
Ariana Mostafa ◽  
Jacob Berger ◽  
Mehmet Y. Aydin ◽  
Fu Sun ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Detection System ◽  
Alternative Pathway ◽  
Rna Extraction ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Complete Agreement ◽  
Rt Pcr ◽  
Viral Transport Medium

The COVID-19 pandemic provides an urgent example where a gap exists between availability of state-of-the-art diagnostics and current needs. As assay protocols and primer sequences become widely known, many laboratories perform diagnostic tests using methods such as RT-PCR or reverse transcription loop mediated isothermal amplification (RT-LAMP). Here, we report an RT-LAMP isothermal assay for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus and demonstrate the assay on clinical samples using a simple and accessible point-of-care (POC) instrument. We characterized the assay by dipping swabs into synthetic nasal fluid spiked with the virus, moving the swab to viral transport medium (VTM), and sampling a volume of the VTM to perform the RT-LAMP assay without an RNA extraction kit. The assay has a limit of detection (LOD) of 50 RNA copies per μL in the VTM solution within 30 min. We further demonstrate our assay by detecting SARS-CoV-2 viruses from 20 clinical samples. Finally, we demonstrate a portable and real-time POC device to detect SARS-CoV-2 from VTM samples using an additively manufactured three-dimensional cartridge and a smartphone-based reader. The POC system was tested using 10 clinical samples, and was able to detect SARS-CoV-2 from these clinical samples by distinguishing positive samples from negative samples after 30 min. The POC tests are in complete agreement with RT-PCR controls. This work demonstrates an alternative pathway for SARS-CoV-2 diagnostics that does not require conventional laboratory infrastructure, in settings where diagnosis is required at the point of sample collection.

scholarly journals Improved Detection of Rhinoviruses in Clinical Samples by Using a Newly Developed Nested Reverse Transcription-PCR Assay

1999 ◽  
Vol 37 (3) ◽  
pp. 524-530 ◽  
Author(s):  
Arno C. Andeweg ◽  
Theo M. Bestebroer ◽  
Martijn Huybreghs ◽  
Tjeerd G. Kimman ◽  
Jan C. de Jong
Keyword(s):  
Reverse Transcription ◽  
Rna Extraction ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Noncoding Regions ◽  
Reverse Transcription Pcr ◽  
Highly Sensitive ◽  
Virus Culture ◽  
Culture Positive

This paper describes the development and evaluation of a new nested reverse transcription (RT)-PCR for the detection of rhinovirus in clinical samples. The nucleotide sequences of the 5′ noncoding regions of 39 rhinoviruses were determined in order to map the most conserved subregions. We designed a set of rhinovirus-specific primers and probes directed to these subregions and developed a new nested RT-PCR. The new assay includes an optimal RNA extraction method and amplicon identification with probe hybridization to discriminate between rhinoviruses and the closely related enteroviruses. It proved to be highly sensitive and specific. When tested on a dilution series of cultured viruses, the new PCR protocol scored positive at 10- to 100-fold-higher dilutions than a previously used nested RT-PCR. When tested on a collection of clinical samples obtained from 1,070 acute respiratory disease patients who had consulted their general practitioners, the new assay demonstrated a rhinovirus in 24% of the specimens, including all culture-positive samples, whereas the previously used PCR assay or virus culture detected a rhinovirus in only 3.5 to 6% of the samples. This new assay should help determine the disease burden associated with rhinovirus infections.

scholarly journals Development of a Conventional RT-PCR Assay for Rapid Detection of Porcine Deltacoronavirus with the Same Detection Limit as a SYBR Green-Based Real-Time RT-PCR Assay

2018 ◽  
Vol 2018 ◽  
pp. 1-7
Author(s):  
Lei Ma ◽  
Fanwen Zeng ◽  
Bihong Huang ◽  
Feng Cong ◽  
Ren Huang ◽  
...  
Keyword(s):  
Detection Limit ◽  
Real Time ◽  
Limit Of Detection ◽  
Sybr Green ◽  
Watery Diarrhea ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Rna Molecules ◽  
Positive Rate

Porcine deltacoronavirus (PDCoV) is a newly discovered coronavirus, which belongs to the family Coronaviridae. It causes watery diarrhea, vomiting, and dehydration in newborn piglets. A sensitive RT-PCR method is urgently required to detect PDCoV infection. In this study, we developed and evaluated a conventional RT-PCR assay and a SYBR green-based real-time RT-PCR assay that targeted the PDCoV n gene. Both assays are specific and have the same limit of detection at 2 × 101 copies of RNA molecules per reaction. Eighty-four clinical samples were subjected to both conventional RT-PCR and real-time RT-PCR, and the same positive rate (41.7%) was achieved, which was much higher than the positive rate (26.2%) using a previously described one-step RT-PCR technique. In summary, a conventional RT-PCR technique was successfully established for the detection of PDCoV with the same detection limit as a SYBR green-based real-time RT-PCR assay.

scholarly journals Microfluidic nano-scale qPCR enables ultra-sensitive detection of SARS-CoV-2

2020 ◽  
Author(s):  
Xin Xie ◽  
Tamara Gjorgjieva ◽  
Zaynoun Attieh ◽  
Mame Massar Dieng ◽  
Marc Arnoux ◽  
...  
Keyword(s):  
Viral Infections ◽  
Limit Of Detection ◽  
False Negative ◽  
False Negative Rate ◽  
Clinical Samples ◽  
Nasopharyngeal Swab ◽  
Rt Pcr ◽  
Viral Loads ◽  
Nano Scale ◽  
Negative Rate

Background: A major challenge in controlling the COVID-19 pandemic is the high false-negative rate of the commonly used standard RT-PCR methods for SARS-CoV-2 detection in clinical samples. Accurate detection is particularly challenging in samples with low viral loads that are below the limit of detection (LoD) of standard one- or two-step RT-PCR methods. Methods: We implement a three-step approach for SARS-CoV-2 detection and quantification that employs reverse transcription, targeted cDNA preamplification and nano-scale qPCR based on the Fluidigm 192.24 microfluidic chip. We validate the method using both positive controls and nasopharyngeal swab samples. Results: Using SARS-CoV-2 synthetic RNA and plasmid controls, we demonstrate that the addition of a preamplification step enhances the LoD of the Fluidigm method by 1,000-fold, enabling detection below 1 copy/μl. We applied this method to analyze 182 clinical NP swab samples previously diagnosed using a standard RT-qPCR protocol (91 positive, 91 negative) and demonstrate reproducible detection of SARS-CoV-2 over five orders of magnitude (< 1 to 106 viral copies/μl). Crucially, we detect SARS-CoV-2 with relatively low viral load estimates (<1 to 40 viral copies/μl) in 17 samples with negative clinical diagnosis, indicating a potential false negative rate of 18.7% by clinical diagnostic procedures. Conclusion: The three-step nano-scale RT-qPCR method can robustly detect SARS-CoV-2 in samples with relatively low viral loads (< 1 viral copy/μl) and has the potential to reduce the false negative rate of standard RT-PCR-based diagnostic tests for SARS-CoV-2 and other viral infections.

scholarly journals 149. Extraction-free RT-PCR to Detect SARS-CoV-2 Variants of Concern

2021 ◽  
Vol 8 (Supplement_1) ◽  
pp. S89-S91
Author(s):  
Brian L Harry ◽  
Yue Qiu ◽  
Ling Lu ◽  
Mara Couto-Rodriguez ◽  
Dorottya Nagy-Szakal ◽  
...  
Keyword(s):  
Viral Load ◽  
Real Time ◽  
Limit Of Detection ◽  
Turnaround Time ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Research Support ◽  
Pcr Assay ◽  
Synthetic Controls ◽  
Novel Coronavirus

Abstract Background SARS-CoV-2 variants of concern (VOC) have challenged real-time reverse transcriptase polymerase chain reaction (RT-PCR) methods for the diagnosis of COVID-19. Methods The CDC 2019-Novel Coronavirus real-time RT-PCR panel was modified to create a single-plex extraction-free proxy RT-PCR assay, VOCFast™. This assay uses the nucleocapsid N1 as well as novel primer/probe pairs to target VOC mutations in the Orf1a and spike (S) genes. For analytical validation of VOCFast, synthetic controls for the Wuhan, alpha/B.1.1.7, beta/B.1.351, and gamma/P.1 strains were tested at various concentrations. Clinical validation was performed using patient anterior nares swab and saliva specimens collected in the Denver, CO area between Nov 2020 and Feb 2021 or in March 2021. Orthogonal next-generation sequencing (NGS) was also performed. Results Similar N1 quantification cycle (Cq) values corresponding to viral load were observed for all strains, suggesting that VOC mutations do not affect performance of the N1 primer/probe. Orf1a-mut and S1-mut primer/probes generated a stable high Cq value for the Wuhan strain. Conversely, Orf1a-mut Cq values were inversely correlated with viral load for all VOC. The S1-mut Cq was inversely correlated with viral load of the alpha strain, but did not reliably amplify beta/gamma VOC. The limit of detection was 8 copies/uL. The first set of COVID-19 patient specimens revealed no amplification using Orf1a-mut whereas 53% of specimens collected in Mar 2021 demonstrated amplification by Orf-1a. Orthogonal testing by the SARS-CoV-2 NGS Assay and COVID-DX software demonstrated that 12/12 alpha strains, 2/2 beta/gamma strains, and 33/33 Wuhan strains were correctly identified by VOCFast. Detection of VOC in clinical specimens and validation by NGS Conclusion The combination of the N1, Orf1a-mut, and S1-mut primers/probes in VOCFast can distinguish the Wuhan, alpha, and beta/gamma strains and it consistent with NGS results. Testing of clinical samples revealed that VOC emerged in Denver, CO in March 2021. Future work to discriminate beta, gamma, and emerging VOC is ongoing. In summary, VOCFast is an extraction-free RT-PCR assay for nasal swab and saliva specimens that can identify VOC with a turnaround time suitable for clinical testing. Disclosures Brian L. Harry, MD PhD, Summit Biolabs Inc. (Grant/Research Support, Shareholder) Mara Couto-Rodriguez, MS, Biotia (Employee) Dorottya Nagy-Szakal, MD PhD, Biotia Inc (Employee, Shareholder) Niamh B. O’Hara, PhD, Biotia (Board Member, Employee, Shareholder) Shi-Long Lu, MD PhD, Summit Biolabs Inc. (Grant/Research Support, Shareholder)

scholarly journals Development and Evaluation of AccuPower® COVID-19 Multiplex Real-Time RT-PCR Kit and AccuPower® SARS-CoV-2 Multiplex Real-Time RT-PCR Kit for SARS-CoV-2 Detection in Sputum, NPS/OPS, Saliva and Pooled Samples

2021 ◽  
Author(s):  
In Bum Suh ◽  
Jaegyun Lim ◽  
Hyo Seon Kim ◽  
Guil Rhim ◽  
Heebum Kim ◽  
...  
Keyword(s):  
Real Time ◽  
Limit Of Detection ◽  
Clinical Performance ◽  
Rna Extraction ◽  
Kappa Coefficient ◽  
N Gene ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Current Standard ◽  
Pooled Samples

Rapid and accurate detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential for the successful control of the current global COVID-19 pandemic. The real-time reverse transcription polymerase chain reaction (Real-time RT-PCR) is the most widely used detection technique. This research describes the development of two novel multiplex real-time RT-PCR kits, AccuPower ® COVID-19 Multiplex Real-Time RT-PCR Kit (NCVM) specifically designed for use with the ExiStation ™48 system (comprised of ExiPrep ™48 Dx and Exicycler ™96 by BIONEER, Korea) for sample RNA extraction and PCR detection, and AccuPower ® SARS-CoV-2 Multiplex Real-Time RT-PCR Kit (SCVM) designed to be compatible with manufacturers` on-market PCR instruments. The limit of detection (LoD) of SCVM was 2 copies/µ L and the LoD of the NCVM was 120 copies/mL for both the gene and the SARS-CoV-2 gene (N gene and RdRp gene). The AccuPower ® kits demonstrated high precision with no cross reactivity to other respiratory-related microorganisms. The clinical performance of AccuPower ® kits was evaluated using the following clinical samples: sputum and nasopharyngeal/oropharyngeal swab (NPS/OPS) samples. Overall agreement of the AccuPower ® kits with a Food and Drug Administration (FDA) approved emergency use authorized commercial kit (STANDARD ™ M nCoV Real-Time Detection kit, SD BIOSENSOR, Korea) was above 95% (Cohen`s kappa coefficient ≥ 0.95), with a sensitivity of over 95%. The NPS/OPS specimen pooling experiment was conducted to verify the usability of AccuPower ® kits on pooled samples and the results showed greater than 90% agreement with individual NPS/OPS samples. The clinical performance of AccuPower ® kits with saliva samples was also compared with NPS/OPS samples and demonstrated over 95% agreement (Cohen`s kappa coefficient > 0.95). This study shows the BIONEER NCVM and SCVM assays are comparable with the current standard confirmation assay and are suitable for effective clinical management and control of SARS-CoV-2.

scholarly journals Lumipulse G SARS-CoV-2 Ag assay evaluation using clinical samples from different testing groups

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Giulia Menchinelli ◽  
Licia Bordi ◽  
Flora Marzia Liotti ◽  
Ivana Palucci ◽  
Maria Rosaria Capobianchi ◽  
...  
Keyword(s):  
Positive Result ◽  
Limit Of Detection ◽  
False Negative ◽  
Clinical Samples ◽  
Nasopharyngeal Swab ◽  
Repeated Testing ◽  
Rt Pcr ◽  
Subgenomic Rna ◽  
Negative Results ◽  
Percent Agreement

Abstract Objectives Compared to RT-PCR, lower performance of antigen detection assays, including the Lumipulse G SARS-CoV-2 Ag assay, may depend on specific testing scenarios. Methods We tested 594 nasopharyngeal swab samples from individuals with COVID-19 (RT-PCR cycle threshold [Ct] values ≤ 40) or non-COVID-19 (Ct values > 40) diagnoses. RT-PCR positive samples were assigned to diagnostic, screening, or monitoring groups of testing. Results With a limit of detection of 1.2 × 104 SARS-CoV-2 RNA copies/ml, Lumipulse showed positive percent agreement (PPA) of 79.9% (155/194) and negative percent agreement of 99.3% (397/400), whereas PPAs were 100% for samples with Ct values of <18 or 18–<25 and 92.5% for samples with Ct values of 25–<30. By three groups, Lumipulse showed PPA of 87.0% (60/69), 81.1% (43/53), or 72.2% (52/72), respectively, whereas PPA was 100% for samples with Ct values of <18 or 18–<25, and was 94.4, 80.0, or 100% for samples with Ct values of 25–<30, respectively. Additional testing of RT-PCR positive samples for SARS-CoV-2 subgenomic RNA showed that, by three groups, PPA was 63.8% (44/69), 62.3% (33/53), or 33.3% (24/72), respectively. PPAs dropped to 55.6, 20.0, or 41.7% for samples with Ct values of 25–<30, respectively. All 101 samples with a subgenomic RNA positive result had a Lumipulse assay’s antigen positive result, whereas only 54 (58.1%) of remaining 93 samples had a Lumipulse assay’s antigen positive result. Conclusions Lumipulse assay was highly sensitive in samples with low RT-PCR Ct values, implying repeated testing to reduce consequences of false-negative results.

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.

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