scholarly journals Evaluation of mobile real-time polymerase chain reaction tests for the detection of severe acute respiratory syndrome coronavirus 2

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Chukwunonso Onyilagha ◽  
Henna Mistry ◽  
Peter Marszal ◽  
Mathieu Pinette ◽  
Darwyn Kobasa ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Real Time ◽  
Point Of Care ◽  
Middle Eastern ◽  
Turnaround Time ◽  
Cross Reactivity ◽  
Clinical Samples ◽  
Diarrhea Virus ◽  
Highly Sensitive ◽  
Transmissible Gastroenteritis

AbstractThe coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), calls for prompt and accurate diagnosis and rapid turnaround time for test results to limit transmission. Here, we evaluated two independent molecular assays, the Biomeme SARS-CoV-2 test, and the Precision Biomonitoring TripleLock SARS-CoV-2 test on a field-deployable point-of-care real-time PCR instrument, Franklin three9, in combination with Biomeme M1 Sample Prep Cartridge Kit for RNA 2.0 (M1) manual extraction system for rapid, specific, and sensitive detection of SARS-COV-2 in cell culture, human, and animal clinical samples. The Biomeme SARS-CoV-2 assay, which simultaneously detects two viral targets, the orf1ab and S genes, and the Precision Biomonitoring TripleLock SARS-CoV-2 assay that targets the 5′ untranslated region (5′ UTR) and the envelope (E) gene of SARS-CoV-2 were highly sensitive and detected as low as 15 SARS-CoV-2 genome copies per reaction. In addition, the two assays were specific and showed no cross-reactivity with Middle Eastern respiratory syndrome coronavirus (MERS-CoV), infectious bronchitis virus (IBV), porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis (TGE) virus, and other common human respiratory viruses and bacterial pathogens. Also, both assays were highly reproducible across different operators and instruments. When used to test animal samples, both assays equally detected SARS-CoV-2 genetic materials in the swabs from SARS-CoV-2-infected hamsters. The M1 lysis buffer completely inactivated SARS-CoV-2 within 10 min at room temperature enabling safe handling of clinical samples. Collectively, these results show that the Biomeme and Precision Biomonitoring TripleLock SARS-CoV-2 mobile testing platforms could reliably and promptly detect SARS-CoV-2 in both human and animal clinical samples in approximately an hour and can be used in remote areas or health care settings not traditionally serviced by a microbiology laboratory.

scholarly journals Novel rapid identification of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) by real-time RT-PCR using BD Max Open System in Taiwan

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e9318 ◽  
Author(s):  
Cherng-Lih Perng ◽  
Ming-Jr Jian ◽  
Chih-Kai Chang ◽  
Jung-Chung Lin ◽  
Kuo-Ming Yeh ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Real Time ◽  
Turnaround Time ◽  
Rapid Identification ◽  
Clinical Samples ◽  
Respiratory Syndrome Virus ◽  
Acid Extraction ◽  
Prevention Measures ◽  
Rt Pcr ◽  
African Region

Coronavirus disease 2019 has become a worldwide pandemic. By April 7, 2020, approximately 1,279,722 confirmed cases were reported worldwide including those in Asia, European Region, African Region and Region of the Americas. Rapid and accurate detection of Severe Acute Respiratory Syndrome Virus 2 (SARS-CoV-2) is critical for patient care and implementing public health measures to control the spread of infection. In this study, we developed and validated a rapid total nucleic acid extraction method based on real‐time RT-PCR for reliable, high‐throughput identification of SARS-CoV-2 using the BD MAX platform. For clinical validation, 300 throat swab and 100 sputum clinical samples were examined by both the BD MAX platform and in-house real-time RT-PCR methods, which showed 100% concordant results. This BD MAX protocol is fully automated and the turnaround time from sample to results is approximately 2.5 h for 24 samples compared to 4.8 h by in-house real-time RT-PCR. Our developed BD MAX RT-PCR assay can accurately identify SARS-CoV-2 infection and shorten the turnaround time to increase the effectiveness of control and prevention measures for this emerging infectious disease.

scholarly journals Making sense of rapid antigen testing in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnostics

Diagnosis ◽  
2020 ◽  
Vol 0 (0) ◽  
Author(s):  
Camilla Mattiuzzi ◽  
Brandon M. Henry ◽  
Giuseppe Lippi
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Point Of Care ◽  
Turnaround Time ◽  
Molecular Testing ◽  
Upper Respiratory Tract ◽  
Making Sense ◽  
Skilled Personnel ◽  
Potential Benefits ◽  
Antigen Testing ◽  
Care Availability

AbstractAlthough the most effective strategy for preventing or containing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreaks relies on early diagnosis, the paramount and unprecedented number of tests needed to fully achieve this target is overwhelming worldwide testing supply and capacity. Molecular detection of SARS-CoV-2 RNA in nasopharyngeal swabs is still considered the reference diagnostic approach. Nonetheless, identification of SARS-CoV-2 proteins in upper respiratory tract specimens and/or saliva by means of rapid (antigen) immunoassays is emerging as a promising screening approach. These tests have some advantages compared to molecular analysis, such as point of care availability, no need of skilled personnel and dedicated instrumentation, lower costs and short turnaround time. However, these advantages are counterbalanced by lower diagnostic sensitivity compared to molecular testing, which would only enable to identifying patients with higher SARS-CoV-2 viral load. The evidence accumulated to-date has hence persuaded us to develop a tentative algorithm, which would magnify the potential benefits of rapid antigen testing in SARS-CoV-2 diagnostics.

scholarly journals Analysis and validation of a highly sensitive one-step nested quantitative real-time polymerase chain reaction assay for specific detection of severe acute respiratory syndrome coronavirus 2

2020 ◽  
Vol 17 (1) ◽  
Author(s):  
Yang Zhang ◽  
Chunyang Dai ◽  
Huiyan Wang ◽  
Yong Gao ◽  
Tuantuan Li ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Quantitative Polymerase Chain Reaction ◽  
Clinical Samples ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Qrt Pcr ◽  
Highly Sensitive ◽  
One Step ◽  
Polymerase Chain

Abstract Background Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is posing a serious threat to global public health. Reverse transcriptase real-time quantitative polymerase chain reaction (qRT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. Due to technical limitations, the reported positive rates of qRT-PCR assay of throat swab samples vary from 30 to 60%. Therefore, the evaluation of alternative strategies to overcome the limitations of qRT-PCR is required. A previous study reported that one-step nested (OSN)-qRT-PCR revealed better suitability for detecting SARS-CoV-2. However, information on the analytical performance of OSN-qRT-PCR is insufficient. Method In this study, we aimed to analyze OSN-qRT-PCR by comparing it with droplet digital PCR (ddPCR) and qRT-PCR by using a dilution series of SARS-CoV-2 pseudoviral RNA and a quality assessment panel. The clinical performance of OSN-qRT-PCR was also validated and compared with ddPCR and qRT-PCR using specimens from COVID-19 patients. Result The limit of detection (copies/ml) of qRT-PCR, ddPCR, and OSN-qRT-PCR were 520.1 (95% CI: 363.23–1145.69) for ORF1ab and 528.1 (95% CI: 347.7–1248.7) for N, 401.8 (95% CI: 284.8–938.3) for ORF1ab and 336.8 (95% CI: 244.6–792.5) for N, and 194.74 (95% CI: 139.7–430.9) for ORF1ab and 189.1 (95% CI: 130.9–433.9) for N, respectively. Of the 34 clinical samples from COVID-19 patients, the positive rates of OSN-qRT-PCR, ddPCR, and qRT-PCR were 82.35% (28/34), 67.65% (23/34), and 58.82% (20/34), respectively. Conclusion In conclusion, the highly sensitive and specific OSN-qRT-PCR assay is superior to ddPCR and qRT-PCR assays, showing great potential as a technique for detection of SARS-CoV-2 in patients with low viral loads.

scholarly journals Integrated Extreme Real-Time PCR and High-Speed Melting Analysis in 52 to 87 Seconds

2019 ◽  
Vol 65 (2) ◽  
pp. 263-271 ◽  
Author(s):  
Joseph T Myrick ◽  
Robert J Pryor ◽  
Robert A Palais ◽  
Sean J Ison ◽  
Lindsay Sanford ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
High Speed ◽  
Dna Analysis ◽  
Point Of Care ◽  
Turnaround Time ◽  
Single Nucleotide Variants ◽  
Cycle Times ◽  
Pcr Products ◽  
Melting Analysis

Abstract BACKGROUND Extreme PCR in <30 s and high-speed melting of PCR products in <5 s are recent advances in the turnaround time of DNA analysis. Previously, these steps had been performed on different specialized instruments. Integration of both extreme PCR and high-speed melting with real-time fluorescence monitoring for detection and genotyping is presented here. METHODS A microfluidic platform was enhanced for speed using cycle times as fast as 1.05 s between 66.4 °C and 93.7 °C, with end point melting rates of 8 °C/s. Primer and polymerase concentrations were increased to allow short cycle times. Synthetic sequences were used to amplify fragments of hepatitis B virus (70 bp) and Clostridium difficile (83 bp) by real-time PCR and high-speed melting on the same instrument. A blinded genotyping study of 30 human genomic samples at F2 c.*97, F5 c.1601, MTHFR c.665, and MTHFR c.1286 was also performed. RESULTS Standard rapid-cycle PCR chemistry did not produce any product when total cycling times were reduced to <1 min. However, efficient amplification was possible with increased primer (5 μmol/L) and polymerase (0.45 U/μL) concentrations. Infectious targets were amplified and identified in 52 to 71 s. Real-time PCR and genotyping of single-nucleotide variants from human DNA was achieved in 75 to 87 s and was 100% concordant to known genotypes. CONCLUSIONS Extreme PCR with high-speed melting can be performed in about 1 min. The integration of extreme PCR and high-speed melting shows that future molecular assays at the point of care for identification, quantification, and variant typing are feasible.

scholarly journals Multiplexed Detection of Sepsis Markers in Whole Blood using Nanocomposite Coated Electrochemical Sensors

2020 ◽  
Author(s):  
Uroš Zupančič ◽  
Pawan Jolly ◽  
Pedro Estrela ◽  
Despina Moschou ◽  
Donald E. Ingber
Keyword(s):  
Electrochemical Detection ◽  
Whole Blood ◽  
Electrochemical Sensors ◽  
Point Of Care ◽  
Sample Volume ◽  
Nanocomposite Coating ◽  
Cross Reactivity ◽  
Detection Methods ◽  
Clinical Samples ◽  
Undiluted Serum

ABSTRACTSepsis is a leading cause of mortality worldwide that is difficult to diagnose and manage because this requires simultaneous analysis of multiple biomarkers. Electrochemical detection methods could potentially provide a way to accurately quantify multiple sepsis biomarkers in a multiplexed manner as they have very low limits of detection and require minimal sensor instrumentation; however, affinity-based electrochemical sensors are usually hampered by biological fouling. Here we describe development of an electrochemical detection platform that enables detection of multiple sepsis biomarkers simultaneously by incorporating a recently developed nanocomposite coating composed of crosslinked bovine serum albumin containing a network of reduced graphene oxide nanoparticles that prevents biofouling. Using nanocomposite coated planar gold electrodes, we constructed a procalcitonin sensor and demonstrated sensitive PCT detection in undiluted serum and clinical samples, as well as excellent correlation with a conventional ELISA (adjusted r2 = 0.95). Sensors for two additional sepsis biomarkers — C-reactive protein and pathogen-associated molecular patterns — were developed on the same multiplexed platform and tested in whole blood. Due to the excellent antifouling properties of the nanocomposite coating, all three sensors exhibited specific responses within the clinically significant range without any cross-reactivity in the same channel with low sample volume. This platform enables sensitive simultaneous electrochemical detection of multiple analytes in human whole blood, which can be expanded further to any target analyte with an appropriate antibody pair or capturing probe, and thus, may offer a potentially valuable tool for development of clinical point-of-care diagnostics.GRAPHICAL ABSTRACT

scholarly journals LAMP assay coupled with CRISPR/Cas12a system for portable detection of African swine fever virus

2021 ◽  
Author(s):  
Bo YANG ◽  
zhengwang shi ◽  
Yuan Ma ◽  
Lijuan Wang ◽  
Liyan Cao ◽  
...  
Keyword(s):  
Real Time ◽  
Detection System ◽  
African Swine Fever Virus ◽  
Low Cost ◽  
African Swine Fever ◽  
Lamp Assay ◽  
Cross Reactivity ◽  
Clinical Samples ◽  
Portable Detection ◽  
Real Time Qpcr

African swine fever (ASF) is one of the most severe infectious diseases of pigs. In this study, a LAMP assay coupled with the CRISPR Cas12a system was established in one tube for the detection of the ASFV p72 gene. The single-strand DNA-fluorophore-quencher (ssDNA-FQ) reporters and CRISPR-derived RNA (crRNAs) were screened and selected for the CRISPR detection system. In combination with LAMP amplification assay, the detection limit for the LAMP-CRISPR assay can reach 7 copies/μl of p72 gene per reaction. Furthermore, this method displays no cross-reactivity with other porcine DNA or RNA viruses. The performance of the LAMP-CRISPR assay was compared with real-time qPCR tests for clinical samples, a good consistency between the LAMP-CRISPR assay and real-time qPCR was observed. In the current study, a LAMP coupled with the CRISPR detection method was developed. The method shed a light on the convenient, portable, low cost, highly sensitive and specific detection of ASFV, demonstrating a great application potential for monitoring on-site ASFV in the field.

Evaluation of two point of care technologies for measuring monoclonal antibody therapeutic-A concentrations in blood

Bioanalysis ◽  
2020 ◽  
Vol 12 (20) ◽  
pp. 1449-1458
Author(s):  
Saloumeh K Fischer ◽  
Kathi Williams ◽  
Ian Harmon ◽  
Bryan Bothwell ◽  
Hua Xu ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Real Time ◽  
Point Of Care ◽  
Turnaround Time ◽  
Monitoring Methods ◽  
Sample Collection ◽  
Time Data ◽  
Serum Samples ◽  
Quick Turnaround Time

Aim: Current blood monitoring methods require sample collection and testing at a central lab, which can take days. Point of care (POC) devices with quick turnaround time can provide an alternative with faster results, allowing for real-time data leading to better treatment decisions for patients. Results/Methodology: An assay to measure monoclonal antibody therapeutic-A was developed on two POC devices. Data generated using 75 serum samples (65 clinical & ten spiked samples) show correlative results to the data generated using Gyrolab technology. Conclusion: This case study uses a monoclonal antibody therapeutic-A concentration assay as an example to demonstrate the potential of POC technologies as a viable alternative to central lab testing with quick results allowing for real-time decision-making.

scholarly journals OC 8173 RAPID DETECTION OF MYCOBACTERIUM ULCERANS BY RECOMBINASE POLYMERASE AMPLIFICATION

2019 ◽  
Vol 4 (Suppl 3) ◽  
pp. A2.1-A2
Author(s):  
Michael Frimpong ◽  
Hubert Ahor ◽  
Francisca Sarpong ◽  
Ken Laing ◽  
Mark Wansbrough-Jones ◽  
...  
Keyword(s):  
Sensitivity And Specificity ◽  
Point Of Care ◽  
Clinical Signs ◽  
Buruli Ulcer ◽  
Cross Reactivity ◽  
Current Control ◽  
Mycobacterium Ulcerans ◽  
Recombinase Polymerase Amplification ◽  
Clinical Samples ◽  
Clinical Sensitivity

BackgroundThere are no primary measures to prevent people from contracting Buruli ulcer, mainly due to poor understanding of its epidemiology. The current control strategy emphasises early diagnosis and prompt treatment, with the goal of avoiding the complications associated with advanced stages of the disease. There is no diagnostic test for the disease appropriate for use at the primary health care level where most cases are detected and treated. Diagnosis based on clinical signs is unreliable in inexperienced hands and complicated by infections that have similar presentations. This study was to develop and evaluate the use of recombinase polymerase amplification (RPA) assay for the detection of Mycobacterium ulcerans at the point of patient care.MethodsA specific fragment of IS2404 of M. ulcerans was amplified in 15 min at a constant temperature of 42°C, using the RPA assay and analysed on a portable fluorometre. The’method was tested for sensitivity and specificity with molecular standard of IS2404 DNA fragment, various M.’ulcerans strains, other mycobacteria and environmentally associated bacteria. Additionally, the assay performance as a diagnostic tool was tested with archived DNA from symptomatic patients. All results were compared with that of a highly sensitive IS2404 PCR.ResultsThe detection limit was 50 copies of IS2404 in 15 min using plasmid standard and 125 fg with genomic Mu DNA equivalent 25 genomic copies. The assay was highly specific in detecting all strains of M. ulcerans with no observed cross reactivity with other mycobacteria and common skin colonising bacteria. The clinical sensitivity and specificity of the BU-RPA assay using clinical samples was 86% and 100% respectively.ConclusionWe have developed a real-time isothermal RPA assay for the detection of M. ulcerans as a cheaper alternative to PCR. Combining this assay with a simple extraction protocol will maximise its use as point-of-care test for Buruli ulcer.

scholarly journals Multiplex Real-Time Reverse-Transcription Polymerase Chain Reaction Assays for Diagnostic Testing of Severe Acute Respiratory Syndrome Coronavirus 2 and Seasonal Influenza Viruses: A Challenge of the Phase 3 Pandemic Setting

2020 ◽  
Author(s):  
Fabiola Mancini ◽  
Fabrizio Barbanti ◽  
Maria Scaturro ◽  
Stefano Fontana ◽  
Angela Di Martino ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Severe Acute Respiratory Syndrome ◽  
Real Time ◽  
Reverse Transcription ◽  
Seasonal Influenza ◽  
Influenza Viruses ◽  
Activity Period ◽  
Clinical Samples ◽  
Chain Reaction ◽  
Polymerase Chain

Abstract Background Pandemic coronavirus disease 2019 (COVID-19) disease represents a challenge for healthcare structures. The molecular confirmation of samples from infected individuals is crucial and therefore guides public health decision making. Clusters and possibly increased diffuse transmission could occur in the context of the next influenza season. For this reason, a diagnostic test able to discriminate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from influenza viruses is urgently needed. Methods A multiplex real-time reverse-transcription polymerase chain reaction (PCR) assay was assessed using 1 laboratory protocol with different real-time PCR instruments. Overall, 1000 clinical samples (600 from samples SARS-CoV-2–infected patients, 200 samples from influenza-infected patients, and 200 negative samples) were analyzed. Results The assay developed was able to detect and discriminate each virus target and to intercept coinfections. The limit of quantification of each assay ranged between 5 and 10 genomic copy numbers, with a cutoff value of 37.7 and 37.8 for influenza and SARS-CoV-2 viruses, respectively. Only 2 influenza coinfections were detected in COVID-19 samples. Conclusions This study suggests that multiplex assay is a rapid, valid, and accurate method for the detection of SARS-CoV-2 and influenza viruses in clinical samples. The test may be an important diagnostic tool for both diagnostic and surveillance purposes during the seasonal influenza activity period.

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