scholarly journals SARS-CoV-2 Detection Using Reverse Transcription Strand Invasion Based Amplification And A Portable Compact Size Instrument

2021 ◽  
Author(s):  
Maiken Worsøe Rosenstierne ◽  
Shreya Joshi ◽  
E. Thomas Danielsen ◽  
Helen Webb ◽  
Dac Mui Luong ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Health System ◽  
Reverse Transcription ◽  
Acid Extraction ◽  
Analytical Sensitivity ◽  
Clinical Sensitivity ◽  
Portable Detection ◽  
Compact Size ◽  
Strand Invasion ◽  
Detection Instrument

Abstract Rapid nucleic-acid based tests that can be performed by non-professionals outside laboratory settings could help the containment of the pandemic SARS-CoV-2 virus and may potentially prevent further widespread lockdowns. Here, we present a novel compact portable detection instrument (the Egoo Health System) for extraction-free detection of SARS-CoV-2 using isothermal Reverse Transcription Strand Invasion Based Amplification (RT-SIBA). The SARS-CoV-2 RT-SIBA assay can be performed directly on crude oropharyngeal swabs without nucleic acid extraction with a reaction time of 30 minutes. The Egoo health system uses a capsule system, which is automatically sealed tight in the Egoo instrument after applying the sample, resulting in a closed system optimal for molecular isothermal amplification. The performance of the Egoo Health System is comparable to the PCR instrument with an analytical sensitivity of 25 viral RNA copies per SARS-CoV-2 RT-SIBA reaction and a clinical sensitivity and specificity between 87.0-98.4% and 96.6-98.2% respectively.

scholarly journals SARS-CoV-2 detection using reverse transcription strand invasion based amplification and a portable compact size instrument

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Maiken W. Rosenstierne ◽  
Shreya Joshi ◽  
E. Thomas Danielsen ◽  
Helen Webb ◽  
Dac Mui Luong ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Health System ◽  
Reverse Transcription ◽  
Acid Extraction ◽  
Analytical Sensitivity ◽  
Clinical Sensitivity ◽  
Portable Detection ◽  
Compact Size ◽  
Strand Invasion ◽  
Detection Instrument

AbstractRapid nucleic-acid based tests that can be performed by non-professionals outside laboratory settings could help the containment of the pandemic SARS-CoV-2 virus and may potentially prevent further widespread lockdowns. Here, we present a novel compact portable detection instrument (the Egoo Health System) for extraction-free detection of SARS-CoV-2 using isothermal reverse transcription strand invasion based amplification (RT-SIBA). The SARS-CoV-2 RT-SIBA assay can be performed directly on crude oropharyngeal swabs without nucleic acid extraction with a reaction time of 30 min. The Egoo Health system uses a capsule system, which is automatically sealed tight in the Egoo instrument after applying the sample, resulting in a closed system optimal for molecular isothermal amplification. The performance of the Egoo Health System is comparable to the PCR instrument with an analytical sensitivity of 25 viral RNA copies per SARS-CoV-2 RT-SIBA reaction and a clinical sensitivity and specificity between 87.0–98.4% and 96.6–98.2% respectively.

scholarly journals Development and Validation of Ribosomal RNA-Targeted Reverse Transcription Real-Time PCR Assays for the Sensitive and Rapid Diagnostics of High Consequence Pathogens

2021 ◽  
Vol 12 ◽  
Author(s):  
Veronika Merold ◽  
Kim Silberreis ◽  
Kilian Stoecker
Keyword(s):  
Real Time ◽  
Ribosomal Rna ◽  
Reverse Transcription ◽  
Real Time Pcr ◽  
Clinical Sample ◽  
Target Cells ◽  
Acid Extraction ◽  
Analytical Sensitivity ◽  
Rapid Diagnostics ◽  
Clinical Sensitivity

Real-time PCR (rtPCR) has become an essential tool in clinical microbiology and has been used for the acute diagnostics of many pathogens. Key performance indicators of rtPCR assays are their specificity as well as their analytical and clinical sensitivity. One way to maximize the sensitivity of such diagnostic rtPCRs is the use of genomic targets, which are present in several copies in the target cells. Here, we use the naturally pre-amplified ribosomal RNA as target for specific and highly sensitive reverse-transcription rtPCR detection of two high consequence pathogens, Yersinia pestis and Francisella tularensis. We determined their analytical sensitivity and illustrate that the newly designed assays are superior compared with other previous published rtPCR assays. Furthermore, we used spiked clinical sample matrices to evaluate their clinical applicability. Finally, we demonstrate that these assays can be applied on heat-inactivated samples without the need of time-consuming nucleic acid extraction.

scholarly journals Limited-Resource Preparable Chitosan Magnetic Particles for Extracting Amplification-Ready Zeptomole-Range Nucleic Acid from Complex Biofluid

2021 ◽  
Author(s):  
Sayantan Tripathy ◽  
Arunansu Talukdar ◽  
Goutam Pramanik ◽  
P. V. Rajesh ◽  
Souradyuti Ghosh
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Real Time Pcr ◽  
Magnetic Particles ◽  
Limited Resource ◽  
Nucleic Acid Amplification ◽  
Acid Extraction ◽  
Analytical Sensitivity ◽  
Nucleic Acid Extraction ◽  
Spin Column

<b>Layman Summary: </b>Nucleic acid extraction is a key prerequisite for any nucleic acid amplification test (NAAT) or isothermal NAAT (iNAAT) based molecular diagnosis assays.<b> </b>Existing methods utilizes spin column system for nucleic acid extraction which are unsuitable for limited resource settings. Our work explores two methods for chitosan coated magnetic particle preparation that can be executed within 6 h from commonly available chemicals with nothing but a magnetic stirrer and water bath and doable by a minimally trained person. We will also investigated the compatibility of the extracted nucleic acid with downstream NAATs such as real time LAMP, colorimetric LAMP, and real time PCR. In the process, we established the analytical sensitivity of the overall method.<div><br><div><b>Characterization methods</b>: SEM, XRD, EDX, FT-IR</div><div><br></div><div><b>Bioanalytical methods:</b> Real time LAMP, Colorimetric LAMP, Real time PCR</div></div>

Comparison of nucleic acid extraction and reverse transcription-qPCR approaches for detection of GI and GII noroviruses in drinking water

2014 ◽  
Vol 199 ◽  
pp. 76-85 ◽  
Author(s):  
Shannon M. Griffin ◽  
Nichole E. Brinkman ◽  
Elizabeth J. Hedrick ◽  
Eric R. Rhodes ◽  
G. Shay Fout
Keyword(s):  
Drinking Water ◽  
Nucleic Acid ◽  
Reverse Transcription ◽  
Acid Extraction ◽  
Nucleic Acid Extraction

scholarly journals Enhanced analytical sensitivity of a quantitative PCR for CMV using a modified nucleic-acid extraction procedure

2000 ◽  
Vol 14 (1) ◽  
pp. 32-37 ◽  
Author(s):  
Andrea Ferreira-Gonzalez ◽  
Saul Yanovich ◽  
Michael R. Langley ◽  
Lisa A. Weymouth ◽  
David S. Wilkinson ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Quantitative Pcr ◽  
Extraction Procedure ◽  
Acid Extraction ◽  
Analytical Sensitivity ◽  
Nucleic Acid Extraction

scholarly journals Evaluation of Three Automated Nucleic Acid Extraction Systems for Identification of Respiratory Viruses in Clinical Specimens by Multiplex Real-Time PCR

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Yoonjung Kim ◽  
Mi-Soon Han ◽  
Juwon Kim ◽  
Aerin Kwon ◽  
Kyung-A Lee
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
False Negative ◽  
Virus Detection ◽  
Turnaround Time ◽  
Nasopharyngeal Swab ◽  
Acid Extraction ◽  
Analytical Sensitivity ◽  
Nucleic Acid Extraction ◽  
Negative Results

A total of 84 nasopharyngeal swab specimens were collected from 84 patients. Viral nucleic acid was extracted by three automated extraction systems: QIAcube (Qiagen, Germany), EZ1 Advanced XL (Qiagen), and MICROLAB Nimbus IVD (Hamilton, USA). Fourteen RNA viruses and two DNA viruses were detected using the Anyplex II RV16 Detection kit (Seegene, Republic of Korea). The EZ1 Advanced XL system demonstrated the best analytical sensitivity for all the three viral strains. The nucleic acids extracted by EZ1 Advanced XL showed higher positive rates for virus detection than the others. Meanwhile, the MICROLAB Nimbus IVD system was comprised of fully automated steps from nucleic extraction to PCR setup function that could reduce human errors. For the nucleic acids recovered from nasopharyngeal swab specimens, the QIAcube system showed the fewest false negative results and the best concordance rate, and it may be more suitable for detecting various viruses including RNA and DNA virus strains. Each system showed different sensitivity and specificity for detection of certain viral pathogens and demonstrated different characteristics such as turnaround time and sample capacity. Therefore, these factors should be considered when new nucleic acid extraction systems are introduced to the laboratory.

scholarly journals Development of Xpert® BCR-ABL Ultra, an Automated and Standardized Multiplex Assay with Required Performance Characteristics for BCR-ABL1 Quantitative Measurement on an International Reporting Scale

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2793-2793 ◽  
Author(s):  
Gwo-Jen Day ◽  
Christina Lockwood ◽  
Jacqueline E. Payton ◽  
Geoffrey L. Uy ◽  
Charles Schiffer ◽  
...  
Keyword(s):  
Dynamic Range ◽  
Limit Of Detection ◽  
Standard Of Care ◽  
Acid Extraction ◽  
Analytical Sensitivity ◽  
Limit Of Quantification ◽  
Clinical Sensitivity ◽  
Real Time Quantitative Pcr ◽  
International Scale ◽  
Assess Treatment Response

Abstract Monitoring BCR-ABL transcript levels in patients on TKI therapy using real-time quantitative PCR is standard of care in the management of CML, and achieving sensitivity to 4.5 logs below baseline is important in patients being considered for TKI discontinuation. In order to accurately assess treatment response, it is essential to ensure that variability in testing methodologies is tightly controlled. This has resulted in substantial efforts to standardize BCR-ABL molecular measurement across laboratories by introducing an International Scale (IS). Here we describe the development and analytical evaluation of Xpert® BCR-ABL Ultra, an automated cartridge-based BCR-ABL monitoring assay incorporating lot-specific standardization to the International Scale as defined by WHO. The BCR-ABL Ultra cartridge simplifies the generation of highly accurate and reproducible assay results by integrating nucleic acid extraction, target amplification, and quantification of the BCR-ABL b3a2 or b2a2 transcripts relative to ABL in peripheral blood specimens. The assay turn-around-time is approximately 2.5 hours and has a %BCR-ABL/ABL(IS) dynamic range of ~6 logs, including two key milestones for the assessment of clinical response at ~10% (IS) (SD=~0.08Log) and ~0.0033% (IS) (SD=~0.25Log) (CMR4.5). Lot-to-lot standardization is achieved using a secondary standard panel of controls derived from and aligned to the first WHO international genetic reference panel. The secondary standards are produced at Cepheid as part of the assay kit manufacturing process and applied to every production lot of cartridges in order to improve assay reliability and reporting comparability. Analytical sensitivity studies using spiked-in CML cell lines demonstrated a limit of detection (LoD) and limit of quantification (LoQ) of 0.003% BCR-ABL/ABL (IS) with a limit of blank (LoB) of 0.00017%. In an analytical specificity study, assay results were negative in 100% of 70 known CML-negative samples, including 20 AML or ALL patients and 50 healthy donors. A study was further conducted to estimate the lower limit of clinical sensitivity using blood from 12 CML patients on TKI therapy who had achieved and maintained MMR with reporting below 0.05% (IS). Eleven out of twelve low CML subjects were detected in at least 19 out of 20 replicates tested per subject over a range of 0.038% (SD=~0.17Log) to 0.0011% (IS) (SD=~0.4Log). In conclusion, the Xpert BCR-ABL Ultra assay demonstrates sensitivity in the range necessary to make clinical decisions regarding the continuation of TKI therapy in patients achieving CMR4.5, and every production lot of cartridges is assigned a unique conversion factor guaranteeing alignment to the WHO International Scale. Disclosures Uy: Novartis: Research Funding.

scholarly journals Gargle-Direct: Extraction-Free Detection of SARS-CoV-2 using Real-time PCR (RT-qPCR) of Saline Gargle Rinse Samples

2020 ◽  
Author(s):  
Vijay J. Gadkar ◽  
David M. Goldfarb ◽  
Virginia Young ◽  
Nicole Watson ◽  
Linda Hoang ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Standard Method ◽  
Molecular Diagnostic ◽  
Heat Inactivation ◽  
Acid Extraction ◽  
Analytical Sensitivity ◽  
Sample Type ◽  
Mouth Rinse ◽  
Nucleic Acid Extraction ◽  
Single Tube

ABSTRACTBackgroundSaline mouth rinse/gargle samples have recently been shown to be a suitable option for swab-independent self-collection for SARS-CoV-2 diagnosis. We sought to evaluate a simplified process for direct reverse transcriptase PCR (RT-qPCR) testing of this novel sample type and to compare performance with routine RT-qPCR using automated nucleic acid extraction.MethodsClinical saline mouth rinse/gargle samples were subjected to automated nucleic acid extraction (“standard method”), followed by RT-qPCR using three assays including the FDA authorized US-CDC’s N1/N2 assay, which was the reference standard for determining sensitivity/specificity. For extraction-free workflow, an aliquot of each gargle sample underwent viral heat inactivation at 65 °C for 20 minutes followed by RT-qPCR testing, without an intermediate extraction step. An in-house validated RT-qPCR lab developed test (LDT), targeting the SARS-CoV-2’s S/ORF8 genes (SORP triplex assay) and the N1/N2 US-CDC assay was used to evaluate the extraction-free protocol. To improve the analytical sensitivity, we developed a single-tube hemi-nested (STHN) version of the SORP triplex assay.ResultsA total of 38 SARS-CoV-2 positive and 75 negative saline mouth rinse/gargle samples were included in this evaluation. A 100% concordance in detection rate was obtained between the standard method and the extraction-free approach for the SORP assay. An average increase of +2.63 to +5.74 of the cycle threshold (CT) values was observed for both the SORP and N1/N2 assay when extraction-free was compared between the standard method. The average ΔCT [ΔCT=CT(Direct PCR)-CT(Extracted RNA)], for each of the gene targets were: S (ΔCT= +4.24), ORF8 (ΔCT=+2.63), N1 (ΔCT=+2.74) and N2 (ΔCT=+5.74). The ΔCT for the STHN SORP assay was +1.51 and −2.05 for the S and ORF8 targets respectively, when extracted method was compared to the standard method.ConclusionOur Gargle-Direct SARS-CoV-2 method is operationally simple, minimizes pre-analytical sample processing and is potentially implementable by most molecular diagnostic laboratories. The empirical demonstration of single-tube hemi-nested RT-qPCR, to specifically address and alleviate the widely-acknowledged problem of reduced analytical sensitivity of detection of extraction-free templates, should help diagnostic laboratories in choosing Gargle-Direct protocol for high-throughput testing.

scholarly journals Development of a reverse transcription recombinase polymerase amplification assay for rapid and direct visual detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)

PLoS ONE ◽  
2021 ◽  
Vol 16 (1) ◽  
pp. e0245164
Author(s):  
Yee Ling Lau ◽  
Ilyiana binti Ismail ◽  
Nur Izati binti Mustapa ◽  
Meng Yee Lai ◽  
Tuan Suhaila Tuan Soh ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Sensitivity And Specificity ◽  
Reverse Transcription ◽  
Cross Reactivity ◽  
Recombinase Polymerase Amplification ◽  
Analytical Sensitivity ◽  
Clinical Sensitivity ◽  
Primary Means ◽  
Amplification Assay ◽  
Recombinase Polymerase Amplification Assay

Rapid diagnosis is an important intervention in managing the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) outbreak. Real time reverse transcription polymerase chain reaction (RT-qPCR) remains the primary means for diagnosing the new virus strain but it is time consuming and costly. Recombinase polymerase amplification (RPA) is an isothermal amplification assay that does not require a PCR machine. It is an affordable, rapid, and simple assay. In this study, we developed and optimized a sensitive reverse transcription (RT)-RPA assay for the rapid detection of SARS-CoV-2 using SYBR Green I and/or lateral flow (LF) strip. The analytical sensitivity and specificity of the RT-RPA assay were tested by using 10-fold serial diluted synthetic RNA and genomic RNA of similar viruses, respectively. Clinical sensitivity and specificity of the RT-RPA assay were carried out using 78 positive and 35 negative nasopharyngeal samples. The detection limit of both RPA and RT-qPCR assays was 7.659 and 5 copies/μL RNA, respectively with no cross reactivity with other viruses. The clinical sensitivity and specificity of RT-RPA were 98% and 100%, respectively. Our study showed that RT-RPA represents a viable alternative to RT-qPCR for the detection of SARS-CoV-2, especially in areas with limited infrastructure.

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