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 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>

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.

Effects of diverse materials-based methods on DNA extraction for Clostridium difficle from stool samples

2019 ◽  
Vol 9 (5) ◽  
pp. 509-516 ◽  
Author(s):  
Ziqi Xiao ◽  
Gaojian Yang ◽  
Deng Yan ◽  
Song Li ◽  
Zhu Chen ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Dna Extraction ◽  
Magnetic Beads ◽  
Diagnostic Technique ◽  
Proteinase K ◽  
Molecular Diagnostic ◽  
Acid Extraction ◽  
Nucleic Acid Extraction ◽  
Spin Column ◽  
Stool Samples

Nosocomial infections, including Clostridium difficile infection (CDI), and their fatality rates have increased in the past few decades. Despite emerging molecular diagnostic technologies with rapid, accurate outcomes, nucleic acid extraction from stool samples remains the first limiting step before downstream applications. Commercial nucleic acid extraction kits greatly decrease labor and time requirements, and also provide nucleic acid preparations with higher quality and purity for enzyme digestion analysis or genotyping. The magnetic bead based technique is a novel method compared with the conventional spin-column method, and currently has widespread use in nucleic acid extraction. We evaluated five DNA extraction kits with magnetic beads using materials with various properties (particle size, concentration of magnetic beads, grinding beads) and reagents (proteinase K, lysozyme, isopropanol, and absolute ethanol) to determine the cost, hands-on time, number of essential operations, and quality and purity of the DNA preparations, compared with those obtained using the QIAamp Fast DNA Stool Mini Kit. The six DNA extraction kits yielded A260/280 ratios ranging from 0.85 to 1.9 (average 1.57), and concentrations from 3.70 to 108.09 ng/μL (average 34.64 ng/μL). All the DNA samples had acceptable downstream application effects, except for those obtained using the TIANGEN Magnetic Soil and Stool DNA Kit. However, gel electrophoresis analysis of the DNA samples resulted in a light strip on the gel, indicating that the proteinaceous contaminant may not have been removed completely. A rapid and accurate molecular diagnostic technique could allow for more suitable treatment and prognosis outcomes for inpatients, depending, in large part, on the quality and purity of DNA preparations, which are frequently neglected. Our study focused on the quality of commercial kits with a primary focus on the treatment of stool samples and molecular diagnostic applications.

scholarly journals Utility of Nucleic Acid Extraction Free COVID-19 Real Time PCR Protocol in Resource Limited Setting: A Pilot Study

2021 ◽  
Author(s):  
Santosh Karade ◽  
Pratik Thosani ◽  
Prashant Patil ◽  
Kavita Bala Anand ◽  
Sourav Sen ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Pilot Study ◽  
Real Time ◽  
Gold Standard ◽  
Rna Extraction ◽  
Molecular Diagnostic ◽  
Acid Extraction ◽  
Rt Pcr ◽  
Nucleic Acid Extraction ◽  
Resource Limited

Introduction: Coronavirus Disease (COVID-19), a respiratory infection, caused by severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2), was first identified in Wuhan, Hubei province, China in December 2019. Alarming increase in the number of cases has put tremendous pressure on existing health resources. Real Time Reverse Transcriptase Polymerase Chain Reaction (RT-PCR), a molecular diagnostic method, is considered gold standard for diagnosis of SARS-CoV-2 infection. It involves RNA extraction as the preliminary step. Innovations to cut down cost and time involved in SARS-CoV-2 testing are need of hour. Aim: The aim of this study was to assess the feasibility of Nucleic Acid Extraction Free (NEF) protocol for COVID-19 diagnosis in resource limited settings. Materials and Methods: In this pilot study a panel of 148 Nasopharyngeal (NP) samples was subjected to the novel NEF RT-PCR protocol and results were compared to gold standard RT-PCR on RNA extracted from NP specimen. The cycle threshold value for each target was tabulated in MS Excel Spreadsheet and data analysis was performed using Statistical Package for Social Sciences (SPSS) software version 15.0. Results: Out of 148 collected samples, 120 showed amplification of E and RdRp targets by RNA extraction-based RT-PCR. Overall sensitivity and specificity observed for NEF protocol was 43.94% and 96.42%, respectively. Conclusion: Further refinement in the protocol would be required to improve the sensitivity of NEF protocol and widespread use in laboratories.

scholarly journals An Automated and Miniaturized Rotating-Disk Device for Rapid Nucleic Acid Extraction

Micromachines ◽  
2019 ◽  
Vol 10 (3) ◽  
pp. 204 ◽  
Author(s):  
Rui Tong ◽  
Lijuan Zhang ◽  
Chuandeng Hu ◽  
Xuee Chen ◽  
Qi Song ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Sample Preparation ◽  
Magnetic Beads ◽  
Rotating Disk ◽  
Molecular Diagnostic ◽  
Acid Extraction ◽  
Nucleic Acid Extraction ◽  
Time Saving ◽  
Central Controller ◽  
Acid Sample

The result of molecular diagnostic and detection greatly dependent on the quality and integrity of the isolated nucleic acid. In this work, we developed an automated miniaturized nucleic acid extraction device based on magnetic beads method, consisting of four components including a sample processing disc and its associated rotary power output mechanism, a pipetting module, a magnet module and an external central controller to enable a customizable and automated robust nucleic acid sample preparation. The extracted nucleic acid using 293T cells were verified using real-time polymerase chain reaction (PCR) and the data implies a comparable efficiency to a manual process, with the advantages of performing a flexible, time-saving (~10 min), and simple nucleic acid sample preparation.

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>

scholarly journals A Single-Tube Nucleic Acid Extraction, Amplification, and Detection Method Using Aluminum Oxide

2006 ◽  
Vol 8 (1) ◽  
pp. 16-21 ◽  
Author(s):  
Shale Dames ◽  
L. Kathryn Bromley ◽  
Mark Herrmann ◽  
Marc Elgort ◽  
Maria Erali ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Aluminum Oxide ◽  
Detection Method ◽  
Acid Extraction ◽  
Nucleic Acid Extraction ◽  
Single Tube

scholarly journals Single-Tube Method for Nucleic Acid Extraction, Amplification, Purification, and Sequencing

2004 ◽  
Vol 50 (10) ◽  
pp. 1755-1761 ◽  
Author(s):  
Rebecca L Margraf ◽  
Sam Page ◽  
Maria Erali ◽  
Carl T Wittwer
Keyword(s):  
Nucleic Acid ◽  
Reverse Transcription ◽  
Solid Phase ◽  
Sequence Data ◽  
Hcv Rna ◽  
Acid Extraction ◽  
Nucleic Acid Extraction ◽  
Single Tube ◽  
Reverse Transcription Pcr ◽  
Conventional Methods

Abstract Background: The hepatitis C virus (HCV) genotype determines patient prognosis and duration of treatment, but sequencing of the gene is lengthy and labor-intensive. We used a commercially available nucleic acid extraction system to develop a single-tube extraction-to-sequencing (STETS) method for HCV genotyping. Methods: HCV RNA was purified and amplified in tubes coated with a solid-phase matrix that irreversibly bound nucleic acid during the extraction step. After reverse transcription-PCR, the amplicon was adsorbed to the original extraction matrix for purification and use in the subsequent sequencing reactions. Results: The STETS method generated genotyping-quality sequence for a range of HCV titers from 500 to 6 000 000 IU/mL. If a viral sample was detected during real-time reverse transcription-PCR, it could be sequenced and genotyped. Read lengths &gt;600 bases were observed with the STETS method. Mixed infections were detected and genotyped if at least 15% of the minor species was present. Combining the STETS method with consecutive sequencing provided a means of performing both forward and reverse sequencing in a single tube. Conclusions: A single-tube nucleic acid extraction-to-sequencing method, which requires less time and labor than conventional methods, generates HCV sequence data that are equivalent to conventional methods and can be used to genotype HCV.

scholarly journals Differing Effects of Standard and Harsh Nucleic Acid Extraction Procedures on Diagnostic Helminth Real-Time PCRs Applied to Human Stool Samples

Pathogens ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 188
Author(s):  
Tanja Hoffmann ◽  
Andreas Hahn ◽  
Jaco J. Verweij ◽  
Gérard Leboulle ◽  
Olfert Landt ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Real Time Pcr ◽  
Acid Extraction ◽  
Nucleic Acid Extraction ◽  
Pcr Assay ◽  
Bead Beating ◽  
Stool Samples ◽  
Pcr Assays ◽  
Human Stool

This study aimed to assess standard and harsher nucleic acid extraction schemes for diagnostic helminth real-time PCR approaches from stool samples. A standard procedure for nucleic acid extraction from stool and a procedure including bead-beating as well as proteinase K digestion were compared with group-, genus-, and species-specific real-time PCR assays targeting helminths and nonhelminth pathogens in human stool samples. From 25 different in-house and commercial helminth real-time PCR assays applied to 77 stool samples comprising 67 historic samples and 10 external quality assessment scheme samples positively tested for helminths, higher numbers of positive test results were observed after bead-beating-based nucleic acid extraction for 5/25 (20%) real-time PCR assays irrespective of specificity issues. Lower cycle threshold values were observed for one real-time PCR assay after the standard extraction scheme, and for four assays after the bead-beating-based scheme. Agreement between real-time PCR results after both nucleic acid extraction strategies according to Cohen’s kappa ranged from poor to almost perfect for the different assays. Varying agreement was observed in eight nonhelminth real-time PCR assays applied to 67 historic stool samples. The study indicates highly variable effects of harsh nucleic acid extraction approaches depending on the real-time PCR assay used.

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