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

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.

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

Clinical Chemistry ◽

10.1373/clinchem.2004.035808 ◽

2004 ◽

Vol 50 (10) ◽

pp. 1755-1761 ◽

Cited By ~ 4

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 >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.

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

Pathogens ◽

10.3390/pathogens10020188 ◽

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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Forensic evaluation of two nucleic acid extraction systems and validation of a RT-qPCR protocol for identification of SARS-CoV-2 in post-mortem nasopharyngeal swabs

Forensic Science International ◽

10.1016/j.forsciint.2021.110775 ◽

2021 ◽

pp. 110775

Author(s):

Pedro A. Barrio ◽

Amparo Fernández-Rodríguez ◽

Pablo Martín ◽

Coro Fernández ◽

Lourdes Fernández ◽

...

Keyword(s):

Nucleic Acid ◽

Forensic Evaluation ◽

Acid Extraction ◽

Post Mortem ◽

Nucleic Acid Extraction

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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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Synergistic use of electroosmotic flow and magnetic forces for nucleic acid extraction

The Analyst ◽

10.1039/c9an02191d ◽

2020 ◽

Vol 145 (6) ◽

pp. 2412-2419 ◽

Cited By ~ 1

Author(s):

Rachel N. Deraney ◽

Lindsay Schneider ◽

Anubhav Tripathi

Keyword(s):

Nucleic Acid ◽

Electric Field ◽

Microfluidic Chip ◽

Applied Electric Field ◽

Electroosmotic Flow ◽

Acid Extraction ◽

Nucleic Acid Extraction ◽

Magnetic Forces ◽

Extraction And Purification

NA extraction and purification utilitzing a microfluidic chip with applied electric field to induce electroosmotic flow opposite the magnetic NA-bound bead mix.

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Evaluation of simple nucleic acid extraction methods for the detection of SARS-CoV-2 in nasopharyngeal and saliva specimens during global shortage of extraction kits

Journal of Clinical Virology ◽

10.1016/j.jcv.2020.104519 ◽

2020 ◽

Vol 129 ◽

pp. 104519 ◽

Cited By ~ 2

Author(s):

Allen Wing-Ho Chu ◽

Wan-Mui Chan ◽

Jonathan Daniel Ip ◽

Cyril Chik-Yan Yip ◽

Jasper Fuk-Woo Chan ◽

...

Keyword(s):

Nucleic Acid ◽

Extraction Methods ◽

Acid Extraction ◽

Nucleic Acid Extraction

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Comparison of 2 highly automated nucleic acid extraction systems for quantitation of human cytomegalovirus in whole blood

Diagnostic Microbiology and Infectious Disease ◽

10.1016/j.diagmicrobio.2010.08.011 ◽

2011 ◽

Vol 69 (2) ◽

pp. 161-166 ◽

Cited By ~ 15

Author(s):

Catherine Mengelle ◽

Jean-Michel Mansuy ◽

Isabelle Da Silva ◽

Chistian Davrinche ◽

Jacques Izopet

Keyword(s):

Nucleic Acid ◽

Human Cytomegalovirus ◽

Whole Blood ◽

Acid Extraction ◽

Nucleic Acid Extraction

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NEATTILL: A simplified procedure for nucleic acid extraction from arrayed tissue for TILLING and other high-throughput reverse genetic applications

Plant Methods ◽

10.1186/1746-4811-6-3 ◽

2010 ◽

Vol 6 (1) ◽

pp. 3 ◽

Cited By ~ 24

Author(s):

Yellamaraju Sreelakshmi ◽

Soni Gupta ◽

Reddaiah Bodanapu ◽

Vineeta Chauhan ◽

Mickey Hanjabam ◽

...

Keyword(s):

Nucleic Acid ◽

High Throughput ◽

Acid Extraction ◽

Reverse Genetic ◽

Nucleic Acid Extraction ◽

Simplified Procedure

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

10.26434/chemrxiv.14579127 ◽

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>

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