Extraction of viral nucleic acids: Comparison of five automated nucleic acid extraction platforms

2012 ◽  
Vol 54 (3) ◽  
pp. 255-259 ◽  
Author(s):  
Jens Verheyen ◽  
Rolf Kaiser ◽  
Michael Bozic ◽  
Monika Timmen-Wego ◽  
Barbara K. Maier ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Acid Extraction ◽  
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 Quantification of SARS-CoV-2 and cross-assembly phage (crAssphage) from wastewater to monitor coronavirus transmission within communities

2020 ◽  
Author(s):  
Hyatt Green ◽  
Maxwell Wilder ◽  
Frank A. Middleton ◽  
Mary Collins ◽  
Ariana Fenty ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Cumulative Incidence ◽  
Small Volume ◽  
Acid Extraction ◽  
Total Nucleic Acid ◽  
Nucleic Acid Extraction ◽  
Wastewater Infrastructure ◽  
Scalable Methods ◽  
Human Specific

Wastewater surveillance of SARS-CoV-2 has become an attractive tool for combating the spread of COVID-19 by assessing the presence or levels of the virus shed in a population. However, the methods to quantify viral RNA and to link those quantities to the level of infection within the community vary. In this study, we sought to identify and optimize scalable methods for recovery of viral nucleic acids from wastewater and attempted to use a constitutive member of the gut virome, human-specific crAssphage, to help account for unknown levels of SARS-CoV-2 decay and dilution in the wastewater infrastructure. Results suggest that ultracentrifugation of a small volume of wastewater through a 50% sucrose cushion followed by total nucleic acid extraction yielded quantifiable virus in an area with a modest number of COVID-19 cases. Further, the ratio of log10(SARS-CoV-2):log10(crAssphage) appears to be associated with the cumulative incidence of COVID-19 in the Syracuse, NY area. In areas where ultracentrifuges are available, these methods may be used to link SARS-CoV-2 quantities in wastewater to levels of transmission within communities with sewer service.

Research note: Rapid isolation of total RNA and genomic DNA from Hakea actities

2002 ◽  
Vol 29 (8) ◽  
pp. 1015 ◽  
Author(s):  
Michael G. Mason ◽  
Susanne Schmidt
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Genomic Dna ◽  
Rna Extraction ◽  
Nutrient Concentrations ◽  
Cluster Roots ◽  
Acid Extraction ◽  
Native Plant ◽  
Nucleic Acid Extraction ◽  
Total Rna

Tissues of the Australian native plant species Hakea actities (Proteaceae) contain numerous metabolites and structural compounds that hinder the isolation of nucleic acids. Separate RNA and genomic DNA extraction procedures were developed to isolate high quality nucleic acids from H. actities. Total RNA was extracted from leaves, roots and cluster roots of H. actities grown in low nutrient levels. Cluster root formation in H. actities only occurs when the plants are grown in low nutrient concentrations. However, under these conditions, nucleic acid extraction becomes increasingly difficult. The new procedures are faster than many of the published nucleic acid extraction protocols, and avoid the use of hazardous chemicals. The RNA extraction method was used successfully on another Australian species and a crop species, suggesting that the procedure is useful for molecular studies of a broad range of plants.

scholarly journals A lab-on-a-chip for preconcentration of bacteria and nucleic acid extraction

RSC Advances ◽  
2018 ◽  
Vol 8 (36) ◽  
pp. 20124-20130 ◽  
Author(s):  
M. Hügle ◽  
G. Dame ◽  
O. Behrmann ◽  
R. Rietzel ◽  
D. Karthe ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Gel Electrophoresis ◽  
Lab On A Chip ◽  
Free Flow ◽  
Acid Extraction ◽  
Nucleic Acid Extraction

A lab-on-a-chip combining free-flow electrophoretic preconcentration and thermoelectric lysis of bacteria as well as purification of nucleic acids by gel-electrophoresis.

scholarly journals Evaluation of commercial methods to separate nucleic acids from intestinal tissues of pigs for diagnosis of porcine epidemic diarrhea

2019 ◽  
Vol 10 (4) ◽  
pp. 477-483
Author(s):  
D. М. Masiuk ◽  
V. S. Nedzvetsky ◽  
A. V. Kokariev ◽  
O. V. Danchuk ◽  
T. O. Vasilenko ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Solid Phase ◽  
Rna Extraction ◽  
Acid Extraction ◽  
Nucleic Acid Extraction ◽  
Viral Dna ◽  
Tissue Mass ◽  
Porcine Epidemic Diarrhea ◽  
Commercial Kits

The article presents the results of evaluating commercial methods for extracting nucleic acids from pig intestinal tissues for the diagnosis of PED. The study was based on samples of small intestine tissues and faeces from 3–5 day old pigs which died from PED. Nucleic acid extraction was performed using commercial kits with different nucleic acid separation strategies based on: silicon-sorbent; silicate membrane fixed in a microcentrifuge column and magnetic balls. The studies were conducted in two stages. The first was a comparison of the results of the amplification of the obtained nucleic acid extracts from the homogenate of the intestines of piglets by using the above-mentioned commercial kits for the extraction of nucleic acids. For this purpose, samples of homogenate were used which in weight corresponded to the guideline for the application of the test kits. The second step was directed to determining the efficiency of extraction of DNA and RNA from homogenate samples with a weight of 10, 50, 100 and 200 mg. Determination of the optimal methodological strategy of nucleic acid extraction for the diagnosis of porcine epidemic diarrhea by PCR has been investigated. The results of the PCR studies of RNA of the PED virus and a unique pig DNA fragment indicate that the extraction of nucleic acids by commercial kits has different levels of efficiency and depends on different factors. According to the research, it was found that the most important of them are the adsorption capacity of the solid-phase sorbent, its configuration and nature, which binds RNA and DNA molecules, the type of sample from which extraction takes place, its volume, or the tissue mass used for extraction. Based on the obtained results, it has been found that the most effective PED virus RNA extraction is by “ArtBioTech”, “Bio Extract Column”, and “Viral DNA/RNA Extraction Kit”, and pig genomic DNA extraction by the “ArtBioTech” and “Viral DNA / RNA extraction Kit”.

scholarly journals Nucleic acid extraction and sequencing from low-biomass synthetic Mars analog soils for in situ life detection

2018 ◽  
Author(s):  
Angel Mojarro ◽  
Julie Hachey ◽  
Ryan Bailey ◽  
Mark Brown ◽  
Robert Doebler ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Origin Of Life ◽  
Acid Extraction ◽  
Nanopore Sequencing ◽  
Nucleic Acid Extraction ◽  
Life On Mars ◽  
Life Detection ◽  
Mars Analog ◽  
Life On Earth

AbstractRecent studies regarding the origin of life and Mars-Earth meteorite transfer simulations suggest that biological informational polymers, such as nucleic acids (DNA and RNA), have the potential to provide unambiguous evidence of life on Mars. To this end, we are developing a metagenomics-based life-detection instrument which integrates nucleic acid extraction and nanopore sequencing: The Search for Extra-Terrestrial Genomes (SETG). Our goal is to isolate and sequence nucleic acids from extant or preserved life on Mars in order to determine if a particular genetic sequence (1) is distantly-related to life on Earth indicating a shared-ancestry due to lithological exchange, or (2) is unrelated to life on Earth suggesting a convergent origin of life on Mars. In this study, we validate prior work on nucleic acid extraction from cells deposited in Mars analog soils down to microbial concentrations observed in the driest and coldest regions on Earth. In addition, we report low-input nanopore sequencing results equivalent to 1 ppb life-detection sensitivity achieved by employing carrier sequencing, a method of sequencing sub-nanogram DNA in the background of a genomic carrier.

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.

scholarly journals Evaluation of an Automated High-Throughput Liquid-Based RNA Extraction Platform on Pooled Nasopharyngeal or Saliva Specimens for SARS-CoV-2 RT-PCR

Viruses ◽  
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.

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

The Analyst ◽  
2020 ◽  
Vol 145 (6) ◽  
pp. 2412-2419 ◽  
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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