scholarly journals Multiplexed detection of respiratory pathogens with a portable analyzer in a “raw-sample-in and answer-out” manner

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Nan Li ◽  
Minjie Shen ◽  
Jiajia Liu ◽  
Li Zhang ◽  
Huili Wang ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Limit Of Detection ◽  
Solid Phase ◽  
Bacterial Species ◽  
Magnetic Bead ◽  
Acid Extraction ◽  
Respiratory Pathogens ◽  
Nucleic Acid Extraction ◽  
Multiplexed Detection ◽  
Portable Analyzer

AbstractCoronavirus disease 2019 (COVID-19) has emerged, rapidly spread and caused significant morbidity and mortality worldwide. There is an urgent public health need for rapid, sensitive, specific, and on-site diagnostic tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In this study, a fully integrated and portable analyzer was developed to detect SARS-CoV-2 from swab samples based on solid-phase nucleic acid extraction and reverse transcription loop-mediated isothermal amplification (RT-LAMP). The swab can be directly inserted into a cassette for multiplexed detection of respiratory pathogens without pre-preparation. The overall detection process, including swab rinsing, magnetic bead-based nucleic acid extraction, and 8-plex real-time RT-LAMP, can be automatically performed in the cassette within 80 min. The functionality of the cassette was validated by detecting the presence of a SARS-CoV-2 pseudovirus and three other respiratory pathogens, i.e., Klebsiella pneumoniae, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia. The limit of detection (LoD) for the SARS-CoV-2 pseudovirus was 2.5 copies/μL with both primer sets (N gene and ORF1ab gene), and the three bacterial species were successfully detected with an LoD of 2.5 colony-forming units (CFU)/μL in 800 μL of swab rinse. Thus, the analyzer developed in this study has the potential to rapidly detect SARS-CoV-2 and other respiratory pathogens on site in a “raw-sample-in and answer-out” manner.

scholarly journals Optimization of magnetic bead-based nucleic acid extraction for SARS-CoV-2 testing using readily available reagents

2021 ◽  
Author(s):  
Simon Haile ◽  
Aidan M. Nikiforuk ◽  
Pawan K. Pandoh ◽  
David D. W. Twa ◽  
Duane E. Smailus ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Diagnostic Testing ◽  
Magnetic Bead ◽  
Acid Extraction ◽  
Flexible Systems ◽  
Nucleic Acid Extraction ◽  
Extraction Protocol

AbstractThe COVID-19 pandemic has highlighted the need for generic reagents and flexible systems in diagnostic testing. Magnetic bead-based nucleic acid extraction protocols using 96-well plates on open liquid handlers are readily amenable to meet this need. Here, one such approach is rigorously optimized to minimize cross-well contamination while maintaining sensitivity.Article SummaryA scalable, non-proprietary, magnetic bead-based automated nucleic acid extraction protocol optimised for minimum cross-well contamination

scholarly journals “FastCheckFLI PPR-like”—A Molecular Tool for the Fast Genome Detection of PPRV and Differential Diagnostic Pathogens

Viruses ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 1227
Author(s):  
Sabrina Halecker ◽  
Thomas C. Mettenleiter ◽  
Martin Beer ◽  
Bernd Hoffmann
Keyword(s):  
Nucleic Acid ◽  
Internal Control ◽  
High Speed ◽  
Magnetic Bead ◽  
Time Frame ◽  
Differential Diagnostics ◽  
Acid Extraction ◽  
Molecular Test ◽  
Nucleic Acid Extraction ◽  
Mouth Disease Virus

To assist the global eradication of peste des petits ruminants virus (PPRV), a molecular test for the rapid and reliable detection of PPRV was developed which additionally enables the detection of pathogens relevant for differential diagnostics. For this purpose, the necessary time frame of a magnetic bead-based nucleic acid extraction protocol was markedly shortened to 7 min and 13 s. The optimized extraction was run on a BioSprint 15 platform. Furthermore, a high-speed multi-well RT-qPCR for the genome detection of PPRV and additional important pathogens such as Foot-and-mouth disease virus, Parapoxvirus ovis, Goatpox virus, and Mycoplasma capricolum subsp. capripneumoniae was established and combined with suitable internal control assays. The here-described qPCR is based on a lyophilized master mix and takes only around 30 to 40 min. Several qPCR cyclers were evaluated regarding their suitability for fast-cycling approaches and for their diagnostic performance in a high-speed RT-qPCR. The final evaluation was conducted on the BioRad CFX96 and also on a portable Liberty16 qPCR cycler. The new molecular test designated as “FastCheckFLI PPR-like”, which is based on rapid nucleic acid extraction and high-speed RT-qPCR, delivered reliable results in less than one hour, allowing its use also in a pen-side scenario.

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 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 >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.

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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