scholarly journals Rapid and affordable size-selected PacBio single-molecule real-time sequencing template library construction using the bead-beating DNA extraction method

2017 ◽  
Vol 4 (3) ◽  
pp. 79 ◽  
Author(s):  
Kengo Kato ◽  
Masanori Hashino ◽  
Tamaki Ito ◽  
Mari Matsui ◽  
Satowa Suzuki ◽  
...  
Keyword(s):  
Real Time ◽  
Dna Extraction ◽  
Single Molecule ◽  
Extraction Method ◽  
Library Construction ◽  
Bead Beating ◽  
Dna Extraction Method ◽  
Template Library

Influence of Primer Sequences and DNA Extraction Method on Detection of Non-O157 Shiga Toxin–Producing Escherichia coli in Ground Beef by Real-Time PCR Targeting the eae, stx, and Serogroup-Specific Genes†

2012 ◽  
Vol 75 (11) ◽  
pp. 1939-1950 ◽  
Author(s):  
JAMIE L. WASILENKO ◽  
PINA M. FRATAMICO ◽  
NEELAM NARANG ◽  
GLENN E. TILLMAN ◽  
SCOTT LADELY ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Dna Extraction ◽  
Shiga Toxin ◽  
Real Time Pcr ◽  
Extraction Method ◽  
Ground Beef ◽  
Dna Extraction Method ◽  
Big Six ◽  
Pcr Targeting

Non-O157 Shiga toxin–producing Escherichia coli (STEC) infections, particularly those caused by the “big six” or “top six” non-O157 serogroups (O26, O45, O103, O111, O121, and O145) can result in severe illness and complications. Because of their significant public health impact and the notable prevalence of STEC in cattle, methods for detection of the big six non-O157 STEC in ground beef have been established. Currently, the U.S. Department of Agriculture, Food Safety and Inspection Service detection methods for screening beef samples for non-O157 STEC target the stx1, stx2, and eae virulence genes, with the 16S rRNA gene as an internal control, in a real-time PCR multiplex assay. Further, the serogroup is determined by PCR targeting genes in the E. coli O-antigen gene clusters of the big six non-O157 serogroups. The method that we previously reported was improved so that additional stx variants, stx1d, stx2e, and stx2g, are detected. Additionally, alignments of the primers targeting the eae gene were used to improve the detection assay so that eae subtypes that could potentially be of clinical significance would also be detected. Therefore, evaluation of alternative real-time PCR assay primers and probes for the stx and eae reactions was carried out in order to increase the stx and eae subtypes detected. Furthermore, a Tris-EDTA DNA extraction method was compared with a previously used procedure that was based on a commercially available reagent. The Tris-EDTA DNA extraction method significantly decreased the cycle threshold values for the stx assay (P < 0.0001) and eae assay (P < 0.0001), thereby increasing the ability to detect the targets. The use of different stx primers and probes increased the subtypes detected to include stx1d, stx2e, and stx2g, and sequence data showed that modification of the eae primer should allow the known eae subtypes to be detected.

scholarly journals A low-cost pipeline for soil microbiome profiling

2020 ◽  
Author(s):  
Anita Bollmann-Giolai ◽  
Michael Giolai ◽  
Darren Heavens ◽  
Iain Macaulay ◽  
Jacob Malone ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Genomic Dna ◽  
Extraction Method ◽  
Low Cost ◽  
Sequence Variants ◽  
Genomic Dna Extraction ◽  
Library Construction ◽  
Dna Extraction Method ◽  
Amplicon Library ◽  
Commercial Kits

AbstractBackgroundCommon bottlenecks in environmental microbiome studies are the consumable and personnel costs necessary for genomic DNA extraction and sequencing library construction. This is harder for challenging environmental samples such as soil, which is rich in PCR inhibitors. To address this, we have established a low-cost genomic DNA extraction method for inhibitor rich samples alongside an Illumina-compatible 16S and ITS rRNA gene amplicon library preparation workflow that uses common laboratory equipment. We evaluated the performance of our genomic DNA extraction method against two leading commercial soil genomic DNA kits (MoBio PowerSoil® and MP Biomedicals™ FastDNA™ SPIN) and a recently published non-commercial extraction method by Zou et al. (2017). Our benchmarking experiment used four different soil types (coniferous, broad leafed, and mixed forest plus a standardised cereal crop compost mix) assessing the quality and quantity of the extracted genomic DNA by analysing sequence variants of 16S V4 and ITS rRNA amplicons.ResultsWe found that our genomic DNA extraction method compares well to both commercially available genomic DNA extraction kits in DNA quality and quantity. The MoBio PowerSoil® kit, which relies on silica column-based DNA extraction with extensive washing delivered the cleanest genomic DNA e.g. best A260:A280 and A260:A230 absorbance ratios. The MP Biomedicals™ FastDNA™ SPIN kit, which uses a large amount of binding material, yielded the most genomic DNA. Our method fits between the two commercial kits, producing both good yields and clean genomic DNA with fragment sizes of approximately 10 kb. Comparative analysis of detected amplicon sequence variants shows that our method correlates well with the two commercial kits.ConclusionHere we present a low-cost genomic DNA extraction method for inhibitor rich sample types such as soil that can be coupled to an Illumina-compatible simple two step amplicon library construction workflow for 16S V4 and ITS marker genes. Our method delivers high quality genomic DNA at a fraction of the cost of commercial kits and enables cost-effective, large scale amplicon sequencing projects. Notably our extracted gDNA molecules are long enough to be suitable for downstream techniques such as full gene sequencing or even metagenomics shotgun approaches using long reads (PacBio or Nanopore), 10x Genomics linked reads, Dovetail genomics etc.

Rapid detection of porcine DNA in processed food samples using a streamlined DNA extraction method combined with the SYBR Green real-time PCR assay

2020 ◽  
Vol 309 ◽  
pp. 125654 ◽  
Author(s):  
Lee Lee Tan ◽  
Siti Aminah Ahmed ◽  
Siew Kit Ng ◽  
Marimuthu Citartan ◽  
Carsten A. Raabe ◽  
...  
Keyword(s):  
Real Time ◽  
Dna Extraction ◽  
Real Time Pcr ◽  
Rapid Detection ◽  
Extraction Method ◽  
Food Samples ◽  
Sybr Green ◽  
Processed Food ◽  
Pcr Assay ◽  
Dna Extraction Method

scholarly journals Effects of DNA extraction method on Porcine circovirus-2 real-time polymerase chain reaction quantification in swine lymph node samples

2011 ◽  
Vol 23 (6) ◽  
pp. 1189-1196 ◽  
Author(s):  
Silvia Faccini ◽  
Arrigo D. Nigrelli ◽  
Giuliana Franzini ◽  
Carlo Rosignoli ◽  
Ilaria Barbieri ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Lymph Node ◽  
Real Time ◽  
Dna Extraction ◽  
Extraction Method ◽  
Porcine Circovirus ◽  
Quantitative Real Time Pcr ◽  
Chain Reaction ◽  
Dna Extraction Method ◽  
Polymerase Chain

Quantitative real-time polymerase chain reaction (PCR) has become an important tool for Porcine circovirus-2 (PCV-2) research and diagnosis. However, significant differences in detection limit and quantification data, among laboratories and quantitative real-time PCR methods, have been demonstrated. New efforts are required for providing more accurate and comparable results. The current study is an evaluation of the effects of DNA extraction procedures on PCV-2 quantification in lymph node samples. Differences, greater than 1 log10 copies/g, were shown among PCV-2 loads detected after different extraction procedures. The work highlighted the critical role of the DNA extraction method in PCV-2 quantification by quantitative real-time PCR. This important aspect should be evaluated when comparing data from different laboratories or different studies. The PCV-2 quantification data should not be considered comparable before demonstrating the equivalence of the DNA extraction methods performed.

scholarly journals Storage media and not extraction method has the biggest impact on recovery of bacteria from the oral microbiome

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Xiaoyan Zhou ◽  
Shanika Nanayakkara ◽  
Jin-Long Gao ◽  
Ky-Anh Nguyen ◽  
Christina J. Adler
Keyword(s):  
Dna Extraction ◽  
Dental Plaque ◽  
Extraction Method ◽  
Storage Conditions ◽  
Oral Microbiome ◽  
Rrna Gene ◽  
Oral Microbiota ◽  
Bead Beating ◽  
Storage Media ◽  
Dna Extraction Method

Abstract Next Generation sequencing has greatly progressed the exploration of the oral microbiome’s role in dental diseases, however, there has been little focus on the effect of sample storage conditions and their interaction with DNA extraction method. Dental plaque samples collected from 20 healthy participants were pooled and stored in either 75% ethanol or Bead solution for up to 6-months at −80 °C, prior to DNA extraction with either QIAamp (non-bead beating) or PowerSoil (bead-beating) kit, followed by Illumina sequencing of 16S rRNA gene. We found that storage media and not extraction method had the biggest influence on the diversity and abundance of the oral microbiota recovered. Samples stored in Bead solution, independent of the extraction kit, retrieved higher diversity (PowerSoil p = 1.64E-07, QIAamp p = 0.0085) and had dissimilar overall ecologies as indicated by lower level of shared diversity (PowerSoil p = 0.0000237, QIAamp p = 0.0088). Comparatively, samples stored in Bead solution and extracted with PowerSoil recovered a higher abundance of Streptococcus species. These data indicate that Bead solution can preserve the oral microbiome in dental plaque reliably, for periods of up to 6-months at −80 °C, and is compatible, with either a bead-beating or non-bead beating DNA extraction method.

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