scholarly journals Development of Rapid Extraction Method of Mycobacterium avium Subspecies paratuberculosis DNA from Bovine Stool Samples

Diagnostics ◽  
2019 ◽  
Vol 9 (2) ◽  
pp. 36 ◽  
Author(s):  
Sören Hansen ◽  
Marco Roller ◽  
Lamia Alslim ◽  
Susanne Böhlken-Fascher ◽  
Kim Fechner ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Mycobacterium Avium ◽  
Extraction Method ◽  
Magnetic Beads ◽  
Mycobacterium Avium Subspecies Paratuberculosis ◽  
Rapid Identification ◽  
Cold Chain ◽  
Silica Membrane ◽  
Molecular Assays ◽  
Stool Samples

The rapid identification of Mycobacterium avium subspecies paratuberculosis (MAP) infected animals within the herd is essential for preventing the spread of the disease as well as avoiding human exposure. Although culture is seen as the gold standard, there are various molecular assays available i.e., polymerase chain reaction (PCR) or isothermal amplification technique (recombinase polymerase amplification (RPA)) for the detection of MAP. The accuracy of the molecular assays is highly dependent on the DNA extraction method. In order to establish a rapid point of need system for the detection of MAP DNA from stool samples, we developed a rapid DNA extraction protocol (MAP DNA SpeedXtract) specified for use in combination with the RPA. The whole procedure from “sample in” to “result out” was conducted in a mobile suitcase laboratory. The DNA extraction is based on reverse purification by magnetic beads, which reduces the required technical demand. The MAP DNA SpeedXtract was performed within 25 min and only three pipetting steps were needed. The amplification and detection time were 20 min in RPA. The sensitivity and specificity of the developed protocol in comparison with the lab-based silica membrane column extraction and real-time PCR were 90.9% (n = 22) and 100% (n = 23), respectively. In conclusion, we established a rapid and reliable protocol for the extraction and detection of MAP DNA. All reagents are cold chain independent. The entire setup is ideal for point of need identification of MAP infected cases.

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.

A systematic evaluation of stool DNA preparation protocols for colorectal cancer screening via analysis of DNA methylation biomarkers

2021 ◽  
Vol 59 (1) ◽  
pp. 91-99
Author(s):  
Shengnan Jin ◽  
Qian Ye ◽  
Yanping Hong ◽  
Wenqing Dai ◽  
Chengliang Zhang ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Dna Methylation ◽  
Dna Extraction ◽  
Genomic Dna ◽  
Magnetic Beads ◽  
Systematic Evaluation ◽  
Stool Samples ◽  
Human Genomic ◽  
Tumor Dna ◽  
Human Genomic Dna

AbstractObjectivesColorectal cancer (CRC) screening using stool samples is now in routine use where tumor DNA methylation analysis for leading markers such as NDRG4 and SDC2 is an integral part of the test. However, processing stool samples for reproducible and efficient extraction of human genomic DNA remains a bottleneck for further research into better biomarkers and assays.MethodsWe systematically evaluated several factors involved in the processing of stool samples and extraction of DNA. These factors include: stool processing (solid and homogenized samples), preparation of DNA from supernatant and pellets, and DNA extraction with column and magnetic beads-based methods. Furthermore, SDC2 and NDRG4 methylation levels were used to evaluate the clinical performance of the optimal protocol.ResultsThe yield of total and human genomic DNA (hgDNA) was not reproducible when solid stool scraping is used, possibly due to sampling variations. More reproducible results were obtained from homogenized stool samples. Magnetic beads-based DNA extraction using the supernatant from the homogenized stool was chosen for further analysis due to better reproducibility, higher hgDNA yield, lower non-hgDNA background, and the potential for automation. With this protocol, a combination of SDC2 and NDRG4 methylation signals with a linear regression model achieved a sensitivity and specificity of 81.82 and 93.75%, respectively.ConclusionsThrough the systematic evaluation of different stool processing and DNA extraction methods, we established a reproducible protocol for analyzing tumor DNA methylation markers in stool samples for colorectal cancer screening.

scholarly journals Electrochemical Detection of Serum Antibodies Against Mycobacterium avium Subspecies paratuberculosis

2021 ◽  
Vol 8 ◽  
Author(s):  
Kaoru Hatate ◽  
J. Hunter Rice ◽  
Karsten Parker ◽  
J. Jayne Wu ◽  
Amy Turner ◽  
...  
Keyword(s):  
Electrochemical Detection ◽  
Mycobacterium Avium ◽  
Magnetic Beads ◽  
Mycobacterium Avium Subspecies Paratuberculosis ◽  
Diagnostic System ◽  
New Method ◽  
Economic Losses ◽  
Dairy Herds ◽  
Redox Active ◽  
Active Probe

Mycobacterium avium subsp. paratuberculosis (MAP) causes a chronic inflammatory intestinal disease, called Johne's disease (JD) in many ruminants. In the dairy industry, JD is responsible for significant economic losses due to decreased milk production and premature culling of infected animals. Test-and-cull strategy in conjunction with risk management is currently recommended for JD control in dairy herds. However, current diagnostic tests are labor-intensive, time-consuming, and/or too difficult to operate on site. In this study, we developed a new method for the detection of anti-M. paratuberculosis antibodies from sera of M. paratuberculosis-infected animals. M. paratuberculosis antigen-coated magnetic beads were sequentially reacted with bovine serum followed by a horseradish peroxidase (HRP)-labeled secondary antibody. The reaction of HRP with its substrate was then quantitatively measured electrochemically using a redox-active probe, ferrocyanide. After optimization of electrochemical conditions and concentration of the redox-active probe, we showed that the new electrochemical detection method could distinguish samples of M. paratuberculosis-infected cattle from those of uninfected cattle with greater separation between the two groups of samples when compared with a conventional colorimetric testing method. Since electrochemical detection can be conducted with an inexpensive, battery-operated portable device, this new method may form a basis for the development of an on-site diagnostic system for JD.

scholarly journals Use of untargeted magnetic beads to capture Mycobacterium smegmatis and Mycobacterium avium paratuberculosis prior detection by mycobacteriophage D29 and Real-Time-PCR

2021 ◽  
Author(s):  
Gabriel Rojas-Ponce ◽  
Dominic Sauvageau ◽  
Roger Zemp ◽  
Herman W. Barkema ◽  
Stephane Evoy
Keyword(s):  
Mycobacterium Avium ◽  
Mycobacterium Smegmatis ◽  
Magnetic Beads ◽  
Mycobacterium Avium Subspecies Paratuberculosis ◽  
Milk Proteins ◽  
Proteinase K ◽  
Mycobacterium Avium Paratuberculosis ◽  
Simple Tool ◽  
Mycobacterial Diseases ◽  
Rule Out

Dynabeads® M-280 Tosylactivated (untargeted magnetic beads) were evaluated to capture Mycobacterium smegmatis and Mycobacterium avium subspecies paratuberculosis (MAP) from spiked feces, milk, and urine. Untargeted magnetic beads added to the spiked samples were slightly mixed for 1 hour and separated in a magnetic rack for further detection. Beads recovered more M. smegmatis cells from PBS suspension that the centrifugation method; these results were confirmed by the recovery of 96.31% of 1.68 x 104 CFU/mL viable M. smegmatis by beads and 0% by centrifugation. Likewise, the F57-qPCR detection of MAP cells, after being recovered by beads and centrifugation, were different; cycle threshold (Ct) was lower (p<0.05) for the detection of MAP cells recovered by beads than centrifugation. Magnetic separation of MAP cells from milk, urine, and feces specimens were detected by amplifying F57 and IS900 sequences. Ct values demonstrated that beads captured no less than 109 CFU/mL from feces and no less than 104 CFU/mL of MAP cells from milk and urine suspensions. Milk proteins were denatured by Proteinase k before capturing MAP cells by magnetic beads. M. smegmatis coupled to magnetic beads were infected by mycobacteriophage D29; plaque former units were observed clearly from urine containing 2 x 105 and 2 x 103 CFU/mL M. smegmatis in 24 hours. The results of this study encourage further effort to rule out the use of untargeted beads as a simple tool for diagnosis of Johne′s disease and other mycobacterial diseases such as tuberculosis.

scholarly journals A High-Throughput Approach for Identification of Nontuberculous Mycobacteria in Drinking Water Reveals Relationship between Water Age and Mycobacterium avium

mBio ◽  
2018 ◽  
Vol 9 (1) ◽  
Author(s):  
Sarah-Jane Haig ◽  
Nadine Kotlarz ◽  
John J. LiPuma ◽  
Lutgarde Raskin
Keyword(s):  
Drinking Water ◽  
Dna Extraction ◽  
High Throughput ◽  
Mycobacterium Avium ◽  
Single Molecule ◽  
Distribution System ◽  
Extraction Method ◽  
Nontuberculous Mycobacteria ◽  
Water Age ◽  
Total Dna

ABSTRACT Nontuberculous mycobacteria (NTM) frequently detected in drinking water (DW) include species associated with human infections, as well as species rarely linked to disease. Methods for improved the recovery of NTM DNA and high-throughput identification of NTM are needed for risk assessment of NTM infection through DW exposure. In this study, different methods of recovering bacterial DNA from DW were compared, revealing that a phenol-chloroform DNA extraction method yielded two to four times as much total DNA and eight times as much NTM DNA as two commercial DNA extraction kits. This method, combined with high-throughput, single-molecule real-time sequencing of NTM rpoB genes, allowed the identification of NTM to the species, subspecies, and (in some cases) strain levels. This approach was applied to DW samples collected from 15 households serviced by a chloraminated distribution system, with homes located in areas representing short (<24 h) and long (>24 h) distribution system residence times. Multivariate statistical analysis revealed that greater water age (i.e., combined distribution system residence time and home plumbing stagnation time) was associated with a greater relative abundance of Mycobacterium avium subsp. avium, one of the most prevalent NTM causing infections in humans. DW from homes closer to the treatment plant (with a shorter water age) contained more diverse NTM species, including Mycobacterium abscessus and Mycobacterium chelonae. Overall, our approach allows NTM identification to the species and subspecies levels and can be used in future studies to assess the risk of waterborne infection by providing insight into the similarity between environmental and infection-associated NTM. IMPORTANCE An extraction method for improved recovery of DNA from nontuberculous mycobacteria (NTM), combined with single-molecule real-time sequencing (PacBio) of NTM rpoB genes, was used for high-throughput characterization of NTM species and in some cases strains in drinking water (DW). The extraction procedure recovered, on average, eight times as much NTM DNA and three times as much total DNA from DW as two widely used commercial DNA extraction kits. The combined DNA extraction and sequencing approach allowed high-throughput screening of DW samples to identify NTM, revealing that the relative abundance of Mycobacterium avium subsp. avium increased with water age. Furthermore, the two-step barcoding approach developed as part of the PacBio sequencing method makes this procedure highly adaptable, allowing it to be used for other target genes and species.

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