Bacterial Genomic DNA Extraction Research Articles

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Oysters are filter feeders that bioaccumulate bacteria in water while feeding. To evaluate the bacterial genomic DNA extracted from retail oyster tissues, including the gills and digestive glands, four isolation methods were used. Genomic DNA extraction was performed using the Allmag™ Blood Genomic DNA (Allrun, Shanghai, China), the MiniBEST Bacterial Genomic DNA Extraction kits (Takara, Dalian, China), and the phenol-chloroform and boiling lysis methods. The concentration of the genomic DNA was measured using a spectrophotometer. The purity of the genomic DNA was evaluated by PCR amplification of 16S rDNA followed by determining the cloning efficiency of the amplicon into the pMD19-T vector. Furthermore, the bacterial DNA quality was also evaluated by PCR assays using a pair of species-specific primers for Vibrio parahaemolyticus. Our results showed that the two commercial kits produced the highest purity of DNA, but with the lowest yields. The phenol-chloroform method produced the highest yield although it was time-consuming. The boiling lysis method was simple and cost effective; however, it was only suitable to isolate genomic DNA from bacteria present in retail samples following an enrichment step. The two commercial kits were good candidates for genomic DNA extraction from retail oyster tissues without enrichment.

Comparative analysis of the distribution of segmented filamentous bacteria in humans, mice and chickens

Segmented filamentous bacteria (SFB) are indigenous gut commensal bacteria. They are commonly detected in the gastrointestinal tracts of both vertebrates and invertebrates. Despite the significant role they have in the modulation of the development of host immune systems, little information exists regarding the presence of SFB in humans. The aim of this study was to investigate the distribution and diversity of SFB in humans and to determine their phylogenetic relationships with their hosts. Gut contents from 251 humans, 92 mice and 72 chickens were collected for bacterial genomic DNA extraction and subjected to SFB 16S rRNA-specific PCR detection. The results showed SFB colonization to be age-dependent in humans, with the majority of individuals colonized within the first 2 years of life, but this colonization disappeared by the age of 3 years. Results of 16S rRNA sequencing showed that multiple operational taxonomic units of SFB could exist in the same individuals. Cross-species comparison among human, mouse and chicken samples demonstrated that each host possessed an exclusive predominant SFB sequence. In summary, our results showed that SFB display host specificity, and SFB colonization, which occurs early in human life, declines in an age-dependent manner.

Potential application of superparamagnetic nanoparticles for extraction of bacterial genomic DNA from contaminated food and environmental samples

Isolation of high-molecular-weight DNA is essential for many molecular biology applications. Owing to the presence of polymerase chain reaction (PCR) inhibitors, there is a scarcity of suitable protocols for PCR-ready DNA extraction from food and natural environments. The conventional chemical methods of DNA extraction are time consuming and laborious and the yield is very low. Thus the aim of this research was to develop a simple, rapid, cost-effective method of genomic DNA extraction from food (milk and fruit juice) and environmental (pond water) samples and to detect bacterial contaminants present in those samples. This approach is efficient for both Gram-positive and Gram-negative bacteria from all the studied samples. Herein super paramagnetic bare iron oxide nanoparticles were implemented for bacterial genomic DNA isolation. The method was also compared to the conventional phenol-chloroform method in the context of quality, quantity and timing process. This method took only half an hour or less to obtain high-molecular-weight purified DNA from minimum bacterial contamination. Additionally, the method was directly compatible to PCR amplification. The problem of availability of suitable generalized methods for DNA isolation from various samples including food and environmental has been solved by a nanobiotechnological approach that may prove to be extremely useful in biotechnological applications.

Antimicrobial Drug Resistance in Corynebacterium diphtheriae mitis

Antimicrobial Drug Resistance in Corynebacterium diphtheriae mitis

A Completely Automated Sample Preparation Instrument and Consumable Device for Isolation and Purification of Nucleic Acids

Molecular diagnostic analysis and life science studies are dependent on the ability to effectively prepare samples for analysis. We report the development of a system that enables robust sample preparation of nucleic acids. To enable completely automated sample preparation, a consumable cartridge and consumable module system were developed to emulate every step of the sample preparation process. This included enzyme and reagent addition, temperature-controlled incubations, noncontact mixing of enzymes and reagents, buffer exchanges, and sample elution. Using this system, completely automated methods were developed for the purification of viral RNA and DNA from plasma and whole blood and of bacterial genomic DNA from water and whole blood. Extracted nucleic acids were detected and quantified using real-time PCR. The data indicate that automated viral DNA extraction was more efficient than sample extractions performed using a manual process, whereas automated total RNA extraction from the same samples was equivalent to controls. Additionally, we found that the process for bacterial genomic DNA extraction from either water or whole blood was equivalent to the manual extraction processes. We conclude the instrument, consumable cartridge, and reagent system enables easy, cost-effective, and robust sample preparation regardless of the experience of the operator.

Evaluation and optimisation of bacterial genomic DNA extraction for no-culture techniques applied to vinegars

Direct genomic DNA extraction from vinegars was set up and suitability for PCR assays performed by PCR/DGGE and sequencing of 16S rRNA gene. The method was tested on 12 intermediary products of special vinegars, fruit vinegars and condiments produced from different raw materials and procedures. DNAs extraction was performed on pellets by chemical, enzymatic, resin mediated methods and their modifications. Suitable yield and DNA purity were obtained by modification of a method based on the use of PVP/CTAB to remove polyphenolic components and esopolysaccharides. By sequencing of bands from DGGE gel, Gluconacetobacter europaeus, Acetobacter malorum/cerevisiae and Acetobacter orleanensis were detected as main species in samples having more than 4% of acetic acid content. From samples having no acetic acid content, sequences retrieved from excised bands revealed high similarity with prokaryotes with no function on vinegar fermentation: Burkholderia spp., Cupriavidus spp., Lactococcus lactis and Leuconostoc mesenteroides. The method was suitable to be applied for no-culture study of vinegars containing polyphenols and esopolysaccharides allowing a more complete assessment of vinegar bacteria.

A method for obtaining DNA from compost

An effective cell lysis method for extraction of bacterial genomic DNA from compost was developed in this study. Enzymatic disruption method, physical-chemical combination method, and commercial kit method were used to extract DNA from compost samples and were compared by analyzing DNA yield and efficient cell lysis. The results showed that all the three methods can be used to extract high-quality DNA from compost, but the enzymatic method had better cell lysis efficiency and DNA yields than others without the use of special equipment and expensive spending. Comparison of different methods for lysing gram-positive bacteria Bacillus subtilis indicated that the enzymatic cell lysis is superior for destroying the gram-positive cell wall. Spin-bind DNA column was used for DNA purification, and the purity of the purified sample was checked by polymerase chain reaction to amplify a region of the 16S rRNA. Results indicated that the part of 16S rRNA were amplified from all the purified DNA samples, and all the amplification products could be digested by the restriction enzyme HhaI.

Identification of an Isolate of Pseudomonas aeroginosa Deposited in PTCC as a PHA Producer Strain: Comparison of Three Different Bacterial Genomic DNA Extraction Methods

Identification of an Isolate of Pseudomonas aeroginosa Deposited in PTCC as a PHA Producer Strain: Comparison of Three Different Bacterial Genomic DNA Extraction Methods

Development of a molecular methodology to quantify Staphylococcus epidermidis in surgical wash-out samples from prosthetic joint replacement surgery.

Prosthetic joint infection is a serious complication of total joint arthroplasty that causes great morbidity in affected individuals. The most common cause of prosthesis associated infections are members of Staphylococcus spp., including Staphylococcus epidermidis. Culture has served as the gold standard for diagnosis, despite obvious shortcomings in terms of sensitivity and time. Bacterial genomic DNA extraction methodologies were evaluated for optimal recovery of genomic DNA from sterilised wash-out samples, spiked with S. epidermidis. Real time Polymerase chain reaction (PCR) assays targeting the S. epidermidis specific gseA gene were designed to reliably detect and quantify S. epidermidis. Sixty post-operative wash-out samples from primary hip and knee arthroplasties were taken aseptically. All were shown to be culture negative using the culture-dependent approach. These were samples were subjected to S. epidermidis-specific real time PCR. Standard curve showed good linearity. Sensitivity limit of the assay was <10CFU S. epidermidis per sample. Reproducibility of the assay was confirmed. S. epidermidis was not identified in any of these samples using the novel species specific SYBR Green real time PCR technique. Results indicated that wash-out samples were true negatives and did not harbour S. epidermidis. To support this, patients displayed no symptoms of infection. To illustrate the full effectiveness of the novel real time PCR assay, a larger number of samples need to be tested (>1,000 patients).

Real-Time Multiplex SYBR Green I–Based PCR Assay for Simultaneous Detection of Salmonella Serovars and Listeria monocytogenes

A multiplex SYBR Green I–based PCR assay has been developed for simultaneous detection of Salmonella serovars and Listeria monocytogenes using a LightCycler. Primers were designed to amplify an 85-bp sequence from the gene encoding a fimbrinlike protein (fimI) of Salmonella Enteritidis and a 98-bp sequence from the hemolysin gene (hly) of L. monocytogenes. These primers allowed the amplification of PCR products having distinct melting temperature values, resulting in the formation of two distinct peaks representing the two targets. Background signals, resulting from primer-dimer formation in the late cycles of PCR, are eliminated through the acquisition of data at a high temperature (>75°C), but several degrees lower than required for detection of the specific PCR products. A rapid and simple method for the extraction of bacterial genomic DNA from liquid culture, coupled with duplex PCR using LightCycler SYBR Green–based PCR assays, detected the presence of 2.5 cells and 1 cell of Salmonella serovars and L. monocytogenes, respectively, within an hour. Following overnight enrichment, target DNA was present in sufficient quantities in 1μl of culture to enable direct detection with the LightCycler.

Rapid extraction of bacterial genomic DNA with guanidium thiocyanate

A method is described for the rapid isolation and purification of bacterial genomic DNA. A total of 215 bacterial strains representing species of Campylobacter, Corynebacterium, Escherichia, Legionella, Neisseria, Staphylococcus and Streptococcus, were lysed with guanidium thiocyanate. DNA was prepared using just three other reagents and one high-speed centrifugation step. The method, which was applicable to both Gram-positive and Gram-negative bacteria, eliminated endogenous nuclease activity and avoided the need for phenol, RNase and protease treatments. The DNA was of high purity, high molecular mass and double-stranded.