Deoxyribonucleic Acid Extraction Research Articles

Evaluation of Bacteriological Profile in the Apical Root Segment of the Patients with Primary Apical Periodontitis.

Apical periodontitis usually results from bacterial accumulation and contamination occurring in the root-canal system, and extending beyond the apical foramen to involve the periapical tissues. Literature has a paucity of the studies that stress on the division and analysis of the pulp canal segments. The reason for this disparity might be the technique used for collecting the samples from the pulp canals. Hence, we carried out the present study to evaluate the microbial flora in the apical part of the roots with necrotic pulp canals. The present study included the assessment of 40 freshly extracted teeth that had necrotized pulpal tissue along with the presence of periapical periodontal lesions. Removal of the soft tissue lesions attached to the root portion of the teeth along with apical periodontal lesions was done with the help of scalpel blade, after rinsing them with a sterile solution of saline. Thorough cleaning of the root surfaces was done with hydrogen peroxide followed by rapid disinfection with the help of sodium hypochlorite at varying concentrations. Sectioning of the root portion of all the specimens with the help of a disk was done perpendicular to the long axis of the teeth at a distance of roughly 5 to 6 mm from the teeth's apicalmost point. Cryotubes were used for transferring the specimens of apical portions containing 1 mL of buffer and were subjected to immediate frozen processing at a temperature of -20°C. A 10 K-type file was used for the initial collection of the samples followed by subsequent incubation of the files and paper pints in the incubation cabinet. Subsequent deoxyribonucleic acid (DNA) extraction from the samples was done following the procedure described by Siqueira et al. Paster et al's modification of the reverse-capture checkerboard assay was used in the present study. Semiquantitative data were used for overcoming the difficulties arising due to obtaining the counts of the polymerase chain reaction (PCR)-based analysis of specimens. A positive result for the 16S ribosomal ribonucleic acid (rRNA) gene primer was observed only in two examined specimens of all the samples of the apical portion of the root canals in the present study. Negative result was shown by all the control group specimens, which were sterile samples. Presence of bacteria was confirmed by PCR in 38 out of 40 examined specimens. Amount of bacterial taxa, out of these 24 samples, ranged up to 6. Pseudoramibacter alactolyticus, Porphyromonas endodontalis, Dialister oral species, Bacteroidetes species, Streptococcus species, Olsenella uli, Synergistes species, Fusobacterium nucleatum, Parvimonas micra, Treponema denticola, and Filifactor alocis were the specific species detected. Bacteroidetes species was the only species that were detected at levels at or above 105. Heavy bacterial infections were noticed in more than 45% of the cases at the periradicular part of the root canals. Microbial flora of the apical segment of the root with necrotized pulp tissue comprises a vast variety of pathogenic bacteria. For better prognosis of the treatment of such cases, adequate knowledge of the microbial flora of the root, especially the apical portion is necessary.

Diseño de soluciones químicas para la extracción de ácido desoxirribonucleico en productos biotecnológicos

<p>Una de las técnicas utilizadas para la identificación de los organismos genéticamente modificados o transgénicos, es la reacción en cadena de la polimerasa. Para ello, se extrae de la muestra de interés, el ácido desoxirribonucleico con calidad amplificable y a concentraciones adecuadas; mediante el empleo de métodos químico-físicos, mecánicos o enzimáticos. Sin embargo, para la obtención de mejores resultados, se realiza la combinación de estos métodos. Tal es el caso del procedimiento basado en el uso del detergente catiónico bromuro de cetil trimetil amonio; ampliamente reportado en literatura temática. Con el objetivo de obtener alternativas al uso de este reactivo, para la extracción de ácido desoxirribonucleico, se diseñaron nuevas variantes; que combinan el uso de sustancias químicas como: Urea, Guanidio, Tween 20, Sarcosil y Dodecil Sulfato de Sodio y se siguieron los procedimientos establecidos para la extracción de ácidos nucleicos según el método CTAB. Al comparar los valores de las concentraciones de ácido nucleico extraído de las muestras de arroz, trigo, maíz, soya, tabaco y plátano, se obtienen concentraciones similares de ácido desoxirribonucleico de calidad amplificable, en los métodos diseñados, respecto al método que emplea Bromuro de cetil trimetil amonio. Sin embargo, al realizar un análisis económico, se determinó que con la aplicación de urea y Dodecil Sulfato de Sodio, se obtiene un ahorro económico del 48.3% en costo de reactivos; equivalente a 0.5 dólar por cada muestra analizada. Estos resultados manifiestan, la posibilidad de emplear métodos alternativos, al CTAB, para la extracción de ácido desoxirribonucleico en matrices complejas. </p>

Análisis comparativo de diferentes métodos de extracción de DNA y su eficiencia de genotipificación en población mexicana

Los estudios de variabilidad genetica han reportado inconsistencias de resultados entre poblaciones, en gran medida, debido a que los metodos de extraccion de acido desoxirribonucleico (DNA, por sus siglas en ingles) y las tecnicas de genotipificacion son altamente variables entre ellas, lo que conduce a la asignacion erronea de genotipos. El objetivo de este estudio es comparar distintas tecnicas de extraccion de DNA y de genotipificacion de polimorfismos. Para llevar a cabo el estudio, se analizo el DNA de 10 muestras sanguineas correspondientes a individuos mestizos mexicanos, y se purifico por los metodos de fenol-cloroformo-alcohol isoamilico (FCI), gradiente de sales (GS), gradiente de sacarosa (GSC), DNAzol® y DNeasy Blood & Tissue Kit ®. Se genotipificaron los polimorfismos GSTT1 *0 y GSTM1*0 por reaccion en cadena de la polimerasa (PCR, por sus siglas en ingles) multiple, CYP1A1*2C por RFLP’s y AhR Arg554Lys por PCR tiempo real. Los resultados mostraron diferencias significativas (p < 0.001) en cantidad, pureza e integridad entre los distintos metodos. En los analisis moleculares se observo que el metodo de FCI presenta inhibidores de la reaccion de PCR, ya que no fue posible amplificar los fragmentos por PCR multiple y PCR punto final, aunque si hubo amplificacion en el PCR tiempo real. Los metodos GS y GSC amplificaron todas las muestras en las tres modalidades de PCR y mostraron resultados concordantes, mientras que solo el 80% de las muestras extraidas mediante DNAzol ® y DNeasy ® amplificaron, y los resultados no fueron concordantes para DNAzol ® (50%) y DNeasy ® (80%) en el analisis de PCR - RFLP’s. Los metodos de extraccion GS y GSC mostraron mayor recuperacion de DNA, con parametros de calidad e integridad optimos para los analisis moleculares por PCR

Determination of the staphylococcal cassette chromosome in methicillin-resistant Staphylococcus aureus strains isolated from various clinical samples

Aim ― The present study aimed to detect mecA and staphylococcal cassette chromosome mec (SCCmec) types in methicillin-resistant Staphylococcus aureus (MRSA) isolates obtained from various clinical samples in two university hospitals. It was also aimed to make comparison amongst the isolates. Materials and Methods ― A total of 99 MRSA strains isolated from various clinical samples between 2011-2015 were included in the study. Bacterial deoxyribonucleic acid (DNA) was extracted from Staphylococcus aureus strains using GF-1 DNA extraction Kit (Vivantis, Malaysia). mecA gene were detected, and SCCmec cassette types were determined by multiplex polymerase chain reaction (PCR) first, and following specific PCR. Specific MRSA strains such as COL type I, PER3 type Ia, and HU25 type IIIa were used as the quality control strains for optimization of multiplex PCR. The amplification products were electrophoresed using agarose gel electrophoresis in TAE buffer (mixture of tris base, acetic acid and ethylenediaminetetraacetic acid). Results ― mecA gene was detected in 60 Staphylococcus aureus isolates, and these were identificated as MRSA. Amongst the MRSA strains, SCCmec type III was the most frequent cassette type (42 isolates, 70.0%). SCCmec type I was detected in 27 isolates (45.0%), type II was in 26 isolates (43.3%), and type V in 23 isolates (38.3%). Conclusion ― In the present study, the most frequent cassette was detected as SCCmec type III in concordance with the studies conducted in Turkey and in some regions in the world. In conclusion, determination of epidemiological and molecular characteristics of MRSA strains has critical importance because of the difficulties in the treatment and of the nosocomial infections and epidemics they caused. The data obtained would contribute to the preventions in terms of epidemiology.

Fast-Track, One-Step E. coli Detection: A Miniaturized Hydrogel Array Permits Specific Direct PCR and DNA Hybridization while Amplification.

A timesaving and convenient method for bacterial detection based on one-step, one-tube deoxyribonucleic acid (DNA) hybridization on hydrogel array while target gene amplification is described. The hydrogel array is generated by a fast one-pot synthesis, where N,N'-dimethylacrylamide/polyethyleneglycol(PEG1900 )-bisacrylamide mixture polymerizes via radical photoinitiation by visible light within 20 min concomitant with in situ capture probe immobilization. These DNA-functionalized hydrogel droplets arrayed on a planar glass surface are placed in the polymerase chain reaction (PCR) mixture during the thermal amplification cycles. The bacterial cells can be implemented in a direct PCR reaction, omitting the need for prior template DNA extraction. The resulting fluorescence signal is immediately detectable after the end of the PCR (1 h) following one short washing step by microscopy. Therefore a valid signal can be reached within 1.5 h including 10 min for pipetting and placement of the tubes and chips. The performance of this novel hydrogel DNA array was successfully proven with varying cell numbers down to a limit of 10(1) Escherichia coli cells.

Development of a method for DNA extraction from oil palm leaves and the effects of pH and ionic strength on nucleic acid quantification

Oil palm (Elaeis guineensis Jacq.) has benefited little from molecular marker technologies although the long generation time and the unpredictable variations due to allogamy make it a high priority for improving breeding efficiency. Progress would require an efficient, low cost and high-throughput deoxyribonucleic acid (DNA) extraction method adapted for oil palm. A method for genomic DNA extraction from oil palm leaves that is simple and cost-effective was developed without the use of liquid nitrogen. A260/280 ratios between 1.987 and 2.078 were obtained. Furthermore, we could demonstrate that nucleic acid concentration and yield were inversely proportional to the pH and ionic strength of the solvent. The measured values varied significantly between 572 ng/µl and 496 ng/µl. DNA extracted with this method is stable and can be reproducibly amplified by PCR.

Electrospun nanofibres in agriculture and the food industry: a review.

The interesting characteristics of electrospun nanofibres, such as high surface-to-volume ratio, nanoporosity, and high safety, make them suitable candidates for use in a variety of applications. In the recent decade, electrospun nanofibres have been applied to different potential fields such as filtration, wound dressing, drug delivery, etc. and a significant number of review papers have been published in these fields. However, the use of electrospun nanofibres in agriculture is comparatively novel and is still in its infancy. In this paper, the specific applications of electrospun nanofibres in agriculture and food science, including plant protection using pheromone-loaded nanofibres, plant protection using encapsulation of biocontrol agents, preparation of protective clothes for farm workers, encapsulation of agrochemical materials, deoxyribonucleic acid extraction in agricultural research studies, pre-concentration and measurement of pesticides in crops and environmental samples, preparation of nanobiosensors for pesticide detection, encapsulation of food materials, fabrication of food packaging materials, and filtration of beverage products are reviewed and discussed. This paper may help researchers develop the use of electrospun nanofibres in agriculture and food science to address some serious problems such as the intensive use of pesticides. © 2016 Society of Chemical Industry.

Phenotypic and genotypic detection of methicillin-resistant Staphylococcus aureus in hunting dogs in Maiduguri metropolitan, Borno State, Nigeria.

Aim:To determine the presence of MRSA in hunting dogs in Maiduguri metropolitan.Materials and Methods:Phenotypic methods used includes microscopic technique, colony morphology study, catalase-coagulase tests, and the use of mannitol salt agar test, oxacillin resistance screening agar base, and antibiotic susceptibility testing methods. Genotypic approach was used for deoxyribonucleic acid extraction, and the presence of nuc and mecA gene was detected using polymerase chain reaction (PCR) techniques.Results:Examination of 416 swab samples from nasal and perineal region of dogs revealed a total of 79.5% of S. aureus, where 62.5% of the isolates were MRSA. Molecular analysis revealed that 7nuc genes specific for S. aureus from 20 presumptive MRSA assay were all mecA PCR negative. The isolates were sensitive to gentamicin and ciprofloxacin but proved resistant to cefoxitin and oxacillin.Conclusion:High isolation rate of MRSA was found in hunting dogs. Significant level (p<0.05) of MRSA was isolated in the nasal cavity of hunting dogs than its perineum. Only nuc genes were detected from the MRSA isolates.

Blood Group Determination using DNA extracted from Exfoliated Primary Teeth at Various Time Durations and Temperatures: A PCR Study.

AimTo determine polymerase chain reaction (PCR)-based blood group on tooth pulp obtained from teeth stored for 1 month, 6 months, and 1 year following extraction and to evaluate the stability of deoxyribonucleic acid (DNA) in primary tooth subjected to a temperature of 200°C ± 5°C for 15 minutes.Materials and methodsDental pulp tissue was collected from 40 exfoliated primary teeth stored for various time durations and temperature and preserved at 4°C till DNA extraction was carried out. Deoxyribonucleic acid was extracted using silica membrane-based spin-column procedure of QIAamp DNA minikit from BioRad. Deoxyribonucleic acid was subjected to PCR amplification and monoplex allele-specific PCR primers for ABO genotyping.Statistical analysis usedThe data were analyzed by comparison (based on percentage).ResultsIn our study, overall, 85% samples showed a DNA yield. Cent percent results were obtained for samples studied at the end of 1 month followed by 90 and 80% for samples studied for 6 months and 1 year respectively. Heated samples showed 70% result.ConclusionPolymerase chain reaction was found to be an effective method for blood group determination for teeth stored at various time durations and temperatures. However, as the time interval increased, the number of positive results obtained decreased.How to cite this articlePai RK, Bhat SS, Salman A, Hegde S. Blood Group Determination using DNA extracted from Exfoliated Primary Teeth at Various Time Durations and Temperatures: A PCR Study. Int J Clin Pediatr Dent 2016;9(4):308-312.

The effect of deoxyribonucleic acid extraction methods from lymphoid tissue on the purity, content, and amplifying ability.

Background:Nowadays, definitive diagnosis of numerous diseases is based on the genetic and molecular findings. Therefore, preparation of fundamental materials for these evaluations is necessary. Deoxyribonucleic acid (DNA) is the first material for the molecular pathology and genetic analysis, and better results need more pure DNA. Furthermore, higher concentration of achieved DNA causes better results and higher amplifying ability for subsequent steps. We aim to evaluate five DNA extraction methods to compare DNA intimacy including purity, concentration, and amplifying ability with each other.Materials and Methods:The lymphoid tissue DNA was extracted from formalin-fixed, paraffin embedded (FFPE) tissue through five different methods including phenol-chloroform as the reference method, DNA isolation kit (QIAamp DNA FFPE Tissue Kit, Qiagen, Germany), proteinase K and xylol extraction and heat alkaline plus mineral oil extraction as authorship innovative method. Finally, polymerase chain reaction (PCR) and real-time PCR method were assessed to compare each following method consider to DNA purity and its concentration.Results:Among five different applied methods, the highest mean of DNA purity was related to heat alkaline method. Moreover, the highest mean of DNA concentration was related to heat alkaline plus mineral oil. Furthermore, the best result in quantitative PCR was in proteinase K method that had the lowest cycle threshold averages among the other extraction methods.Conclusion:We concluded that our innovative method for DNA extraction (heat alkaline plus mineral oil) achieved high DNA purity and concentration.

Dentistry to the rescue of missing children: A review.

Today's society is becoming increasingly unsafe for children: we frequently hear about new incidents of missing children, which lead to emotional trauma for the loved ones and expose systemic failures of law and order. Parents can take extra precautions to ensure the safety of their children by educating them about ways to protect themselves and keep important records of the child such as updated color photographs, fingerprints, deoxyribonucleic acid (DNA) samples, etc., handy. However, in spite of all efforts, the problem of missing children still remains. Developments in the field of dentistry have empowered dentists with various tools and techniques to play a pivotal role in tracing a missing child. One such tool is Toothprints, a patented arch-shaped thermoplastic dental impression wafer developed by Dr. David Tesini, a paediatric dentist from Massachusetts. Toothprints enables a unique identification of the missing children not only through the bite impression but also through salivary DNA. Besides the use of Toothprints, a dentist can assist investigating agencies in identifying the missing children in multiple ways, including postmortem dental profiling, labeled dental fixtures, DNA extraction from teeth, and serial number engraving on the children's teeth. More importantly, all these tools cause minimal inconvenience to the individual, making a dentist's role in tracking a missing child even more significant. Thus, the simple discipline of maintaining timely dental records with the help of their dentists can save potential hassles for the parents in the future.

Single-tube nested PCR assay with in-house DNA extraction for Mycobacterium tuberculosis detection in blood and urine.

Molecular analyses are auxiliary tools for detecting Koch's bacilli in clinical specimens from patients with suspected tuberculosis (TB). However, there are still no efficient diagnostic tests that combine high sensitivity and specificity and yield rapid results in the detection of TB. This study evaluated single-tube nested polymerase chain reaction (STNPCR) as a molecular diagnostic test with low risk of cross contamination for detecting Mycobacterium tuberculosis in clinical samples. Mycobacterium tuberculosis deoxyribonucleic acid (DNA) was detected in blood and urine samples by STNPCR followed by agarose gel electrophoresis. In this system, reaction tubes were not opened between the two stages of PCR (simple and nested). STNPCR demonstrated good accuracy in clinical samples with no cross contamination between microtubes. Sensitivity in blood and urine, analyzed in parallel, was 35%-62% for pulmonary and 41%-72% for extrapulmonary TB. The specificity of STNPCR was 100% in most analyses, depending on the type of clinical sample (blood or urine) and clinical form of disease (pulmonary or extrapulmonary). STNPCR was effective in detecting TB, especially the extrapulmonary form for which sensitivity was higher, and had the advantage of less invasive sample collection from patients for whom a spontaneous sputum sample was unavailable. With low risk of cross contamination, the STNPCR can be used as an adjunct to conventional methods for diagnosing TB.

Transgênicos: a visão de alunos do ensino fundamental de uma escola do município de Jaciara-MT sobre os alimentos geneticamente modificados

Este trabalho foi realizado com o intuito de sensibilizar e informar os alunos do Ensino fundamental da Escola Estadual Santo Antônio, localizada na cidade de Jaciara-MT, sobre os organismos geneticamente modificados (OGMs) ou simplesmente, os transgênicos. Para isso, mostrou-se aos educandos a presença dos transgênicos na vida cotidiana das pessoas, principalmente na alimentação. Dentro deste contexto, explicou-se os conceitos relacionados ao assunto, tais como o entendimento sobre a estrutura do DNA (ácido desoxirribonucleico) e transgenia. Buscou-se o entendimento do assunto por meio de aulas expositivas e dialogadas, onde foram aplicados questionários, com o objetivo de avaliar o conhecimento prévio dos alunos. Também foram realizadas atividades lúdicas (como jogos de quebra-cabeça, cartas e montagens) e práticas (extração do DNA da cebola). Vale ressaltar que em cada atividade, eram abertos os debates, verificando a sensação e reação dos discentes, analisando o nível de aprendizagem. Como resultado, percebeu-se que a inserção a atividades práticas, lúdicas e dinâmicas, torna o aprendizado mais eficiente e prazeroso, fazendo com que o momento da aula seja melhor aproveitado, evidenciando a obtenção de resultados satisfatórios dentro do ensino de Ciências, e quais as vantagens de trabalhá-las na Educação Básica.

MPTH-14PROGNOSTIC ROLE OF METHYLATION STATUS OF MGMT PROMOTER ON GLIOBLASTOMA PATIENTS ESTIMATED QUANTITATIVELY BY PYROSEQUENCING

OBJECTIVE: This study was aimed to determine whether pyrosequencing might be useful to determine methylation status of MGMT promoter using relatively old archival tissue samples of glioblastomas as a clinical biomarker. Additionally, we examined other prognostic factors for survival of glioblastoma patients. METHODS: The available study set included FFPE tissue from 104 patients at our two hospitals from 1997 to 2012, of which all were diagnosed as glioblastoma. With review of medical records, clinicopathological data were collected. Deoxyribonucleic acid (DNA) extraction and bisulfite conversion was performed from three (10 µm thickness) slices of FFPE material using the QIAamp DNA FFPE extraction kit and EpiTect bisulfite kit (Qiagen, Hilden, Germany), respectively. RESULTS: Among them, methylated MGMT promoter was found in 43 (41.3%) of samples. The average percentage of methylation was 14.0 ± 16.8%, and methylated cases were 39.0 ± 14.7%; that of each year did not show a linear increasing or decreasing pattern according to the age of the FFPE block (p = 0.687). In the multivariate analysis, age (p = 0.048), performance status (p = 0.002), extent of surgery (p < 0.05), method of adjuvant therapy (p < 0.05), and methylation status estimated by MSP (p = 0.034) and pyrosequencing (p = 0.001) were independently associated with overall survival. Additionally, patients with more methylated cases (≥ 39%) had longer survival than those with less methylated cases (≥ 9% to < 39%) (HR of 7.529, 95% CI 3.956-11.102; p = 0.016). CONCLUSION: In this study, status and extent of methylation in MGMT gene promoters analyzed by pyrosequencing were found to be associated with overall survival in glioblastoma patients. Pyrosequencing is a quantitative method overcoming the problems of MSP, and is a simple technique for accurate analysis of DNA sequences.

Metodologies used in the extraction of deoxyribonucleic acid

The objective was to do a review the literature regarding the methodologies used in the extraction process of deoxyribonucleic acid. The nucleic acids may be extracted from various organic materials. Different methodologies are used for extraction, which should ensure the quality of the samples according to the objectives of the research and studied species, such as proteinase K, phenol / chloroform, hair bulbs, cards for blood, among other commercial kits and protocols adapted

Rapid detection of health-care-associated bloodstream infection in critical care using multipathogen real-time polymerase chain reaction technology: a diagnostic accuracy study and systematic review.

There is growing interest in the potential utility of real-time polymerase chain reaction (PCR) in diagnosing bloodstream infection by detecting pathogen deoxyribonucleic acid (DNA) in blood samples within a few hours. SeptiFast (Roche Diagnostics GmBH, Mannheim, Germany) is a multipathogen probe-based system targeting ribosomal DNA sequences of bacteria and fungi. It detects and identifies the commonest pathogens causing bloodstream infection. As background to this study, we report a systematic review of Phase III diagnostic accuracy studies of SeptiFast, which reveals uncertainty about its likely clinical utility based on widespread evidence of deficiencies in study design and reporting with a high risk of bias. Determine the accuracy of SeptiFast real-time PCR for the detection of health-care-associated bloodstream infection, against standard microbiological culture. Prospective multicentre Phase III clinical diagnostic accuracy study using the standards for the reporting of diagnostic accuracy studies criteria. Critical care departments within NHS hospitals in the north-west of England. Adult patients requiring blood culture (BC) when developing new signs of systemic inflammation. SeptiFast real-time PCR results at species/genus level compared with microbiological culture in association with independent adjudication of infection. Metrics of diagnostic accuracy were derived including sensitivity, specificity, likelihood ratios and predictive values, with their 95% confidence intervals (CIs). Latent class analysis was used to explore the diagnostic performance of culture as a reference standard. Of 1006 new patient episodes of systemic inflammation in 853 patients, 922 (92%) met the inclusion criteria and provided sufficient information for analysis. Index test assay failure occurred on 69 (7%) occasions. Adult patients had been exposed to a median of 8 days (interquartile range 4-16 days) of hospital care, had high levels of organ support activities and recent antibiotic exposure. SeptiFast real-time PCR, when compared with culture-proven bloodstream infection at species/genus level, had better specificity (85.8%, 95% CI 83.3% to 88.1%) than sensitivity (50%, 95% CI 39.1% to 60.8%). When compared with pooled diagnostic metrics derived from our systematic review, our clinical study revealed lower test accuracy of SeptiFast real-time PCR, mainly as a result of low diagnostic sensitivity. There was a low prevalence of BC-proven pathogens in these patients (9.2%, 95% CI 7.4% to 11.2%) such that the post-test probabilities of both a positive (26.3%, 95% CI 19.8% to 33.7%) and a negative SeptiFast test (5.6%, 95% CI 4.1% to 7.4%) indicate the potential limitations of this technology in the diagnosis of bloodstream infection. However, latent class analysis indicates that BC has a low sensitivity, questioning its relevance as a reference test in this setting. Using this analysis approach, the sensitivity of the SeptiFast test was low but also appeared significantly better than BC. Blood samples identified as positive by either culture or SeptiFast real-time PCR were associated with a high probability (> 95%) of infection, indicating higher diagnostic rule-in utility than was apparent using conventional analyses of diagnostic accuracy. SeptiFast real-time PCR on blood samples may have rapid rule-in utility for the diagnosis of health-care-associated bloodstream infection but the lack of sensitivity is a significant limiting factor. Innovations aimed at improved diagnostic sensitivity of real-time PCR in this setting are urgently required. Future work recommendations include technology developments to improve the efficiency of pathogen DNA extraction and the capacity to detect a much broader range of pathogens and drug resistance genes and the application of new statistical approaches able to more reliably assess test performance in situation where the reference standard (e.g. blood culture in the setting of high antimicrobial use) is prone to error. The systematic review is registered as PROSPERO CRD42011001289. The National Institute for Health Research Health Technology Assessment programme. Professor Daniel McAuley and Professor Gavin D Perkins contributed to the systematic review through their funded roles as codirectors of the Intensive Care Foundation (UK).

Prevalence of Herpesvirus and Correlation with Clinical Parameters in Indian Subjects with Chronic Periodontitis.

The identification of new uncultured species and viruses supports the possibility of combination of the herpes-virus-bacterial periodontal infection for periodontitis. The paucity of data and studies with larger sample size in Indian subjects provides an unclear picture of the presence of the herpesvirus in this population. This was a cross-sectional study consisting of 100 each in the healthy group and chronic periodontitis (CP) group. The subgingival plaque was collected and polymerase chain reaction was performed post deoxyribonucleic acid (DNA) extraction by using specific primers for human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV). The data were analyzed using Fisher's exact test, Mann-Whitney U test and Spearman's coefficient correlation. Human cytomegalovirus and EBV viruses were significantly higher in the CP group as compare to the healthy group. A higher percentage of those with CMV positive had EBV also positive (28.3%) compared to only 9.1% of CMV negative being EBV positive in the CP group. When both the healthy and CP group in total was compared, there was a significant correlation with all clinical parameters. Both the viruses dominated in disease as compared to health were similar to the earlier findings. The CP group had higher pocket depth and clinical attachment loss in the virus positive subjects. These findings could suggest that virus serves as a prelude to the disease and the combination of the two viruses could play a role in the pathogenesis.

Isolation of granulosa cells from follicular fluid; applications in biomedical and molecular biology experiments.

Background:Recently, a lot of research has been conducted to investigate the molecular mechanisms of the low quality of oocytes with granulosa cells (GCs). GCs are one of the major cell types found in follicular fluid and purification of these cells from the follicular fluid is very important for further studies. Although, there are different techniques of purification, a method for separation of highly-pure and minimally-damaged cells is necessary. In this paper, we presented a novel method for high purification of GCs with a large quantity and high purity.Materials and Methods:Follicular fluid was collected from patients who referred for in vitro fertilization and GCs in follicular fluid were extracted by Ficoll, Percoll and Red blood cell lysing buffer (RLB) methods. Then purity of extracted GCs was assessed by flow cytometry and morphological properties of GCs were observed by differential interference contrast microscopy. The purity of deoxyribonucleic acid and ribonucleic acid extracts was examined by NanoDrop 1000, pre-restriction fragment length polymorphism and electrophoresis techniques. Quality and quantity of extracting GCs were affected during the cell separation procedures.Results:Our results showed that each of purification method can affect quality and quantity of extracted cells.Conclusion:RLB method for extraction of GCs was shown to be a convenient procedure in comparison with Ficoll and Percoll methods.

Active cigarette smoking, variants in carcinogen metabolism genes and breast cancer risk among pre- and postmenopausal women in Ontario, Canada.

Cigarette smoking is strongly associated with various diseases including many cancers; however, evidence regarding breast cancer risk remains inconclusive with some studies reporting no association, and others an increased risk with long duration and early initiation of smoking. Genetic variation in carcinogen-metabolizing enzymes may modify these associations. Breast cancer cases were identified from the Ontario Cancer Registry (OCR) during 2003-2004 and population controls through random digit dialing methods. All subjects completed self-administered questionnaires. Subsequently, saliva samples were obtained from cases (N = 1,776) and controls (N = 1,839) for deoxyribonucleic acid (DNA) extraction. Multivariate logistic regression was used to estimate odds ratio (OR) and 95% confidence intervals (CI) for active smoking variables, and interactions were assessed between smoking and 36 carcinogen-metabolizing candidate gene variants. No statistically significant association was found between active smoking and breast cancer risk among all women nor when stratified by menopausal status; however, nonsignificant increased premenopausal breast cancer risk was observed among current smokers and women smoking before first pregnancy. Several statistically significant interactions were observed between smoking and genetic variants (CYP1A2 1548C>T, CYP1A1 3801T>C, CYP1B1 4326G>C, NAT1 c.-85-1014T>A, UGT1A7 W208R 622T>C, SOD2 c.47T>C, GSTT1 deletion). However, in analyses stratified by these genotypes, smoking ORs had wide confidence intervals (and with few exceptions included 1.0) making interpretations difficult. Active smoking was not associated with breast cancer risk, although several significant interactions were observed between smoking, carcinogen-metabolizing genetic variants, and breast cancer risk.

Phylogenetic Relationships of Sub-Populations of Barbus sharpeyi fish in southwest of Iran using mitochondrial DNA with PCR- Sequencing method

This study was conducted to Investigate cytochrome oxidase I (COI) Region from Barbus sharpeyi mitochondrial DNA (mtDNA), In Other to study genetic and phylogenetic relationships. A total number of 31 samples of fishery farms, jrahi river in shadegan and karkhe river in hovaizeh in southwest of Iran were studied. After DNA(Deoxyribo Nucleic Acid ) extraction, COI Region from mtDNA (aprox. 610 bp) was amplified using COI – 625R and COI – 625F primers. A total number of 25 Amplified PCR)Polymerase Chain Reaction) products was sequenced. Then sequenced samples was Investigated using BioEdit 5.0.9, MEGA5 (Molecular Evolutionary Genetics Analysis Version 5.0.) and Arlequin 3.5 softwares. The average nucleotide Diversity was 0.0286, 0.0785 and 0.0072 respectively. This result showed a low genetic diversity within sub-populations in B.sharpeyi specie. The UPMGA(Unweighted Pair Group Method with Arithmetic Mean) dandrogram which was constructed based on the distance matrix between groups with MEGA5 program. ‏‏‏most part of genetic variance ( prox. %85.5) in B. sharpeyi was within populations. The obtained results at the present study showed a gene flow between Jrahi and Karoon river was happened. This results suggested that for maintaining of genetic diversity of B. sharpeyi sub-populations at appropriate levels , the exchange and transfer of barbus stocking between Jrahi and other rivers should be avoided and should be used new reproductive stocks.