scholarly journals Evaluation of Real-Time 16S rDNA PCR and Pyrosequencing for Routine Identification of Bacteria in Joint Fluid and Tissue Specimens

2011 ◽  
Vol 01 (01) ◽  
pp. 1-6 ◽  
Author(s):  
Naomi J. Gadsby ◽  
Alev Onen ◽  
Sally-Anne Phillips ◽  
Luke Tysall ◽  
Steffen J. Breusch ◽  
...  
Keyword(s):  
Real Time ◽  
16S Rdna ◽  
Joint Fluid ◽  
16S Rdna Pcr ◽  
Routine Identification ◽  
Identification Of Bacteria ◽  
Tissue Specimens

Rapid screening by real-time 16S rDNA PCR for bacterial contamination of blood products

2008 ◽  
Vol 46 (7) ◽  
Author(s):  
Hendrik W. Reesink ◽  
Tamimount Mohammadi ◽  
Rubyn N.I. Pietersz ◽  
Paul H.M. Savelkoul
Keyword(s):  
Real Time ◽  
16S Rdna ◽  
Bacterial Contamination ◽  
Rapid Screening ◽  
Blood Products ◽  
16S Rdna Pcr

DETECTING MYCOBACTERIUM TUBERCULOSIS RAPIDLY AND MYCOBACTERIUM TUBERCULOSIS STRAINS WITHOUT IS6110 SEQUENCE BY PCR ASSAY

2012 ◽  
pp. 15-19
Author(s):  
Thi Chau Anh Nguyen ◽  
Hoang Bach Nguyen ◽  
Hai Duong Huynh ◽  
Nu Xuan Thanh Le ◽  
Xuan Cuong Le ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
16S Rdna ◽  
False Negative ◽  
Clinical Samples ◽  
Tuberculosis Complex ◽  
Pcr Assay ◽  
Complex Objects ◽  
Method Performance ◽  
Gene Coding ◽  
16S Rdna Pcr

Background: The Nested IS6110 PCR is used for detecting tuberculosis, however IS6110 sequence is not present in the genome of all strains of M.tuberculosis, the result may be false negative. The gene coding 16S ribosome always contains a short sequence specific to M. tuberculosis complex. Objects: Performance of the 16S Real-time PCR to detect M. tuberculosis and combining to the nested IS6110 PCR to determine the rate of Mtb strains without IS6110 from clinical samples. Materials and method: Performance of 16S rDNA PCR by commercial kit of Viet A Inc. for all 480 samples, the samples which were positive with the 16S rDNA PCR were retested in IS6110 PCR assay by in-house kit. Results: The Realtime 16S rDNA PCR detected 258 cases (53.8%) of tuberculosis. There were 3 (1.2 %) M. tuberculosis strains which do not harbor IS6110 sequence in genome. Conclusion: The IS6110 nested PCR can be applied more widely than the 16S rDNA realtime PCR. In case of using IS6110 PCR assay, results may show a low proportion of false negative. Combining 16S rDNA PCR with the IS6110 based PCR allowed detection of deletion of IS6110 sequence in M. tuberculosis isolates.

Isolation of Enterobacter sakazakii from Stable Flies, Stomoxys calcitrans L. (Diptera: Muscidae)†

2006 ◽  
Vol 69 (3) ◽  
pp. 671-673 ◽  
Author(s):  
F. MRAMBA ◽  
A. BROCE ◽  
L. ZUREK
Keyword(s):  
16S Rdna ◽  
Foodborne Pathogen ◽  
Animal Manure ◽  
Stomoxys Calcitrans ◽  
Fecal Coliforms ◽  
Stable Fly ◽  
Enterobacter Sakazakii ◽  
Stable Flies ◽  
16S Rdna Pcr ◽  
Glucose Agar

Enterobacter sakazakii is an opportunistic foodborne pathogen that causes meningitis, enterocolitis, and sepsis, primarily in immunocompromised infants. Previously, it was suggested that stable flies, Stomoxys calcitrans, were a vector or reservoir of this pathogen. In our study, by means of a culturing approach combined with 16S rDNA PCR–restriction fragment length polymorphism genotyping and sequencing, we screened 928 individual stable flies collected in Kansas and Florida. Two stable flies (0.2%) were positive for E. sakazakii. In addition, 411 (44%) stable flies carried bacteria-forming red colonies (presumably enterics) on a violet red bile glucose agar (mean count = 6.4 × 104 CFU per fly), and 120 (13%) stable flies carried fecal coliforms (mean count = 8.7 × 103 CFU per fly). Sequencing of 16S rDNA showed that enterics from violet red bile glucose agar were represented by several genera, including Escherichia, Shigella, Providencia, Enterobacter, Pantoea, Proteus, Serratia, and Morganella. Our study shows that stable flies carry bacteria typically present in animal manure (a developmental site of stable fly larvae), which indicates that the natural reservoir of E. sakazakii is the digestive tract or manure of domestic animals. The low prevalence of E. sakazakii associated with stable flies suggests that stable flies do not play a major role as a reservoir or vector of this pathogen.

scholarly journals Identification, Detection, and Enumeration of Human Bifidobacterium Species by PCR Targeting the Transaldolase Gene

2002 ◽  
Vol 68 (5) ◽  
pp. 2420-2427 ◽  
Author(s):  
Teresa Requena ◽  
Jeremy Burton ◽  
Takahiro Matsuki ◽  
Karen Munro ◽  
Mary Alice Simon ◽  
...  
Keyword(s):  
Real Time ◽  
16S Rdna ◽  
Quantitative Pcr ◽  
Gene Sequence ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Bifidobacterium Longum ◽  
Bifidobacterium Adolescentis ◽  
Fecal Samples ◽  
Real Time Quantitative Pcr ◽  
Pcr Targeting

ABSTRACT Methods that enabled the identification, detection, and enumeration of Bifidobacterium species by PCR targeting the transaldolase gene were tested. Bifidobacterial species isolated from the feces of human adults and babies were identified by PCR amplification of a 301-bp transaldolase gene sequence and comparison of the relative migrations of the DNA fragments in denaturing gradient gel electrophoresis (DGGE). Two subtypes of Bifidobacterium longum, five subtypes of Bifidobacterium adolescentis, and two subtypes of Bifidobacterium pseudocatenulatum could be differentiated using PCR-DGGE. Bifidobacterium angulatum and B. catenulatum type cultures could not be differentiated from each other. Bifidobacterial species were also detected directly in fecal samples by this combination of PCR and DGGE. The number of species detected was less than that detected by PCR using species-specific primers targeting 16S ribosomal DNA (rDNA). Real-time quantitative PCR targeting a 110-bp transaldolase gene sequence was used to enumerate bifidobacteria in fecal samples. Real-time quantitative PCR measurements of bifidobacteria in fecal samples from adults correlated well with results obtained by culture when either a 16S rDNA sequence or the transaldolase gene sequence was targeted. In the case of samples from infants, 16S rDNA-targeted PCR was superior to PCR targeting the transaldolase gene for the quantification of bifidobacterial populations.

scholarly journals Comparison of different conditions for DNA extraction in sputum - a pilot study

2019 ◽  
Vol 14 ◽  
Author(s):  
Martina Oriano ◽  
Leonardo Terranova ◽  
Antonio Teri ◽  
Samantha Sottotetti ◽  
Luca Ruggiero ◽  
...  
Keyword(s):  
Real Time ◽  
16S Rdna ◽  
Dna Extraction ◽  
Real Time Pcr ◽  
Alpha Diversity ◽  
Diversity Indices ◽  
Extraction Yield ◽  
Chronic Respiratory Diseases ◽  
Dna Yield ◽  
Selection Of

Background: The analysis of microbiome in respiratory samples is a topic of great interest in chronic respiratory diseases. The method used to prepare sputum samples for microbiome analysis is very heterogeneous. The selection of the most suitable methodology for DNA extraction is fundamental to have the most representative data. The objective of this study was to compare different conditions for DNA extraction from sputum in adult patients with bronchiectasis. Methods: Five sputum samples from bronchiectasis patients were collected at the Policlinico Hospital in Milan, Italy. Eighteen conditions for DNA extraction were compared, including two enzyme-based (Roche and Zymo) and one beads-based (Mobio) technique. These techniques were tested with/without Dithiothreitol (DTT) and with/without lysostaphin (0.18 and 0.36 mg/mL) step. DNA was quantified, tested using Real-time PCR for 16S rDNA and S. aureus and, then, microbiome was evaluated. Results: Although 16S rDNA was similarly detected across all the different techniques, Roche kit gave the highest DNA yield. The lowest Ct values for Real-time PCR for S. aureus was identified when lysostaphin was added. Considering genera from microbiome, alpha diversity indices did not show any significant differences between techniques, while relative abundances were more similar in presence of DTT. Conclusions: None of the conditions emerged to be superior to the others even if enzyme-based kits seem to be needed in order to have a higher extraction yield.

scholarly journals Towards accurate exclusion of neonatal bacterial meningitis: a feasibility study of a novel 16S rDNA PCR assay

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Arthur Abelian ◽  
Thomas Mund ◽  
Martin D. Curran ◽  
Stuart A. Savill ◽  
Nipa Mitra ◽  
...  
Keyword(s):  
Bacterial Meningitis ◽  
16S Rdna ◽  
Feasibility Study ◽  
Pcr Assay ◽  
16S Rdna Pcr
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