scholarly journals Detection of Streptococcus mutans Genomic DNA in Human DNA Samples Extracted from Saliva and Blood

2011 ◽  
Vol 2011 ◽  
pp. 1-6 ◽  
Author(s):  
Alexandre R. Vieira ◽  
Kathleen B. Deeley ◽  
Nicholas F. Callahan ◽  
Jacqueline B. Noel ◽  
Ida Anjomshoaa ◽  
...  
Keyword(s):  
Real Time ◽  
Streptococcus Mutans ◽  
Real Time Pcr ◽  
Genomic Dna ◽  
Bacterial Colonization ◽  
Human Saliva ◽  
Caries Experience ◽  
Multifactorial Disease ◽  
Pcr Assay ◽  
Human Dna

Caries is a multifactorial disease, and studies aiming to unravel the factors modulating its etiology must consider all known predisposing factors. One major factor is bacterial colonization, and Streptococcus mutans is the main microorganism associated with the initiation of the disease. In our studies, we have access to DNA samples extracted from human saliva and blood. In this report, we tested a real-time PCR assay developed to detect copies of genomic DNA from Streptococcus mutans in 1,424 DNA samples from humans. Our results suggest that we can determine the presence of genomic DNA copies of Streptococcus mutans in both DNA samples from caries-free and caries-affected individuals. However, we were not able to detect the presence of genomic DNA copies of Streptococcus mutans in any DNA samples extracted from peripheral blood, which suggests the assay may not be sensitive enough for this goal. Values of the threshold cycle of the real-time PCR reaction correlate with higher levels of caries experience in children, but this correlation could not be detected for adults.

Multiplex Real-Time PCR Detection of Fumonisin-Producing and Trichothecene-Producing Groups of Fusarium Species

2004 ◽  
Vol 67 (3) ◽  
pp. 536-543 ◽  
Author(s):  
B. H. BLUHM ◽  
M. A. COUSIN ◽  
C. P. WOLOSHUK
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Genomic Dna ◽  
Fusarium Species ◽  
Fusarium Spp ◽  
Pcr Assay ◽  
Traditional Methods ◽  
Microbiological Methods ◽  
Pcr Products ◽  
Its Pcr

Some species of Fusarium can produce mycotoxins during food processing procedures that facilitate fungal growth, such as the malting of barley. The objectives of this study were to develop a 5′ fluorogenic (Taqman) real-time PCR assay for group-specific detection of trichothecene- and fumonisin-producing Fusarium spp. and to identify Fusarium graminearum and Fusarium verticillioides in field-collected barley and corn samples. Primers and probes were designed from genes involved in mycotoxin biosynthesis (TRI6 and FUM1), and for a genus-specific internal positive control, primers and a probe were designed from Fusarium rDNA sequences. Real-time PCR conditions were optimized for amplification of the three products in a single reaction format. The specificity of the assay was confirmed by testing 9 Fusarium spp. and 33 non- Fusarium fungal species. With serial dilutions of purified genomic DNA from F. verticillioides, F. graminearum, or both as the template, the detection limit of the assay was 5 pg of genomic DNA per reaction. The three products were detectable over four orders of magnitude of template concentration (5 pg to 5 ng of genomic DNA per reaction); at 50 ng template per reaction, only the TRI6 and FUM1 PCR products were detected. Barley and corn samples were evaluated for the presence of Fusarium spp. with traditional microbiological methods and with the real-time PCR assay. The 20 barley samples and 1 corn sample that contained F. graminearum by traditional methods of analysis tested positive for the TRI6 and internal transcribedspacer (ITS) PCR products. The five corn samples that tested positive for F. verticillioides by traditional methods also were positive for the FUM1 and ITS PCR products. These results indicate that the described multiplex real-time PCR assay provides sensitive and accurate differential detection of fumonisin- and trichothecene-producing groups of Fusarium spp. in complex matrices.

scholarly journals Quantification of 2,4-Diacetylphloroglucinol-Producing Pseudomonas fluorescens Strains in the Plant Rhizosphere by Real-Time PCR

2007 ◽  
Vol 73 (17) ◽  
pp. 5531-5538 ◽  
Author(s):  
Olga V. Mavrodi ◽  
Dmitri V. Mavrodi ◽  
Linda S. Thomashow ◽  
David M. Weller
Keyword(s):  
Pseudomonas Fluorescens ◽  
Real Time ◽  
Real Time Pcr ◽  
Genomic Dna ◽  
Plant Root ◽  
Population Densities ◽  
Pcr Assay ◽  
Wheat Roots ◽  
Specific Pcr ◽  
Pure Cultures

ABSTRACT A real-time PCR SYBR green assay was developed to quantify populations of 2,4-diacetylphloroglucinol (2,4-DAPG)-producing (phlD +) strains of Pseudomonas fluorescens in soil and the rhizosphere. Primers were designed and PCR conditions were optimized to specifically amplify the phlD gene from four different genotypes of phlD + P. fluorescens. Using purified genomic DNA and genomic DNA extracted from washes of wheat roots spiked with bacteria, standard curves relating the threshold cycles (CT s) and copies of the phlD gene were generated for P. fluorescens strains belonging to genotypes A (Pf-5), B (Q2-87), D (Q8r1-96 and FTAD1R34), and I (FTAD1R36). The detection limits of the optimized real-time PCR assay were 60 to 600 fg (8 to 80 CFU) for genomic DNA isolated from pure cultures of P. fluorescens and 600 fg to 6.0 pg (80 to 800 CFU, corresponding to log 4 to 5 phlD + strain CFU/rhizosphere) for bacterial DNA extracted from plant root washes. The real-time PCR assay was utilized to quantify phlD + pseudomonads in the wheat rhizosphere. Regression analysis of population densities detected by real-time PCR and by a previously described phlD-specific PCR-based dilution endpoint assay indicated a significant linear relationship (P = 0.0016, r 2 = 0.2). Validation of real-time PCR assays with environmental samples was performed with two different soils and demonstrated the detection of more than one genotype in Quincy take-all decline soil. The greatest advantage of the developed real-time PCR is culture independence, which allows determination of population densities and the genotype composition of 2,4-DAPG producers directly from the plant rhizospheres and soil.

scholarly journals Multiplex Real-Time PCR Assay for Simultaneous Identification of Neisseria gonorrhoeae and Its Ciprofloxacin Susceptibility Status

2017 ◽  
Vol 55 (11) ◽  
pp. 3201-3209 ◽  
Author(s):  
Sumudu R. Perera ◽  
Nurul H. Khan ◽  
Irene Martin ◽  
Ali Taheri ◽  
Rajinder P. Parti ◽  
...  
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Real Time ◽  
Sensitivity And Specificity ◽  
Real Time Pcr ◽  
Genomic Dna ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Content Type ◽  
Simultaneous Identification ◽  
Susceptibility Status

ABSTRACTA real-time PCR (RT-PCR) assay was designed for the simultaneous identification ofNeisseria gonorrhoeaeand its ciprofloxacin susceptibility status. A SYBR green-based multiplex RT-PCR format was used; it comprised two different forward primers and a common reverse primer to detect single nucleotide polymorphisms (SNPs) ingyrAofN. gonorrhoeae. The primer pairs were evaluated for their sensitivity and specificity using genomic DNA from 254N. gonorrhoeaeisolates (82 were ciprofloxacin susceptible and 172 were ciprofloxacin resistant) and 23 non-N. gonorrhoeaespecies isolates. The performance of the primers was validated using genomic DNA from 100 differentN. gonorrhoeaeisolates (46 were ciprofloxacin susceptible and 54 were ciprofloxacin resistant) and 52 non-N. gonorrhoeaeisolates. The latter panel was revalidated by testing 99 (46 isolates were ciprofloxacin susceptible and 53 isolates were ciprofloxacin resistant) of theN. gonorrhoeaeisolates and 23 non-N. gonorrhoeaeisolates. These primers detectedN. gonorrhoeaeand its ciprofloxacin susceptibility status with over 99% sensitivity and specificity for all panels tested. This assay has the potential to be an inexpensive and rapid test for the simultaneous identification ofN. gonorrhoeaeand its ciprofloxacin susceptibility status.

scholarly journals Improving molecular detection of Candida DNA in whole blood: comparison of seven fungal DNA extraction protocols using real-time PCR

2008 ◽  
Vol 57 (3) ◽  
pp. 296-303 ◽  
Author(s):  
L. Metwally ◽  
D. J. Fairley ◽  
P. V. Coyle ◽  
R. J. Hay ◽  
S. Hedderwick ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Dna Extraction ◽  
Real Time Pcr ◽  
Whole Blood ◽  
Genomic Dna ◽  
Molecular Techniques ◽  
Diagnostic Methods ◽  
Yeast Cells ◽  
Pcr Assay

The limitations of classical diagnostic methods for invasive Candida infections have led to the development of molecular techniques such as real-time PCR to improve diagnosis. However, the detection of low titres of Candida DNA in blood from patients with candidaemia requires the use of extraction methods that efficiently lyse yeast cells and recover small amounts of DNA suitable for amplification. In this study, a Candida-specific real-time PCR assay was used to detect Candida albicans DNA in inoculated whole blood specimens extracted using seven different extraction protocols. The yield and quality of total nucleic acids were estimated using UV absorbance, and specific recovery of C. albicans genomic DNA was estimated quantitatively in comparison with a reference (Qiagen kit/lyticase) method currently in use in our laboratory. The extraction protocols were also compared with respect to sensitivity, cost and time required for completion. The TaqMan PCR assay used to amplify the DNA extracts achieved high levels of specificity, sensitivity and reproducibility. Of the seven extraction protocols evaluated, only the MasterPure yeast DNA extraction reagent kit gave significantly higher total nucleic acid yields than the reference method, although nucleic acid purity was highest using either the reference or YeaStar genomic DNA kit methods. More importantly, the YeaStar method enabled C. albicans DNA to be detected with highest sensitivity over the entire range of copy numbers evaluated, and appears to be an optimal method for extracting Candida DNA from whole blood.

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