Quantitative identification of fecal water pollution sources by TaqMan real-time PCR assays using Bacteroidales 16S rRNA genetic markers

2010 ◽  
Vol 88 (6) ◽  
pp. 1373-1383 ◽  
Author(s):  
Dae-Young Lee ◽  
Susan C. Weir ◽  
Hung Lee ◽  
Jack T. Trevors
Keyword(s):  
Water Pollution ◽  
16S Rrna ◽  
Real Time ◽  
Genetic Markers ◽  
Real Time Pcr ◽  
Pollution Sources ◽  
Fecal Water ◽  
Quantitative Identification ◽  
Pcr Assays

scholarly journals Diagnostic Accuracy of Real-Time PCR Assays Targeting 16S rRNA and lipl32 Genes for Human Leptospirosis in Thailand: A Case-Control Study

PLoS ONE ◽  
2011 ◽  
Vol 6 (1) ◽  
pp. e16236 ◽  
Author(s):  
Janjira Thaipadunpanit ◽  
Wirongrong Chierakul ◽  
Vanaporn Wuthiekanun ◽  
Direk Limmathurotsakul ◽  
Premjit Amornchai ◽  
...  
Keyword(s):  
16S Rrna ◽  
Diagnostic Accuracy ◽  
Real Time ◽  
Real Time Pcr ◽  
Case Control Study ◽  
Case Control ◽  
Human Leptospirosis ◽  
Pcr Assays ◽  
Control Study

Real-Time PCR Detection and Identification of Prohibited Mammalian and Avian Material in Animal Feeds

2009 ◽  
Vol 72 (5) ◽  
pp. 1055-1062 ◽  
Author(s):  
SAIRA CAWTHRAW ◽  
GINNY C. SAUNDERS ◽  
TREVOR C. MARTIN ◽  
JASON SAWYER ◽  
OTTO WINDL ◽  
...  
Keyword(s):  
16S Rrna ◽  
Real Time ◽  
Real Time Pcr ◽  
Animal Feed ◽  
Meat And Bone Meal ◽  
Bone Meal ◽  
Detection And Identification ◽  
Pcr Assays ◽  
Species Specific ◽  
Feed Samples

A method for the detection and identification of “prohibited” mammalian or avian material in animal feed was developed and assessed through the analysis of DNA. A generic real-time PCR assay was designed to detect the presence of mammalian and avian mitochondrial DNA 16S rRNA genes in animal feed samples. Samples positive with this screening method were further investigated using identification assays to detect the 16S rRNA gene from bovine, ovine, porcine, and avian species and to determine whether the DNA originated from species whose material is prohibited from inclusion in farmed animal feed. An internal positive control was coamplified in the 16S real-time PCR assays to monitor PCR amplification efficiency and avoid potential false-negative results. Using vegetable-based feed standards spiked with meat and bone meal generated with a commercial rendering process, 0.1% meat and bone meal could be detected using the general and species-specific 16S assays. The species-specific assays had 100% specificity for the homologous target species. The 16S real-time PCR assays were evaluated alongside existing tests based on protein evaluation or microscopic examination for a wide range of commercial animal feed samples. In total, 111 (0.76%) of 14,678 samples examined contained prohibited material based on the results from at least one of these tests. However, most positive results did not represent noncompliance because they were associated with samples of pet food, which can legitimately contain material prohibited for use in food for farmed animals. The species-specific 16S assays confirmed the presence of prohibited material in 75% of the 111 samples, whereas the existing protein and microscope tests confirmed the presence of this material in 25 and 54% of the samples, respectively.

scholarly journals Correction: Diagnostic Accuracy of Real-Time PCR Assays Targeting 16S rRNA and lipl32 Genes for Human Leptospirosis in Thailand: A Case-Control Study

PLoS ONE ◽  
2011 ◽  
Vol 6 (2) ◽  
Author(s):  
Janjira Thaipadungpanit ◽  
Wirongrong Chierakul ◽  
Vanaporn Wuthiekanun ◽  
Direk Limmathurotsakul ◽  
Premjit Amornchai ◽  
...  
Keyword(s):  
16S Rrna ◽  
Diagnostic Accuracy ◽  
Real Time ◽  
Real Time Pcr ◽  
Case Control Study ◽  
Case Control ◽  
Human Leptospirosis ◽  
Pcr Assays ◽  
Control Study

scholarly journals Power Analysis for Real-Time PCR Quantification of Genes in Activated Sludge and Analysis of the Variability Introduced by DNA Extraction

2003 ◽  
Vol 69 (11) ◽  
pp. 6597-6604 ◽  
Author(s):  
Hebe M. Dionisi ◽  
Gerda Harms ◽  
Alice C. Layton ◽  
Igrid R. Gregory ◽  
Jack Parker ◽  
...  
Keyword(s):  
16S Rrna ◽  
Activated Sludge ◽  
Real Time ◽  
Real Time Pcr ◽  
Power Analysis ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Pcr Assay ◽  
Pcr Assays

ABSTRACT The aims of this study were to determine the power of discrimination of the real-time PCR assay for monitoring fluctuations in microbial populations within activated sludge and to identify sample processing points where methodological changes are needed to minimize the variability in target quantification. DNA was extracted using a commercially available kit from mixed liquor samples taken from the aeration tank of four bench-scale activated-sludge reactors operating at 2-, 5-, 10-, and 20-day solid retention times, with mixed-liquor volatile suspended solid (MLVSS) values ranging from 260 to 2,610 mg/liter. Real-time PCR assays for bacterial and Nitrospira 16S rRNA genes were chosen because they represent, respectively, a highly abundant and a less-abundant bacterial target subject to clustering within the activated sludge matrix. The mean coefficient of variation in DNA yields (measured as microgram of DNA per milligram of MLVSS) in triplicate extractions of 12 different samples was 12.2%. Based on power analyses, the variability associated with DNA extraction had a small impact on the overall variability of the real-time PCR assay. Instead, a larger variability was associated with the PCR assay. The less-abundant target (Nitrospira 16S rRNA gene) had more variability than the highly abundant target (bacterial 16S rRNA gene), and samples from the lower-biomass reactors had more variability than samples from the higher-biomass reactors. Power analysis of real-time PCR assays indicated that three to five samples were necessary to detect a twofold increase in bacterial 16S rRNA genes, whereas three to five samples were required to detect a fivefold increase in Nitrospira 16S rRNA genes.

scholarly journals Development of Bacteroides 16S rRNA Gene TaqMan-Based Real-Time PCR Assays for Estimation of Total, Human, and Bovine Fecal Pollution in Water

2006 ◽  
Vol 72 (6) ◽  
pp. 4214-4224 ◽  
Author(s):  
Alice Layton ◽  
Larry McKay ◽  
Dan Williams ◽  
Victoria Garrett ◽  
Randall Gentry ◽  
...  
Keyword(s):  
16S Rrna ◽  
Real Time ◽  
Dna Extraction ◽  
Real Time Pcr ◽  
False Positive ◽  
Fecal Contamination ◽  
True Positive ◽  
Fecal Samples ◽  
Positive Identification ◽  
Pcr Assays

ABSTRACT Bacteroides species are promising indicators for differentiating livestock and human fecal contamination in water because of their high concentration in feces and potential host specificity. In this study, a real-time PCR assay was designed to target Bacteroides species (AllBac) present in human, cattle, and equine feces. Direct PCR amplification (without DNA extraction) using the AllBac assay was tested on feces diluted in water. Fecal concentrations and threshold cycle were linearly correlated, indicating that the AllBac assay can be used to estimate the total amount of fecal contamination in water. Real-time PCR assays were also designed for bovine-associated (BoBac) and human-associated (HuBac) Bacteroides 16S rRNA genes. Assay specificities were tested using human, bovine, swine, canine, and equine fecal samples. The BoBac assay was specific for bovine fecal samples (100% true-positive identification; 0% false-positive identification). The HuBac assay had a 100% true-positive identification, but it also had a 32% false-positive rate with potential for cross-amplification with swine feces. The assays were tested using creek water samples from three different watersheds. Creek water did not inhibit PCR, and results from the AllBac assay were correlated with those from Escherichia coli concentrations (r 2 = 0.85). The percentage of feces attributable to bovine and human sources was determined for each sample by comparing the values obtained from the BoBac and HuBac assays with that from the AllBac assay. These results suggest that real-time PCR assays without DNA extraction can be used to quantify fecal concentrations and provide preliminary fecal source identification in watersheds.

DEVELOPMENT OF MULTIPLEX REAL-TIME PCR ASSAY FOR SIX PATHOGENS: RAPID TOOL FOR DIAGNOSIS OF BACTERIAL MENINGITIS

2019 ◽  
pp. 60-66
Author(s):  
Viet Quynh Tram Ngo ◽  
Thi Ti Na Nguyen ◽  
Hoang Bach Nguyen ◽  
Thi Tuyet Ngoc Tran ◽  
Thi Nam Lien Nguyen ◽  
...  
Keyword(s):  
Bacterial Meningitis ◽  
Real Time ◽  
Real Time Pcr ◽  
Diagnostic Methods ◽  
Type B ◽  
E Coli ◽  
Pcr Assays ◽  
Set Up ◽  
Etiological Agents ◽  
Suis Serotype

Introduction: Bacterial meningitis is an acute central nervous infection with high mortality or permanent neurological sequelae if remained undiagnosed. However, traditional diagnostic methods for bacterial meningitis pose challenge in prompt and precise identification of causative agents. Aims: The present study will therefore aim to set up in-house PCR assays for diagnosis of six pathogens causing the disease including H. influenzae type b, S. pneumoniae, N. meningitidis, S. suis serotype 2, E. coli and S. aureus. Methods: inhouse PCR assays for detecting six above-mentioned bacteria were optimized after specific pairs of primers and probes collected from the reliable literature resources and then were performed for cerebrospinal fluid (CSF) samples from patients with suspected meningitis in Hue Hospitals. Results: The set of four PCR assays was developed including a multiplex real-time PCR for S. suis serotype 2, H. influenzae type b and N. meningitides; three monoplex real-time PCRs for E. coli, S. aureus and S. pneumoniae. Application of the in-house PCRs for 116 CSF samples, the results indicated that 48 (39.7%) cases were positive with S. suis serotype 2; one case was positive with H. influenzae type b; 4 cases were positive with E. coli; pneumococcal meningitis were 19 (16.4%) cases, meningitis with S. aureus and N. meningitidis were not observed in any CSF samples in this study. Conclusion: our in-house real-time PCR assays are rapid, sensitive and specific tools for routine diagnosis to detect six mentioned above meningitis etiological agents. Key words: Bacterial meningitis, etiological agents, multiplex real-time PCR

scholarly journals Differing Effects of Standard and Harsh Nucleic Acid Extraction Procedures on Diagnostic Helminth Real-Time PCRs Applied to Human Stool Samples

Pathogens ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 188
Author(s):  
Tanja Hoffmann ◽  
Andreas Hahn ◽  
Jaco J. Verweij ◽  
Gérard Leboulle ◽  
Olfert Landt ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Real Time Pcr ◽  
Acid Extraction ◽  
Nucleic Acid Extraction ◽  
Pcr Assay ◽  
Bead Beating ◽  
Stool Samples ◽  
Pcr Assays ◽  
Human Stool

This study aimed to assess standard and harsher nucleic acid extraction schemes for diagnostic helminth real-time PCR approaches from stool samples. A standard procedure for nucleic acid extraction from stool and a procedure including bead-beating as well as proteinase K digestion were compared with group-, genus-, and species-specific real-time PCR assays targeting helminths and nonhelminth pathogens in human stool samples. From 25 different in-house and commercial helminth real-time PCR assays applied to 77 stool samples comprising 67 historic samples and 10 external quality assessment scheme samples positively tested for helminths, higher numbers of positive test results were observed after bead-beating-based nucleic acid extraction for 5/25 (20%) real-time PCR assays irrespective of specificity issues. Lower cycle threshold values were observed for one real-time PCR assay after the standard extraction scheme, and for four assays after the bead-beating-based scheme. Agreement between real-time PCR results after both nucleic acid extraction strategies according to Cohen’s kappa ranged from poor to almost perfect for the different assays. Varying agreement was observed in eight nonhelminth real-time PCR assays applied to 67 historic stool samples. The study indicates highly variable effects of harsh nucleic acid extraction approaches depending on the real-time PCR assay used.

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