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

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.

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Development and Assessment of a Real-Time PCR Assay for Rapid and Sensitive Detection of a Novel Thermotolerant Bacterium, Lactobacillus thermotolerans, in Chicken Feces

Applied and Environmental Microbiology ◽

10.1128/aem.71.8.4214-4219.2005 ◽

2005 ◽

Vol 71 (8) ◽

pp. 4214-4219 ◽

Cited By ~ 15

Author(s):

Abu Sadeque Md Selim ◽

Piyanuch Boonkumklao ◽

Teruo Sone ◽

Apinya Assavanig ◽

Masaru Wada ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Real Time ◽

Real Time Pcr ◽

Pure Culture ◽

16S Rrna Genes ◽

Rrna Genes ◽

Rrna Gene ◽

Pcr Assay ◽

Chicken Feces

ABSTRACT A new real-time PCR assay was successfully developed using a TaqMan fluorescence probe for specific detection and enumeration of a novel bacterium, Lactobacillus thermotolerans, in chicken feces. The specific primers and probe were designed based on the L. thermotolerans 16S rRNA gene sequences, and these sequences were compared to those of all available 16S rRNA genes in the GenBank database. The assay, targeting 16S rRNA gene, was evaluated using DNA from a pure culture of L. thermotolerans, DNA from the closely related bacteria Lactobacillus mucosae DSM 13345T and Lactobacillus fermentum JCM 1173T, and DNA from other lactic acid bacteria in quantitative experiments. Serial dilutions of L. thermotolerans DNA were used as external standards for calibration. The minimum detection limit of this technique was 1.84 × 103 cells/ml of an L. thermotolerans pure culture. The assay was then applied to chicken feces in two different trials. In the first trial, the cell population was 104 cells/g feces on day 4 and 105 cells/g feces on days 11 to 18. However, cell populations of 106 to 107 cells/g feces were detected in the second trial. The total bacterial count, measured by 4′,6-diamidino-2-phenylindole (DAPI) staining, was approximately 1011 cells/g feces. These results suggest that in general, L. thermotolerans is a normal member of the chicken gut microbiota, although it is present at relatively low levels in the feces.

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Real-Time PCR Screening for 16S rRNA Mutations Associated with Resistance to Tetracycline in Helicobacter pylori

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.49.8.3166-3170.2005 ◽

2005 ◽

Vol 49 (8) ◽

pp. 3166-3170 ◽

Cited By ~ 27

Author(s):

Erik Glocker ◽

Marco Berning ◽

Monique M. Gerrits ◽

Johannes G. Kusters ◽

Manfred Kist

Keyword(s):

Helicobacter Pylori ◽

16S Rrna ◽

Real Time ◽

Real Time Pcr ◽

Tetracycline Resistance ◽

16S Rrna Genes ◽

Rrna Genes ◽

Rrna Gene ◽

Pcr Screening ◽

H Pylori

ABSTRACT The effectiveness of recommended first-line therapies for Helicobacter pylori infections is decreasing due to the occurrence of resistance to metronidazole and/or clarithromycin. Quadruple therapies, which include tetracycline and a bismuth salt, are useful alternative regimens. However, resistance to tetracycline, mainly caused by mutations in the 16S rRNA genes (rrnA and rrnB) affecting nucleotides 926 to 928, are already emerging and can impair the efficacies of such second-line regimens. Here, we describe a novel real-time PCR for the detection of 16S rRNA gene mutations associated with tetracycline resistance. Our PCR method was able to distinguish between wild-type strains and resistant strains exhibiting single-, double, or triple-base-pair mutations. The method was applicable both to DNA extracted from pure cultures and to DNA extracted from fresh or frozen H. pylori-infected gastric biopsy samples. We therefore conclude that this real-time PCR is an excellent method for determination of H. pylori tetracycline resistance even when live bacteria are no longer available.

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Development of Real-Time PCR Assays for Detection of the Streptococcus milleri Group from Cystic Fibrosis Clinical Specimens by Targeting the cpn60 and 16S rRNA Genes

Journal of Clinical Microbiology ◽

10.1128/jcm.02082-09 ◽

2010 ◽

Vol 48 (4) ◽

pp. 1150-1160 ◽

Cited By ~ 16

Author(s):

A. B. Olson ◽

C. D. Sibley ◽

L. Schmidt ◽

M. A. Wilcox ◽

M. G. Surette ◽

...

Keyword(s):

Cystic Fibrosis ◽

16S Rrna ◽

Real Time ◽

Real Time Pcr ◽

16S Rrna Genes ◽

Rrna Genes ◽

Clinical Specimens ◽

Streptococcus Milleri ◽

Streptococcus Milleri Group ◽

Pcr Assays

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Development and Application of a Real-Time PCR Approach for Quantification of Uncultured Bacteria in the Central Baltic Sea

Applied and Environmental Microbiology ◽

10.1128/aem.70.8.4971-4979.2004 ◽

2004 ◽

Vol 70 (8) ◽

pp. 4971-4979 ◽

Cited By ~ 45

Author(s):

Matthias Labrenz ◽

Ingrid Brettar ◽

Richard Christen ◽

Sebastien Flavier ◽

Julia Bötel ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Baltic Sea ◽

Real Time ◽

Relative Abundance ◽

Real Time Pcr ◽

16S Rrna Genes ◽

Rrna Genes ◽

Rrna Gene ◽

Uncultured Bacteria

ABSTRACT We have developed a highly sensitive approach to assess the abundance of uncultured bacteria in water samples from the central Baltic Sea by using a noncultured member of the “Epsilonproteobacteria” related to Thiomicrospira denitrificans as an example. Environmental seawater samples and samples enriched for the target taxon provided a unique opportunity to test the approach over a broad range of abundances. The approach is based on a combination of taxon- and domain-specific real-time PCR measurements determining the relative T. denitrificans-like 16S rRNA gene and 16S rRNA abundances, as well as the determination of total cell counts and environmental RNA content. It allowed quantification of T. denitrificans-like 16S rRNA molecules or 16S rRNA genes as well as calculation of the number of ribosomes per T. denitrificans-like cell. Every real-time measurement and its specific primer system were calibrated using environmental nucleic acids obtained from the original habitat for external standardization. These standards, as well as the respective samples to be measured, were prepared from the same DNA or RNA extract. Enrichment samples could be analyzed directly, whereas environmental templates had to be preamplified with general bacterial primers before quantification. Preamplification increased the sensitivity of the assay by more than 4 orders of magnitude. Quantification of enrichments with or without a preamplification step yielded comparable results. T. denitrificans-like 16S rRNA molecules ranged from 7.1 × 103 to 4.4 × 109 copies ml−1 or 0.002 to 49.7% relative abundance. T. denitrificans-like 16S rRNA genes ranged from 9.0 × 101 to 2.2 ×106 copies ml−1 or 0.01 to 49.7% relative abundance. Detection limits of this real-time-PCR approach were 20 16S rRNA molecules or 0.2 16S rRNA gene ml−1. The number of ribosomes per T. denitrificans-like cell was estimated to range from 20 to 200 in seawater and reached up to 2,000 in the enrichments. The results indicate that our real-time PCR approach can be used to determine cellular and relative abundances of uncultured marine bacterial taxa and to provide information about their levels of activity in their natural environment.

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Monitoring of Microbial Community Inhabiting a Low-Grade Copper Sulphide Ore by Quantitative Real-Time PCR Analysis of 16S rRNA Genes

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.20-21.539 ◽

2007 ◽

Vol 20-21 ◽

pp. 539-542 ◽

Cited By ~ 13

Author(s):

Francisco Remonsellez ◽

F. Galleguillos ◽

Sonestie Janse van Rensburg ◽

G.F. Rautenbach ◽

Pedro A. Galleguillos ◽

...

Keyword(s):

16S Rrna ◽

Real Time ◽

Real Time Pcr ◽

16S Rrna Genes ◽

Rrna Genes ◽

Low Grade ◽

Copper Sulphide ◽

Quantitative Real Time Pcr ◽

Sulphide Ore ◽

Heap Bioleaching

Microbial heap bioleaching is being used as an industrial process to recover copper from low grade ores. It is known that a consortium of different microorganisms participates in this process. Therefore identification and quantification of communities inhabiting heap bioleaching operations is a key step for understanding the dynamics and role of these microorganisms in the process. A quantitative real-time PCR approach was used to investigate the microbial dynamics in this process. To study the microbial population inhabiting a low-grade copper sulphide ore bioleaching industrial heap process at Escondida Mine in Chile, 16S rRNA genetic libraries were constructed using bacterial and archaeal universal primers. Phylogenetic analyses of sequences retrieved from genetic libraries showed that the community is mainly composed by microoganisms related to Acidithiobacillus ferrooxidans (2 strains), Acidithiobacillus thiooxidans, Leptospirillum ferrooxidans, Leptospirillum ferriphilum and the archaea Ferroplasma. Specific primers for real-time PCR determination were designed and tested to amplify each of the sequences obtained by cloning. Standard curves for real time PCR were performed using plasmid DNA from selected clones. This methodology is actually being used to monitor relevant microorganisms inhabiting this low-grade copper sulphide ore bioleaching industrial heap.

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Microflora profiling of root canal utilizing real-time PCR and cloning-sequence analyses based on 16S rRNA genes—differences between before and after root canal treatments

Interface Oral Health Science 2007 ◽

10.1007/978-4-431-76690-2_46 ◽

2008 ◽

pp. 265-266

Author(s):

Yasuhiro Ito ◽

Takuichi Sato ◽

Gen Mayanagi ◽

Keiko Yamaki ◽

Hidetoshi Shimauchi ◽

...

Keyword(s):

16S Rrna ◽

Real Time ◽

Real Time Pcr ◽

Root Canal ◽

16S Rrna Genes ◽

Rrna Genes ◽

Sequence Analyses ◽

Before And After

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Quantification of ammonia-oxidizing bacteria populations in full-scale sewage activated sludge systems and assessment of system variables affecting their performance

Water Science & Technology ◽

10.2166/wst.2006.376 ◽

2006 ◽

Vol 54 (1) ◽

pp. 91-99 ◽

Cited By ~ 17

Author(s):

T. Limpiyakorn ◽

F. Kurisu ◽

O. Yagi

Keyword(s):

Activated Sludge ◽

Real Time ◽

Real Time Pcr ◽

Full Scale ◽

16S Rrna Genes ◽

Rrna Genes ◽

Ammonia Oxidizing Bacteria ◽

Primer Sets ◽

Activated Sludge Systems ◽

Pcr Primer

This study carried out quantification of ammonia-oxidizing bacteria (AOB) populations in 12 full-scale sewage activated sludge systems that were different in ammonia removals and treatment processes during three different seasons. Experiment was divided into 3 parts: 1) analysis of AOB communities by PCR-DGGE-cloning-sequencing of 16S rRNA genes; 2) development of four real-time PCR primer sets for quantification of the particular AOB of interest; and 3) quantification of AOB populations by using the newly developed real-time PCR primer sets. The results suggested that all the primer sets gave good reproducibility and specificity for PCR amplification with the detection limits of 102 copies/PCR reaction. Although the 12 systems were different in several aspects, one of the identified sequence types of Nitrosomonas oligotropha cluster was the dominant AOB in every system and every season studied. However, the other sequence type of this cluster was not significantly involved in ammonia removals in the systems. The occurrence of N. communis cluster in the systems seemed to depend on the remaining oxygen concentrations in the sludge floc and thus the activity of aerobic heterotrophs in the aeration tanks. N. europaea–Nitrosococcus. mobilis solely existed in one A2O system of which the influent contained twice the chloride concentrations than those of other systems.

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PCR and culture for diagnosis of Acanthamoeba keratitis

British Journal of Ophthalmology ◽

10.1136/bjophthalmol-2020-316730 ◽

2020 ◽

pp. bjophthalmol-2020-316730

Author(s):

Helene Yera ◽

Vichita Ok ◽

Fiona Lee Koy Kuet ◽

Naima Dahane ◽

Frédéric Ariey ◽

...

Keyword(s):

Real Time ◽

Internal Control ◽

Real Time Pcr ◽

Latent Class ◽

Rrna Gene ◽

Target Sequence ◽

Small Subunit Rrna ◽

Pcr Assay ◽

Corneal Scraping ◽

Pcr Assays

Background/AimsAcanthamoeba keratitis (AK) is a rare but sight-threatening infection. Molecular diagnosis of corneal scraping has improved the diagnosis of AK. Different molecular targets and conditions have been used in diagnosis thus far. In this study, we prospectively compared the performance of five PCR assays on corneal samples for the diagnosis of AK.Methods1217 corneal scraping samples were obtained from patients, for whom an AK was suspected. Sample processing involved both molecular diagnostics and culture. Acanthamoeba PCR assays detected different regions of the Acanthamoeba nuclear small-subunit rRNA gene: three final point PCR assays using Nelson, ACARNA and JDP1–JDP2 pairs of primers, and two real-time PCR assays using Acant primer-probe. Human DNA and internal control were co-amplified in the real-time PCR assay to ensure scraping quality and the absence of inhibitors. In the absence of a gold standard, the performance of each test was evaluated using latent class analysis. Genotypes of Acanthamoeba isolates were also characterised.ResultsEstimated prevalence of AK was 1.32%. The sensitivity of Acanthamoeba diagnostic PCRs (73.3% to 86.7%) did not differ significantly from that of culture (66.7%), or according to the target sequence or the technology. Sensitivity could be increased to 93.8% or 100% by combining two or three assays, respectively. PCR specificity (99.3% to 100%) differed between the assays. T4 was the predominant Acanthamoeba genotype (84.6%).ConclusionsCulture and a single PCR assay could lead to misdiagnosing AK. A combination of different PCR assays and improved sample quality could increase diagnosis sensitivity.

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Ultrasensitive detection of Bacillus anthracis by real time PCR targeting a polymorphism in multi-copy 16S rRNA genes and their transcripts

10.1101/2021.09.20.21263746 ◽

2021 ◽

Author(s):

Peter Braun ◽

Martin Duy-Thanh Nguyen ◽

Mathias C Walter ◽

Gregor Grass

Keyword(s):

16S Rrna ◽

Bacillus Anthracis ◽

Real Time ◽

Real Time Pcr ◽

Single Copy ◽

Rrna Genes ◽

Rt Pcr ◽

Pcr Assay ◽

Species Specific ◽

Pcr Targeting

The anthrax pathogen Bacillus anthracis poses a significant threat to human health. Identification of B. anthracis is challenging because of the bacterium’s close genetic relationship to other Bacillus cereus group species. Thus, molecular detection is founded on species-specific PCR targeting single-copy genes. Here, we validated a previously recognized multi-copy target, a species-specific SNP present in 2-5 copies in every B. anthracis genome analyzed. For this, a hydrolysis probe-based real time PCR assay was developed and rigorously tested. The assay was specific as only B. anthracis DNA yielded positive results, was linear over 9 log10 units and was sensitive with a limit of detection (LoD) of 2.9 copies/reaction. Though not exhibiting a lower LoD than established single copy PCR targets (dhp61 or PL3), the higher copy number of the B. anthracis–specific 16S rRNA gene allele afforded ≤2 unit lower threshold (Ct) values. To push the detection limit even further, the assay was adapted for reverse transcription PCR on 16S rRNA transcripts. This RT-PCR assay was also linear over 9 log10 units and was sensitive with a LoD of 6.3 copies/reaction. In a dilution-series of experiments, the 16S RT-PCR assay achieved a thousand-fold higher sensitivity than the DNA-targeting assays. For molecular diagnostics, we recommend a real time RT-PCR assay variant in which both DNA and RNA serve as templates (thus, no requirement for DNase treatment). This will at least provide results equaling the DNA-based implementation if no RNA is present but will be superior even at the lowest residual rRNA concentrations.

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