Primer and probe sets for group-specific quantification of the genera Nitrosomonas and Nitrosospira using real-time PCR

Use of quantitative real-time PCR (QPCR) with TaqMan probes is increasingly popular in various environmental works to detect and quantify a specific microorganism or a group of target microorganism. Although many aspects of conducting a QPCR assay have become very easy to perform, a proper design of oligonucleotide sequences comprising primers and a probe is still considered as one of the most important aspects of a QPCR application. This work was conducted to design group specific primer and probe sets for the detection of ammonia oxidizing bacteria (AOB) using a real-time PCR with a TaqMan system. The genera Nitrosomonas and Nitrosospira were grouped into five clusters based on similarity of their 16S rRNA gene sequences. Five group-specific AOB primer and probe sets were designed. These sets separately detect four subgroups of Nitrosomonas (Nitrosomonas europaea-, Nitrosococcus mobilis-, Nitrosomonas nitrosa-, and Nitrosomonas cryotolerans-clusters) along with the genus Nitrosospira. Target-group specificity of each primer and probe set was initially investigated by analyzing potential false results in silico, followed by a series of experimental tests for QPCR efficiency and detection limit. In general, each primer and probe set was very specific to the target group and sensitive to detect target DNA as low as two 16S rRNA gene copies per reaction mixture. QPCR efficiency, higher than 93.5%, could be achieved for all primer and probe sets. The primer and probe sets designed in this study can be used to detect and quantify the beta-proteobacterial AOB in biological nitrification processes and various environments.     

Kinetics of baculovirus replication and release using real-time quantitative polymerase chain reaction.

The study of viral-based processes is hampered by (a) their complex, transient nature, (b) the instability of products, and (c) the lack of accurate diagnostic assays. Here, we describe the use of real-time quantitative polymerase chain reaction to characterize baculoviral infection. Baculovirus DNA content doubles every 1.7 h from 6 h post-infection until replication is halted at the onset of budding. No dynamic equilibrium exists between replication and release, and the kinetics are independent of the cell density at the time of infection. No more than 16% of the intracellular virus copies bud from the cell.     

The use of a quantitative real-time polymerase chain reaction assay for identification and enumeration of Lactobacillus buchneri in silage

Aims:  To detect and quantify Lactobacillus buchneri in plant samples with the aid of polymerase chain reaction (PCR) methods.
Methods and Results:  DNA from silage samples spiked with different amounts of L. buchneri cells was isolated using a lysozyme/sodium dodecyl sulfate lysis and phenol/chloroform extraction method. The DNA served as a template for PCR amplification with primers specific for the bacterium. The primers were developed by comparison of 16S rDNA sequences from different lactic acid bacteria (LAB) and testing for specificity with 11 different strains of LAB. As few as 100 L. buchneri colony-forming units per gram of silage could be detected. Additionally, the technique was successfully applied to quantify the population of L. buchneri in two cultivars of corn with or without inoculation.
Conclusions:  The PCR assay provided a specific and rapid tool for identifying and enumerating L. buchneri in silage samples.
Significance and Impact of the Study:  The use of microbial inoculants for silage production is a safe and environment friendly practice, but the full potential of such additives can only be achieved with a better understanding of the fate and activity of the microbes involved. The current study describes a methodology to detect and enumerate L. buchneri , a micro-organism used as an inoculant.     

Triplex real‐time polymerase chain reaction assay for detection and quantification of norovirus (GI and GII) and sapovirus

To improve detection of norovirus (NoVGI, NoVGII) and sapovirus (SaV), a simultaneous quantitative RT‐PCR method was established. This triplex real‐time PCR method was evaluated using a combination of optimized specific primers and probes. The performance of the developed PCR assay was equivalent to that of monoplex real‐time PCR across a broad dynamic range of 10²–10⁷ copies/assay using plasmid DNA standards. The limit of detection was 10² copies/assay. The quantitative value was comparable with that of monoplex real‐time PCR of stool samples. Our triplex real‐time PCR is useful for detection of NoV and SaV infections.     

Detection of methane and quantification of methanogenic archaea in faeces from young broiler chickens using real-time PCR

AIMS: To detect the presence of methanogens in the faeces of broiler chicks during the first 2 weeks of age. METHODS AND RESULTS: Chicken faecal samples from 120 broiler chicks were incubated for methane gas formation and methanogenic archaea were analysed using real-time PCR. The copy number of the order Methanobacteriales 16S rDNA gene in chicken faeces when the broilers were 3-12 days of age, litter and house flies collected in the bird house ranged from 4.19 to 5.51 log(10) g(-1) wet weight. The number of positive methane culture tubes increased from 25% to 100% as the birds aged. CONCLUSIONS: Methanogens were successfully detected in faecal samples from 3- to 12-day-old broilers, as well as litter and house flies using real-time PCR. The copy number of methanogenic 16S rDNA gene in these samples was also similar to the number observed in litter and house flies. SIGNIFICANCE AND IMPACT OF THE STUDY: The same methanogens consistently appeared in chicken faeces a few days after birth. Detection of the methanogenic bacteria in litter and house flies implicated them as potential environmental sources for methanogen colonization in broiler chicks.     

Measurement of Pseudomonas aeruginosa multidrug efflux pumps by quantitative real-time polymerase chain reaction

Multidrug efflux pumps contribute to multiple antibiotic resistance in Pseudomonas aeruginosa. Pump expression usually has been quantified by Western blotting. Quantitative real-time polymerase chain reaction has been developed to measure mRNA expression for genes of interest. Whether this method correlates with pump protein quantities is unclear. We devised a real-time PCR for mRNA expression of MexAB-OprM and MexXY-OprM multidrug efflux pumps. In laboratory strains differing in MexB and MexY expression and in several clinical isolates, protein and mRNA expression correlated well. Quantitative real-time PCR should be a useful alternative in quantitating expression of multidrug efflux pumps by P. aeruginosa isolates in clinical laboratories.     

Rapid identification of Aerococcus viridans using the polymerase chain reaction and an oligonucleotide probe

A polymerase chain reaction/oligonucleotide probe method was developed for the specific identification of the Gram-positive bacterium Aerococcus viridans. Primers for the enzymatic amplification reaction were designed from specific sequences within the 16S rRNA. The method was also highly sensitive and 10 cfu of A. viridans could be detected in 5 h although the reliability of detection was poor in mixed cultures with Escherichia coli.     

Detection of Salmonella enterica in food using two-step enrichment and real-time polymerase chain reaction

Aims: A new real‐time polymerase chain reaction‐based method was developed for the detection of Salmonella enterica in food. Methods and Results: The method consisted of a novel two‐step enrichment involving overnight incubation in buffered peptone water and a 5‐h subculture in Rappaport–Vassiliadis medium, lysis of bacterial cells and a Salmonella‐specific 5′‐nuclease real‐time PCR with an exogenous internal amplification control. Because a two‐step enrichment was used, the detection limit for dead S. enterica cells in artificially contaminated ice cream and salami samples was high at 10⁷ CFU (25 g)^?1, eliminating potential false‐positive results. When the method was evaluated with a range of 100 naturally contaminated food samples, three positive samples were detected by both the real‐time PCR‐based method and by the standard microbiological method, according to EN ISO 6579. When the real‐time PCR‐based method was evaluated alongside the standard microbiological method according to EN ISO 6579 with 36 food samples artificially contaminated at a level of 10⁰ CFU (25 g)^?1, identical results were obtained from both methods. Conclusions: The real‐time PCR‐based method involving a two‐step enrichment produced equivalent results to EN ISO 6579 on the day after sample receipt. Significance and Impact of the Study: The developed method is suitable for rapid detection of S. enterica in food.     

Gene expression normalization in a dual-compartment system: a real-time quantitative polymerase chain reaction protocol for symbiotic anthozoans

Traditional real-time quantitative polymerase chain reaction protocols cannot be used accurately with symbiotic organisms unless the relative contribution of each symbiotic compartment to the total nucleic acid pool is known. A modified 'universal reference gene' protocol was created for reef-building corals and sea anemones, anthozoans that harbour endosymbiotic dinoflagellates belonging to the genus Symbiodinium. Gene expression values are first normalized to an RNA spike and then to a symbiont molecular proxy that represents the number of Symbiodinium cells extracted and present in the RNA. The latter is quantified using the number of genome copies of heat shock protein-70 (HSP70) amplified in the real-time quantitative polymerase chain reaction. Gene expression values are then normalized to the total concentration of RNA to account for differences in the amount of live tissue extracted among experimental treatments and replicates. The molecular quantification of symbiont cells and effect of increasing symbiont contributions to the nucleic acid pool on gene expression were tested in vivo using differentially infected sea anemones Aiptasia pulchella. This protocol has broad application to researchers who seek to measure gene expression in mixed organism assemblages.     

Molecular karyotyping and aneuploidy detection in Arabidopsis thaliana using quantitative fluorescent polymerase chain reaction

Certain cellular processes are sensitive to changes in gene dosage. Aneuploidy is deleterious because of an imbalance of gene dosage on a chromosomal scale. Identification, classification and characterization of aneuploidy are therefore important for molecular, population and medical genetics and for a deeper understanding of the mechanisms underlying dosage sensitivity. Notwithstanding recent progress in genomic technologies, limited means are available for detecting and classifying changes in chromosome dose. The development of an inexpensive and scalable karyotyping method would allow rapid detection and characterization of both simple and complex aneuploid types. In addition to the problem of karyotyping, genomic and molecular genetic studies of aneuploids and polyploids are complicated by multiple heterozygous combinations possible at loci present in more than two copies. Quantitative scoring of allele genotypes would enable large-scale population genetic experiments in polyploids, and permit genetic analyses on bulked populations in diploid species. Here, we demonstrate that quantitative fluorescent-polymerase chain reaction (QF-PCR) can be used to simultaneously genotype and karyotype aneuploid and polyploid Arabidopsis thaliana. Comparison of QF-PCR with flow cytometric determination of nuclear DNA content indicated near perfect agreement between the methods, but complete karyotype resolution was only possible using QF-PCR. A complex karyotype, determined by QF-PCR, was validated by comparative genomic hybridization to microarrays. Finally, we screened the progeny of tetraploid individuals and found that more than 25% were aneuploid and that our artificially induced tetraploid strain produced fewer aneuploid individuals than a tetraploid strain isolated from nature.     

Non-invasive genetic identification of small mammal species using real-time polymerase chain reaction

DNA identification of non-invasive samples is a potentially useful tool for monitoring small mammal species. Here we describe a novel method for identifying five small mammal species: wood mouse, bank vole, common shrew, pygmy shrew and water shrew. Species-specific real-time polymerase chain reaction primers were designed to amplify fragments of the mitochondrial cytochrome b gene from hair and scat samples. We also amplified nuclear DNA from scats, demonstrating their potential as a source of DNA for population genetic studies.     

Use of real-time PCR for group-specific quantification of aceticlastic methanogens in anaerobic processes: population dynamics and community structures

The TaqMan quantitative PCR (QPCR) method was used to detect and quantify the 16S rRNA genes of aceticlastic methanogens at different taxonomic levels. Three different sets of primers coupled with a TaqMan probe for QPCR assays to detect the 16S rRNA genes of the order Methanosarcinales, as well as the families Methanosarcinaceae and Methanosaetaceae, were separately used. Using these primer and probe sets, the 16S rRNA genes of aceticlastic methanogens in samples from various anaerobic processes (i.e., nine pure cultures, batch experiment, and three different continuous processes including a full-scale digester), were monitored and quantified by QPCR assays. A batch experiment cultivating a mixture of aceticlastic methanogens, was conducted to monitor their population dynamics. Using this group-specific quantification method, the dynamics of a competition between two aceticlastic populations, as modulated by the acetate concentration, could well be described. The target 16S rRNA genes in environmental samples, collected from three different anaerobic processes treating sludge, cheese whey, and synthetic wastewaters, were additionally quantified. The quantified 16S rRNA gene concentrations for all samples successfully represented the community structures of the target methanogens, which were correlated accurately with the operational parameters of the anaerobic processes. It was also successful to demonstrate probe nesting of aceticlastic methanogens at the levels of order and family.     

Quantification of Escherichia coli by kinetic 5'-nuclease polymerase chain reaction (real-time PCR) oriented to sfmD gene

AIMS: A kinetic 5'-nuclease polymerase chain reaction (real-time PCR) for the quantification of Escherichia coli was developed. METHODS AND RESULTS: Specific primers and a fluorogenic probe oriented to sfmD gene, encoding a putative outer membrane export usher protein, were designed. The PCR system was highly specific and sensitive for E. coli, as determined with 37 non-E. coli strains (exclusivity, 100%) and 24 E. coli strains (inclusivity, 100%). When used in real-time PCR, linear calibration lines were obtained in the range from 10(2) to 10(8) CFU ml(-1) for three E. coli strains. Salmonella Enteritidis (10(6) CFU ml(-1)) or Citrobacter freundii (10(6) CFU ml(1)) had no effect on quantification of E. coli by the method. CONCLUSIONS: The developed real-time PCR is suitable for rapid quantification of E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: In connection to an appropriate sample preparation technique, the method is suitable for food safety and technological hygiene applications.