Quantification of bacterial RubisCO genes in soils by cbbL targeted real-time PCR

Soils harbor a high diversity of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) large subunit coding genes (cbbL). Real-time PCR was used to quantify this gene in differently managed agricultural soils and soil microhabitats. We developed primers and a TaqMan probe that target the "red-like" RubisCO gene cbbL. Primers and probe were developed based on cbbL sequences of selected bacterial pure cultures and of environmental clones. The amount of cbbL copies in the investigated soils were detected in the range of 6.8x10(6) to 3.4x10(7) "red-like" cbbL copies/g soil. The cbbL genes could be located entirely in the clay and silt fraction, while the coarse sand fractions revealed no detectable level of bacterial RubisCO genes. These results indicate that bacteria with RubisCO coding genes are numerous and widespread in soils, however the functional implication of this gene in soils is not yet clear.     

Quantification of ammonia-oxidizing bacteria in arable soil by real-time PCR

Real-time PCR was used to quantify populations of ammonia-oxidizing bacteria representing the beta subdivision of the class Proteobacteria in samples of arable soil, both nitrogen fertilized and unfertilized, from Mellby, Sweden. Primers and probes targeting a 16S ribosomal DNA region of the ammonia-oxidizing bacteria were designed and used. In the fertilized soil there were approximately 6.2 x 10(7) ammonia-oxidizing bacteria per g of soil, three times more than the number of bacteria in the unfertilized soil. The lytic efficiency of bead beating in these soils was investigated by using populations of free or loosely attached bacteria, bacteria tightly bound to particles, and bacteria in nonfractionated samples. The shapes of the curves generated in these tests showed that the concentration of template DNA released at various times remained constant after 10 to 100 s of bead beating.     

Quantification of a novel group of nitrate-reducing bacteria in the environment by real-time PCR

Nitrate reduction is performed by phylogenetically diverse bacteria. Analysis of narG (alpha subunit of the membrane bound nitrate reductase) trees constructed using environmental sequences revealed a new cluster that is not related to narG gene from known nitrate-reducing bacteria. In this study, primers targeting this as yet uncultivated nitrate-reducing group were designed and used to develop a real-time SYBR(R) Green PCR assay. The assay was tested with clones from distinct nitrate-reducing groups and applied to various environmental samples. narG copy number was high ranging between 5.08x10(8) and 1.12x10(11) copies per gram of dry weight of environmental sample. Environmental real-time PCR products were cloned and sequenced. Data was used to generate a phylogenetic tree showing that all environmental products belonged to the target group. Moreover, 16S rDNA copy number was quantified in the different environments by real-time PCR using universal primers for Eubacteria. 16S rDNA copy number was similar or slightly higher than that of narG, between 7.12x10(9) and 1.14x10(11) copies per gram of dry weight of environmental sample. Therefore, the yet uncultivated nitrate-reducing group targeted in this study seems to be numerically important in the environment, as revealed by narG high absolute and relative densities across various environments. Further analysis of the density of the nitrate-reducing community as a whole by real-time PCR may provide insights into the correlation between microbial density, diversity and activity.     

The distribution of denitrifying bacteria in soils monitored by DNA-probing

Abstract DNA probes of dissimilatory NO₂⁻- and N₂O-reductases have been used to screen for the distribution of denitrifying bacteria in soils. In control experiments with known organisms, denitrifiers gave positive DNA-DNA hybridization signals in the dot-blot experiments whereas non-denitrifiers did not hybridize in almost all cases. Bacteria from soil of our institute garden were isolated, grown up and assayed for denitrification activity and for DNA-hybridizations with both gene probes. A correlation of about 75% was obtained between activities and signals. The remainder of the isolates gave a weak signal in the dot blots and only half of them expressed denitrification activities in the cultures. The confidence of about 75% was good enough to assay for the distribution of denitrifying bacteria in five different soil-types of the Düsseldorf area. In all cases, the percentage of bacteria which gave no hybridization signal was high in the plant-free, bulk soil, whereas bacteria with strong or very strong signals favourably associated with the roots of plants isolated from the different soils. The result that denitrifying bacteria predominantly occur at the surface of roots was obtained when the samples were taken both in June and November. The probes for either NO₂⁻- or N₂O-reductase gave essentially the same results. For comparison, a gene probe for nitrogenase was included in this investigation. N₂-fixing bacteria showed a tendency to associate with the roots of plants in some but not in all soils. The overall number of bacteria was much higher at the roots of the plants than in the bulk soil in any of the samples assayed in the present investigation.     

Quantification of live and dead probiotic bacteria in lyophilised product by real-time PCR and by flow cytometry

The basic requirement for probiotic bacteria to be able to exert expected positive effects is to be alive; therefore, appropriate quantification methods are crucial. Due to disadvantages of conventional microbiological methods, the bacterial quantification based on the nucleic acid detection is increasingly used. The objective of this study was to evaluate the possibility to use propidium monoazide (PMA) in combination with real-time polymerase chain reaction (PCR) method or LIVE/DEAD BacLight viability kit in combination with flow cytometry (FCM) for determination of probiotic bacteria in a lyophilised product containing Lactobacillus acidophilus LA-5 and Bifidobacterium animalis ssp. lactis BB-12. In addition, the viability of probiotic bacteria in lyophilised product during 3 months storage was investigated. In the product, the results of real-time PCR quantification of PMA-treated cells did not differ significantly from those of non-treated cells, which indicate that most of the bacterial cells retained the membrane integrity although they have lost the culturability. The results obtained by FCM analysis were comparable with those by PMA real-time PCR. In conclusion, the PMA real-time PCR and FCM determination of the viability of probiotic bacteria could complement the plate count method which considers only the culturable part of the population.     

Quantification of tree fine roots by real-time PCR

Plant and Soil - Tree roots contribute large quantities of biomass in forests and are important drivers of different ecosystem processes. However, estimation of root biomass remains a challenge. We...     

Quantification of Gram-negative sulphate-reducing bacteria in rice field soil by 16S rRNA gene-targeted real-time PCR

For the quantification of Gram-negative sulphate reducers in rice fields, 11 real-time PCR assays were established targeting 16S rRNA genes combined with SybrGreen detection. Three of these assays were specific for the "main" groups, i.e. the Desulfovibrionaceae, the Desulfobacteraceae and Desulfobulbus sp., whereas eight assays were developed for subgroups within the first two main groups. The detection limits of the assays were between 2 x 10(5) and 4 x 10(3) targets g(-1) (wet weight) or less than 0.02% of the eubacterial 16S rDNA targets in bulk soil, rhizosphere soil and rice root DNA extracts. Analysis of soil spiked with defined cell numbers of sulphate-reducing bacteria showed good correlation of measured target numbers to amended cells. In rice field bulk and rhizosphere soil, the Desulfobacteraceae were the predominant main group with target numbers of 6.4 x 10(7) (+/-1.0 x 10(7)) and 7.5 x 10(7) (+/-1.7 x 10(7)), respectively. Within this group the Desulforhabdus/Synthrophobacter assemblage and Desulfobacterium sp. were predominant. At the rice roots, the three main groups were abundant in similar numbers (approx. 1.0 x 10(8)) indicating that the relative abundance of the Desulfovibrionaceae and also of Desulfobulbus sp. was increased, relatively to the Desulfobacteraceae. Within the Desulfovibrionaceae the subgroup was predominant that was detected by assay DSV-II. This assay detects many from rice field soil isolated Desulfovibrio-strains and molecular retrieved sequences. Therefore these organisms that were already detected in the rice field environment by isolation and by molecular techniques are indeed best adapted to the conditions provided by the rice roots.     

Quantitative detection of selenate-reducing bacteria by real-time PCR targeting the selenate reductase gene

We designed a primer set to target selenate reductase (SerA) for detecting selenate reducing bacteria (SeRB). Our serA gene-based PCR primer set has high specificity in that it and positively amplified some SeRB, but not denitrifying bacteria (DB). Phylogenetic analysis of serA clone sequences of environmental samples from selenate-reducing membrane biofilm reactor (MBfR) biofilms showed that these sequences were closely grouped and had high similarity to selenate reductase gene sequences from SeRB Thauera selenatis and DB Dechloromonas; however, they were distant to other genes from dimethylsulfoxide (DMSO) enzyme family. Constructing a standard curve targeting the serA gene, we found that the good linearity for the qPCR assay when applied it to quantify SeRB in MBfR biofilms, and the gene copies of SeRB correlated well to the selenate removal percentages. Our results demonstrated the feasibility of using the serA gene-based PCR primer set to detect and quantify SeRB in environmental samples.     

Quantification of tetracycline resistance genes in feedlot lagoons by real-time PCR

A new real-time PCR method is presented that detects and quantifies three tetracycline resistance (Tcr) genes [tet(O), tet(W), and tet(Q)] in mixed microbial communities resident in feedlot lagoon wastewater. Tcr gene real-time TaqMan primer-probe sets were developed and optimized to quantify the Tcr genes present in seven different cattle feedlot lagoons, to validate the method, and to assess whether resistance gene concentrations correlate with free-tetracycline levels in lagoon waters. The method proved to be sensitive across a wide range of gene concentrations and provided consistent and reproducible results from complex lagoon water samples. The log10 of the sum of the three resistance gene concentrations was correlated with free-tetracycline levels (r2 = 0.50, P < 0.001; n = 18), with the geometric means of individual resistance concentrations ranging from 4- to 8.3-fold greater in lagoon samples with above-median tetracycline levels (>1.95 microg/liter by enzyme-linked immunosorbent assay techniques) than in below-median lagoon samples. Of the three Tcr genes tested, tet(W) and tet(Q) were more commonly found in lagoon water samples. Successful development of this real-time PCR assay will permit other studies quantifying Tcr gene numbers in environmental and other samples.     

PCR detection of genes encoding nitrite reductase in denitrifying bacteria

Using consensus regions in gene sequences encoding the two forms of nitrite reductase (Nir), a key enzyme in the denitrification pathway, we designed two sets of PCR primers to amplify cd1- and Cu-nir. The primers were evaluated by screening defined denitrifying strains, denitrifying isolates from wastewater treatment plants, and extracts from activated sludge. Sequence relationships of nir genes were also established. The cd1 primers were designed to amplify a 778 to 799-bp region of cd1-nir in the six published sequences. Likewise, the Cu primers amplified a 473-bp region in seven of the eight published Cu-nir sequences. Together, the two sets of PCR primers amplified nir genes in nine species within four genera, as well as in four of the seven sludge isolates. The primers did not amplify genes of nondenitrifying strains. The Cu primers amplified the expected fragment in all 13 sludge samples, but cd1-nir fragments were only obtained in five samples. PCR products of the expected sizes were verified as nir genes after hybridization to DNA probes, except in one case. The sequenced nir fragments were related to other nir sequences, demonstrating that the primers amplified the correct gene. The selected primer sites for Cu-nir were conserved, while broad-range primers targeting conserved regions of cd1-nir seem to be difficult to find. We also report on the existence of Cu-nir in Paracoccus denitrificans Pd1222.     

Quantification of Microthrix parvicella in activated sludge bacterial communities by real-time PCR

AIMS: This study was to develop a simple and reliable method for quantifying Microthrix parvicella 16S rRNA gene copies and its application to activated sludge samples collected from wastewater treatment plants (WWTP) with and without foaming problems. METHODS AND RESULTS: The relative frequency of M. parvicella was determined by combining real-time PCR assays for quantification of total bacterial 16S rRNA gene copies and M. parvicella 16S rRNA gene copies. The developed method was applied to analyse 32 activated sludge samples obtained from German WWTP. The level of M. parvicella 16S rRNA gene copies in the 18 nonfoaming samples was below 3% of the total number of 16S rRNA gene copies and in the range of 0-18% for the 14 foaming samples. CONCLUSIONS: The described method allows reliable monitoring of the amount of M. parvicella in activated sludge samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The described method may become an important component of a warning system for forthcoming bulking and foaming episodes.     

Quantification of enterococci and human adenoviruses in environmental samples by real-time PCR

Pathogenic bacteria and enteric viruses can be introduced into the environment via human waste discharge. Methods for rapid detection and quantification of human viruses and fecal indicator bacteria in water are urgently needed to prevent human exposure to pathogens through drinking and recreational waters. Here we describe the development of two real-time PCR methods to detect and quantify human adenoviruses and enterococci in environmental waters. For real-time quantification of enterococci, a set of primers and a probe targeting the 23S rRNA gene were used. The standard curve generated using Enterococcus faecalis genomic DNA was linear over a 7-log-dilution series. Serial dilutions of E. faecalis suspensions resulted in a lower limit of detection (LLD) of 5 CFU/reaction. To develop real-time PCR for adenoviruses, degenerate primers and a Taqman probe targeting a 163-bp region of the adenovirus hexon gene were designed to specifically amplify 14 different serotypes of human adenoviruses, including enteric adenovirus serotype 40 and 41. The standard curve generated was linear over a 5-log-dilution series, and the LLD was 100 PFU/reaction using serial dilutions of purified adenoviral particles of serotype 40. Both methods were optimized to be applicable to environmental samples. The real-time PCR methods showed a greater sensitivity in detection of adenoviruses in sewage samples than the viral plaque assay and in detection of enterococci in coastal waters than the bacterial culture method. However, enterococcus real-time PCR overestimated the number of bacteria in chlorinated sewage in comparison with the bacterial culture method. Overall, the ability via real-time PCR to detect enterococci and adenoviruses rapidly and quantitatively in the various environmental samples represents a considerable advancement and a great potential for environmental applications.     

Quantification of human fecal bifidobacterium species by use of quantitative real-time PCR analysis targeting the groEL gene

Quantitative real-time PCR assays targeting the groEL gene for the specific enumeration of 12 human fecal Bifidobacterium species were developed. The housekeeping gene groEL (HSP60 in eukaryotes) was used as a discriminative marker for the differentiation of Bifidobacterium adolescentis, B. angulatum, B. animalis, B. bifidum, B. breve, B. catenulatum, B. dentium, B. gallicum, B. longum, B. pseudocatenulatum, B. pseudolongum, and B. thermophilum. The bifidobacterial chromosome contains a single copy of the groEL gene, allowing the determination of the cell number by quantification of the groEL copy number. Real-time PCR assays were validated by comparing fecal samples spiked with known numbers of a given Bifidobacterium species. Independent of the Bifidobacterium species tested, the proportion of groEL copies recovered from fecal samples spiked with 5 to 9 log(10) cells/g feces was approximately 50%. The quantification limit was 5 to 6 log(10) groEL copies/g feces. The interassay variability was less than 10%, and variability between different DNA extractions was less than 23%. The method developed was applied to fecal samples from healthy adults and full-term breast-fed infants. Bifidobacterial diversity in both adults and infants was low, with mostly ≤3 Bifidobacterium species and B. longum frequently detected. The predominant species in infant and adult fecal samples were B. breve and B. adolescentis, respectively. It was possible to distinguish B. catenulatum and B. pseudocatenulatum. We conclude that the groEL gene is a suitable molecular marker for the specific and accurate quantification of human fecal Bifidobacterium species by real-time PCR.     

Quantification of the carbazole 1,9a-dioxygenase gene by real-time competitive PCR combined with co-extraction of internal standards

The fluorogenic probe assay, competitive polymerase chain reaction (PCR) and co-extraction with internal standard cells were combined to develop a rapid, sensitive, and accurate quantification method for the copy number of a target carbazole 1,9a-dioxygenase gene (carAa) and the cell number of Pseudomonas sp. strain CA10. The internal standard DNA was modified by replacement of a 20-bp long region with one for binding a specific probe in fluorogenic PCR (TaqMan). The resultant DNA fragment was similar to the corresponding region of the intact carAa gene in terms of G+C content. When used as a competitor in the PCR reaction, the internal standard DNA was distinguishable from the target carAa gene by two specific fluorogenic probes with different fluorescence labels, and was automatically detected in a single tube using the ABI7700 sequence detection system. To minimize variations in the efficiency of cell lysis and DNA extraction between the samples, the co-extraction method was combined. A mini-transposon was used to introduce competitor DNA into the genome of other pseudomonads, and the resultant construct was used as the standard cell. After the addition of a fixed amount of the internal standard cells to soil samples, total DNA was extracted (co-extraction). Using this method, the copy number of the carAa gene and the cell number of strain CA10 in soil samples could be quantified rapidly.     

Enumeration and detection of acetic acid bacteria by real-time PCR and nested PCR

Acetic acid bacteria play a negative role in wine making because they increase the volatile acidity of wines. They can survive in the various phases of alcoholic fermentation and it is very important to control their presence and ulterior development. The main objective of the present work is to test fast, sensitive and reliable techniques such as real-time PCR (rt-PCR) and nested PCR for enumerating and detecting the presence of this bacterial group without plating. Primers were designed on the basis of the available 16S rRNA gene sequences and tested successfully with reference acetic acid bacteria strains. The usefulness of rt-PCR was demonstrated by comparing the results with traditional techniques (colony and microscope counting). The results were similar with all the techniques. Optimized rt-PCR enabled numbers between 10(7) and 10(1) cells mL(-1) to be enumerated, while nested PCR detected less than 10 cells mL(-1). Although this latter technique cannot be used for enumeration, it has several advantages in routine laboratory analysis.     

Quantification bias caused by plasmid DNA conformation in quantitative real-time PCR assay

Quantitative real-time PCR (qPCR) is the gold standard for the quantification of specific nucleic acid sequences. However, a serious concern has been revealed in a recent report: supercoiled plasmid standards cause significant over-estimation in qPCR quantification. In this study, we investigated the effect of plasmid DNA conformation on the quantification of DNA and the efficiency of qPCR. Our results suggest that plasmid DNA conformation has significant impact on the accuracy of absolute quantification by qPCR. DNA standard curves shifted significantly among plasmid standards with different DNA conformations. Moreover, the choice of DNA measurement method and plasmid DNA conformation may also contribute to the measurement error of DNA standard curves. Due to the multiple effects of plasmid DNA conformation on the accuracy of qPCR, efforts should be made to assure the highest consistency of plasmid standards for qPCR. Thus, we suggest that the conformation, preparation, quantification, purification, handling, and storage of standard plasmid DNA should be described and defined in the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) to assure the reproducibility and accuracy of qPCR absolute quantification.