Serious Overestimation in Quantitative PCR by Circular (Supercoiled) Plasmid Standard: Microalgal pcna as the Model Gene

Quantitative real-time PCR (qPCR) has become a gold standard for the quantification of nucleic acids and microorganism abundances, in which plasmid DNA carrying the target genes are most commonly used as the standard. A recent study showed that supercoiled circular confirmation of DNA appeared to suppress PCR amplification. However, to what extent to which different structural types of DNA (circular versus linear) used as the standard may affect the quantification accuracy has not been evaluated. In this study, we quantitatively compared qPCR accuracies based on circular plasmid (mostly in supercoiled form) and linear DNA standards (linearized plasmid DNA or PCR amplicons), using proliferating cell nuclear gene (pcna), the ubiquitous eukaryotic gene, in five marine microalgae as a model gene. We observed that PCR using circular plasmids as template gave 2.65-4.38 more of the threshold cycle number than did equimolar linear standards. While the documented genome sequence of the diatom Thalassiosira pseudonana shows a single copy of pcna, qPCR using the circular plasmid as standard yielded an estimate of 7.77 copies of pcna per genome whereas that using the linear standard gave 1.02 copies per genome. We conclude that circular plasmid DNA is unsuitable as a standard, and linear DNA should be used instead, in absolute qPCR. The serious overestimation by the circular plasmid standard is likely due to the undetected lower efficiency of its amplification in the early stage of PCR when the supercoiled plasmid is the dominant template.     

Culture-Independent Identification of Nontuberculous Mycobacteria in Cystic Fibrosis Respiratory Samples

Respiratory tract infections with nontuberculous mycobacteria (NTM) are increasing in prevalence and are a significant cause of lung function decline in individuals with cystic fibrosis (CF). NTM have been detected in culture-independent analyses of CF airway microbiota at lower rates than would be expected based on published prevalence data, likely due to poor lysing of the NTM cell wall during DNA extraction. We compared a standard bacterial lysis protocol with a modified method by measuring NTM DNA extraction by qPCR and NTM detection with bacterial 16S rRNA gene sequencing. The modified method improved NTM DNA recovery from spiked CF sputum samples by a mean of 0.53 log₁₀ copies/mL for M. abscessus complex and by a mean of 0.43 log₁₀ copies/mL for M. avium complex as measured by qPCR targeting the atpE gene. The modified method also improved DNA sequence based NTM detection in NTM culture-positive CF sputum and bronchoalveolar lavage samples; however, both qPCR and 16S rRNA gene sequencing remained less sensitive than culture for NTM detection. We highlight the limitations of culture-independent identification of NTM from CF respiratory samples, and illustrate how alterations in the bacterial lysis and DNA extraction process can be employed to improve NTM detection with both qPCR and 16S rRNA gene sequencing.     

Detection and persistence of fecal Bacteroidales as water quality indicators in unchlorinated drinking water

The results of this study support the use of fecal Bacteroidales qPCR as a rapid method to complement traditional, culture-dependent, water quality indicators in systems where drinking water is supplied without chlorination or other forms of disinfection. A SYBR-green based, quantitative PCR assay was developed to determine the concentration of fecal Bacteroidales 16S rRNA gene copies. The persistence of a Bacteroides vulgatus pure culture and fecal Bacteroidales from a wastewater inoculum was determined in unchlorinated drinking water at 10 °C. B. vulgatus 16S rRNA gene copies persisted throughout the experimental period (200 days) in sterile drinking water but decayed faster in natural drinking water, indicating that the natural microbiota accelerated decay. In a simulated fecal contamination of unchlorinated drinking water, the decay of fecal Bacteroidales 16S rRNA gene copies was considerably faster than the pure culture but similar to that of Escherichia coli from the same wastewater inoculum.     

内参基因加标法定量土壤微生物目标基因绝对拷贝数

【目的】通过荧光定量PCR技术对土壤微生物目标基因进行绝对定量,其定量结果的准确性容易受到DNA提取得率以及腐殖酸抑制性的影响。【方法】采用内参基因加标法,利用构建的突变质粒DNA,对供试水稻土壤样品中的微生物16S r RNA目标基因的绝对拷贝数进行荧光定量PCR检测,用来表征该样品中细菌群落总体丰度。在定量前通过双向引物扩增方法验证突变质粒中的内参基因对供试土壤的特异性。【结果】不同水稻土壤样品的DNA提取量在样品间差异较大。通过内参基因加标法对DNA提取量进行校正,显著提高了16S r RNA基因绝对定量的精确度。不同水稻土壤样品间的变异系数为17.8,与未加标处理相比降低了66.7%。在此基础上,进一步通过内参基因加标法对土壤有机质和含水率均呈现典型空间特征差异的6处亚热带湿地土壤样品中的16S r RNA基因进行绝对定量。16S r RNA基因绝对拷贝数与土壤微生物生物量碳具有显著的线性相关性(R2=0.694,P0.001),表明内参校正后的16S r RNA基因绝对拷贝数可以准确反映单位质量土壤中微生物的丰度。【结论】内参基因加标法可以对DNA提取得率以及腐殖酸对PCR扩增的抑制性进行校正,从而提高绝对定量的准确性。基于内参基因加标法的目标基因绝对定量PCR检测,可作为土壤微生物生物量测量,以及微生物功能基因绝对丰度定量的一种核酸检测方法。     

Multiple copies of 16S rRNA gene affect the restriction patterns and DGGE profile revealed by analysis of genome database

The use of 16S rRNA gene has been a “golden” method to determine the diversity of microbial communities in environmental samples, phylogenetic relationships of prokaryotes and taxonomic position of newly isolated organisms. However due to the presence of multiple heterogeneous 16S rRNA gene copies in many strains, the interpretation of microbial ecology via 16S rRNA sequences is complicated. Purpose of present paper is to demonstrate the extent to which the multiple heterogeneous 16S rRNA gene copies affect RFLP patterns and DGGE profiles by using the genome database. In present genome database, there are 782 bacterial strains in total whose genomes have been completely sequenced and annotated. Among the total strains, 639 strains (82%) possess multiple 16S rRNA gene copies, 415 strains (53%) whose multiple copies are heterogeneous in sequences as revealed by alignment, 236 strains (30%) whose multiple copies show different restrict patterns by CSP6I+HinfI, MspI+RsaI or HhaI as analyzed in silico. Polymorphisms of the multiple copies in certain strains were further characterized by G+C% and phy-logentic distances based on the sequences of V3 region, which are linked to DGGE patters. Polymorphisms of a few strains were shown as examples. Using artificial communities, it is demonstrated that the presence of multiple heterogeneous 16S rRNA gene copies potentially leads to over-estimation of the diversity of a community. It is suggested that care must be taken when interpreting 16S rRNA-based RFLP and DGGE data and profiling an environmental community.     

Application of quantitative real-time PCR for enumeration of total bacterial,archaeal, and yeast populations in kimchi

Kimchi is a Korean traditional fermented food made of brined vegetables, with a variety of spices. Various microorganisms are associated with the kimchi fermentation process. This study was undertaken in order to apply quantitative real-time PCR targeting the 16S and 26S rRNA genes for the investigation of dynamics of bacterial, archaeal, and yeast communities during fermentation of various types of kimchi. Although the total bacterial and archaeal rRNA gene copy numbers increased during kimchi fermentation, the number of yeasts was not significantly altered. In 1 ng of bulk DNA, the mean number of rRNA gene copies for all strains of bacteria was 5.45×10⁶ which was 360 and 50 times greater than those for archaea and yeast, respectively. The total gene copy number for each group of microorganisms differed among the different types of kimchi, although the relative ratios among them were similar. The common dominance of bacteria in the whole microbial communities of various types of kimchi suggests that bacteria play a principal role in the kimchi fermentation process.     

DNA topoisomerase VIII: a novel subfamily of type IIB topoisomerases encoded by free or integrated plasmids in Archaea and Bacteria

Type II DNA topoisomerases are divided into two families, IIA and IIB. Types IIA and IIB enzymes share homologous B subunits encompassing the ATP-binding site, but have non-homologous A subunits catalyzing DNA cleavage. Type IIA topoisomerases are ubiquitous in Bacteria and Eukarya, whereas members of the IIB family are mostly present in Archaea and plants. Here, we report the detection of genes encoding type IIB enzymes in which the A and B subunits are fused into a single polypeptide. These proteins are encoded in several bacterial genomes, two bacterial plasmids and one archaeal plasmid. They form a monophyletic group that is very divergent from archaeal and eukaryotic type IIB enzymes (DNA topoisomerase VI). We propose to classify them into a new subfamily, denoted DNA topoisomerase VIII. Bacterial genes encoding a topoisomerase VIII are present within integrated mobile elements, most likely derived from conjugative plasmids. Purified topoisomerase VIII encoded by the plasmid pPPM1a from Paenibacillus polymyxa M1 had ATP-dependent relaxation and decatenation activities. In contrast, the enzyme encoded by mobile elements integrated into the genome of Ammonifex degensii exhibited DNA cleavage activity producing a full-length linear plasmid and that from Microscilla marina exhibited ATP-independent relaxation activity. Topoisomerases VIII, the smallest known type IIB enzymes, could be new promising models for structural and mechanistic studies.     

Detection and Quantification of Geobacter lovleyi Strain SZ: Implications for Bioremediation at Tetrachloroethene- and Uranium-Impacted Sites

Geobacter lovleyi strain SZ reduces hexavalent uranium, U(VI), to U(IV) and is the first member of the metal-reducing Geobacter group capable of using tetrachloroethene (PCE) as a growth-supporting electron acceptor. Direct and nested PCR with specific 16S rRNA gene-targeted primer pairs distinguished strain SZ from other known chlorinated ethene-dechlorinating bacteria and closely related Geobacter isolates, including its closest cultured relative, G. thiogenes. Detection limits for direct and nested PCR were approximately 1 × 10⁶ and 1 × 10⁴ 16S rRNA gene copies per μl of template DNA, respectively. A quantitative real-time PCR (qPCR) approach increased the sensitivity to as few as 30 16S rRNA gene copies per μl of template DNA but was less specific. Melting curve analysis and comparison of the shapes of amplification plots identified false-positive signals and distinguished strain SZ from G. thiogenes when analyzed separately. These indicators were less reliable when target (strain SZ) DNA and nontarget (G. thiogenes) DNA with high sequence similarity were mixed, indicating that the development of qPCR protocols should not only evaluate specificity but also explore the effects of nontarget DNA on the accuracy of quantification. Application of specific tools detected strain SZ-like amplicons in PCE-dechlorinating consortia, including the bioaugmentation consortium KB-1, and two chlorinated ethene-impacted groundwater samples. Strain SZ-like amplicons were also detected in 13 of 22 groundwater samples following biostimulation at the uranium- and chlorinated solvent-contaminated Integrated Field-Scale Subsurface Research Challenge (IFC) site in Oak Ridge, TN. The numbers of strain SZ-like cells increased from below detection to 2.3 × 10⁷ ± 0.1 × 10⁷ per liter groundwater, suggesting that strain SZ-like organisms contribute to contaminant transformation. The G. lovleyi strain SZ-specific tools will be useful for monitoring bioremediation efforts at uranium- and/or chlorinated solvent-impacted sites such as the Oak Ridge IFC site.     

Quantitative PCR Targeting 16S rRNA and Reductive Dehalogenase Genes Simultaneously Monitors Multiple Dehalococcoides Strains

The 16S rRNA gene provides insufficient information to infer the range of chloroorganic electron acceptors used by different Dehalococcoides organisms. To overcome this limitation and provide enhanced diagnostic tools for growth measurements, site assessment, and bioremediation monitoring, a quantitative real-time PCR (qPCR) approach targeting 16S rRNA genes and three Dehalococcoides reductive dehalogenase (RDase) genes with assigned function (i.e., tceA, bvcA, and vcrA) was designed and evaluated. qPCR standard curves generated for the RDase genes by use of genomic DNA from Dehalococcoides pure cultures correlated with standard curves obtained for both Bacteria- and Dehalococcoides-targeted 16S rRNA genes, suggesting that the RDase genes are useful targets for quantitative assessment of Dehalococcoides organisms. RDase gene probe/primer pairs were specific for the Dehalococcoides strains known to carry the diagnostic RDase gene sequences, and the qPCR method allowed the detection of as few as 1 to 20 and quantification of as few as 50 to 100 tceA, bvcA, or vcrA gene targets per PCR volume. The qPCR approach was applied to dechlorinating enrichment cultures, microcosms, and samples from a contaminated site. In characterized enrichment cultures where known Dehalococcoides strains were enumerated, the sum of the three RDase genes equaled the total Dehalococcoides cell numbers. In site samples and chloroethane-dechlorinating microcosms, the sum of the three RDase genes was much less than that predicted by Dehalococcoides-targeted qPCR, totaling 10 to 30% of the total Dehalococcoides cell numbers. Hence, a large number of Dehalococcoides spp. contain as-yet-unidentified RDase genes, indicating that our current understanding of the dechlorinating Dehalococcoides community is incomplete.     

Archaeal abundance across a pH gradient in an arable soil and its relationship to bacterial and fungal growth rates

Soil pH is one of the most influential factors for the composition of bacterial and fungal communities, but the influence of soil pH on the distribution and composition of soil archaeal communities has yet to be systematically addressed. The primary aim of this study was to determine how total archaeal abundance (quantitative PCR [qPCR]-based estimates of 16S rRNA gene copy numbers) is related to soil pH across a pH gradient (pH 4.0 to 8.3). Secondarily, we wanted to assess how archaeal abundance related to bacterial and fungal growth rates across the same pH gradient. We identified two distinct and opposite effects of pH on the archaeal abundance. In the lowest pH range (pH 4.0 to 4.7), the abundance of archaea did not seem to correspond to pH. Above this pH range, there was a sharp, almost 4-fold decrease in archaeal abundance, reaching a minimum at pH 5.1 to 5.2. The low abundance of archaeal 16S rRNA gene copy numbers at this pH range then sharply increased almost 150-fold with pH, resulting in an increase in the ratio between archaeal and bacterial copy numbers from a minimum of 0.002 to more than 0.07 at pH 8. The nonuniform archaeal response to pH could reflect variation in the archaeal community composition along the gradient, with some archaea adapted to acidic conditions and others to neutral to slightly alkaline conditions. This suggestion is reinforced by observations of contrasting outcomes of the (competitive) interactions between archaea, bacteria, and fungi toward the lower and higher ends of the examined pH gradient.     

Development of a real-time PCR method for quantification of the three genera Dehalobacter, Dehalococcoides, and Desulfitobacterium in microbial communities

We developed standard curves based on plasmids containing a 16S rRNA gene of a member of one of the three genera Dehalobacter, Desulfitobacterium, and Dehalococcoides. A large difference in amplification efficiency between the standard curves was observed ranging from 1.5 to 2.0. The total eubacterial 16S rRNA gene copy number determined in a sample DNA by using eubacterial primers and the three standard curves led to differences in the estimated copy numbers of a factor up to 73. However, the amplification efficiencies for one specific standard curve were the same independent of the PCR primer pair used. This allowed the determination of the abundance of a population expressed as fractional number, hence, the percentage of genus-specific copy numbers within the total eubacterial 16S rRNA gene copy numbers. Determination of the fractional numbers in DNA mixtures of known composition showed the accuracy of this approach. The average difference in threshold value between two 10-fold dilutions of DNA of pure cultures, mixtures thereof and of environmental samples was -3.45+/-0.34, corresponding to an average almost optimal amplification efficiency of 1.95. This indicated that the low amplification efficiency of certain standard curves seemed to be mainly a problem of the plasmid DNA used and not of the 16S rRNA gene of the target genera.     

Specific detection and quantification of the marine flavobacterial genus Zobellia on macroalgae using novel qPCR and CARD-FISH assays

The flavobacterial genus Zobellia is considered as a model to study macroalgal polysaccharide degradation. The lack of data regarding its prevalence and abundance in coastal habitats constitutes a bottleneck to assess its ecological strategies. To overcome this issue, real-time quantitative PCR (qPCR) and fluorescence in situ hybridization (FISH) methods targeting the 16S rRNA gene were optimized to specifically detect and quantify Zobellia on the surface of diverse macroalgae. The newly designed qPCR primers and FISH probes targeted 98 and 100% of the Zobellia strains in silico and their specificity was confirmed using pure bacterial cultures. The dynamic range of the qPCR assay spanned 8 orders of magnitude from 10 to 10⁸ 16S rRNA gene copies and the detection limit was 0.01% relative abundance of Zobellia in environmental samples. Zobellia-16S rRNA gene copies were detected on all surveyed brown, green and red macroalgae, in proportion varying between 0.1 and 0.9% of the total bacterial copies. The absolute and relative abundance of Zobellia varied with tissue aging on the kelp Laminaria digitata. Zobellia cells were successfully visualized in Ulva lactuca and stranded Palmaria palmata surface biofilm using CARD-FISH, representing in the latter 10⁵ Zobellia cells·cm⁻² and 0.43% of total bacterial cells. Overall, qPCR and CARD-FISH assays enabled robust detection, quantification and localization of Zobellia representatives in complex samples, underlining their ecological relevance as primary biomass degraders potentially cross-feeding other microorganisms.     

Identification of short 'eukaryotic' Okazaki fragments synthesized from a prokaryotic replication origin

Although archaeal genomes encode proteins similar to eukaryotic replication factors, the hyperthermophilic archaeon Pyrococcus abyssi replicates its circular chromosome at a high rate from a single origin (oriC) as in Bacteria. In further elucidating the mechanism of archaeal DNA replication, we have studied the elongation step of DNA replication in vivo. We have detected, in two main archaeal phyla, short RNA-primed replication intermediates whose structure and length are very similar to those of eukaryotic Okazaki fragments. Mapping of replication initiation points further showed that discontinuous DNA replication in P. abyssi starts at a well-defined site within the oriC recently identified in this hyperthermophile. Short Okazaki fragments and a high replication speed imply a very efficient turnover of Okazaki fragments in Archaea. Archaea therefore have a unique replication system showing mechanistic similarities to both Bacteria and Eukarya.     

Quantification of microbial communities in near-surface and deeply buried marine sediments on the Peru continental margin using real-time PCR

Deeply buried marine sediments harbour a large fraction of all prokaryotes on Earth but it is still unknown which phylogenetic and physiological microbial groups dominate the deep biosphere. In this study real-time PCR allowed a comparative quantitative microbial community analysis in near-surface and deeply buried marine sediments from the Peru continental margin. The 16S rRNA gene copy numbers of prokaryotes and Bacteria were almost identical with a maximum of 10(8)-10(10) copies cm(-3) in the near-surface sediments. Archaea exhibited one to three orders of magnitude lower 16S rRNA gene copy numbers. The 18S rRNA gene of Eukarya was always at least three orders of magnitude less abundant than the 16S rRNA gene of prokaryotes. The 16S rRNA gene of the Fe(III)- and Mn(IV)-reducing bacterial family Geobacteraceae and the dissimilatory (bi)sulfite reductase gene (dsrA) of sulfate-reducing prokaryotes were abundant with 10(6)-10(8) copies cm(-3) in near-surface sediments but showed lower numbers and an irregular distribution in the deep sediments. The copy numbers of all genes decreased with sediment depth exponentially. The depth gradients were steeper for the gene copy numbers than for numbers of total prokaryotes (acridine orange direct counts), which reflects the ongoing degradation of the high-molecular-weight DNA with sediment age and depth. The occurrence of eukaryotic DNA also suggests DNA preservation in the deeply buried sediments.     

Nosema ceranae infection in honeybee samples from Tuscanian Archipelago (Central Italy) investigated by two qPCR methods

Nosema apis and Nosema ceranae are microsporidian parasite worldwide spread causing an emerging infectious disease of European honeybee Apis mellifera. The Nosema presence was deeply investigated in several countries but low information are presents about islands. In this investigation was evaluated the presence N. ceranae and N. apis in apiaries located in Tuscanian Archipelago islands (Central Italy). For N. ceranae detection, two different Real-Time PCR (qPCR) methods, the 16S rRNA and Hsp70 gene amplification qPCR, were performed on honey bee samples; while, for N. apis only the 16S rRNA qPCR amplification was performed. On all islands, only N. ceranae was present, while N. apis was not found in the samples. The two qPCR showed significant difference (p < 0.040) in N. ceranae spores quantification. The single-copy Hsp70 gene method qPCR assay systematically detected a lower amount of N. ceranae copies compared to the multi-copy 16S rRNA gene method.     

RNomics in Archaea reveals a further link between splicing of archaeal introns and rRNA processing

The bulge–helix–bulge (BHB) motif recognised by the archaeal splicing endonuclease is also found in the long processing stems of archaeal rRNA precursors in which it is cleaved to generate pre-16S and pre-23S rRNAs. We show that in two species, Archaeoglobus fulgidus and Sulfolobus solfataricus, representatives from the two major archaeal kingdoms Euryarchaeota and Crenarchaeota, respectively, the pre-rRNA spacers cleaved at the BHB motifs surrounding pre-16S and pre-23S rRNAs subsequently become ligated. In addition, we present evidence that this is accompanied by circularisation of ribosomal pre-16S and pre-23S rRNAs in both species. These data reveal a further link between intron splicing and pre-rRNA processing in Archaea, which might reflect a common evolutionary origin of the two processes. One spliced RNA species designated 16S-D RNA, resulting from religation at the BHB motif of 16S pre-rRNA, is a highly abundant and stable RNA which folds into a three-stem structure interrupted by two single-stranded regions as assessed by chemical probing. It spans a region of the pre-rRNA 5′ external transcribed spacer exhibiting a highly conserved folding pattern in Archaea. Surprisingly, 16S-D RNA contains structural motifs found in archaeal C/D box small RNAs and binds to the L7Ae protein, a core component of archaeal C/D box RNPs. This supports the notion that it might have an important but still unknown role in pre-rRNA biogenesis or might even target RNA molecules other than rRNA.     

Quantitative Detection of the Free-Living Amoeba Hartmannella vermiformis in Surface Water by Using Real-Time PCR

A real-time PCR-based method targeting the 18S rRNA gene was developed for the quantitative detection of Hartmannella vermiformis, a free-living amoeba which is a potential host for Legionella pneumophila in warm water systems and cooling towers. The detection specificity was validated using genomic DNA of the closely related amoeba Hartmannella abertawensis as a negative control and sequence analysis of amplified products from environmental samples. Real-time PCR detection of serially diluted DNA extracted from H. vermiformis was linear for microscopic cell counts between 1.14 × 10⁻¹ and 1.14 × 10⁴ cells per PCR. The genome of H. vermiformis harbors multiple copies of the 18S rRNA gene, and an average number (with standard error) of 1,330 ± 127 copies per cell was derived from real-time PCR calibration curves for cell suspensions and plasmid DNA. No significant differences were observed between the 18S rRNA gene copy numbers for trophozoites and cysts of strain ATCC 50237 or between the copy numbers for this strain and strain KWR-1. The developed method was applied to water samples (200 ml) collected from a variety of lakes and rivers serving as sources for drinking water production in The Netherlands. Detectable populations were found in 21 of the 28 samples, with concentrations ranging from 5 to 75 cells/liter. A high degree of similarity (≥98%) was observed between sequences of clones originating from the different surface waters and between these clones and the reference strains. Hence, H. vermiformis, which is highly similar to strains serving as hosts for L. pneumophila, is a common component of the microbial community in fresh surface water.