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。     

Molecular quantification of environmental DNA using microfluidics and digital PCR

Real-time PCR has been widely used to evaluate gene abundance in natural microbial habitats. However, PCR-inhibitory substances often reduce the efficiency of PCR, leading to the underestimation of target gene copy numbers. Digital PCR using microfluidics is a new approach that allows absolute quantification of DNA molecules. In this study, digital PCR was applied to environmental samples, and the effect of PCR inhibitors on DNA quantification was tested. In the control experiment using λ DNA and humic acids, underestimation of λ DNA at 1/4400 of the theoretical value was observed with 6.58ngμL(-1) humic acids. In contrast, digital PCR provided accurate quantification data with a concentration of humic acids up to 9.34ngμL(-1). The inhibitory effect of paddy field soil extract on quantification of the archaeal 16S rRNA gene was also tested. By diluting the DNA extract, quantified copy numbers from real-time PCR and digital PCR became similar, indicating that dilution was a useful way to remedy PCR inhibition. The dilution strategy was, however, not applicable to all natural environmental samples. For example, when marine subsurface sediment samples were tested the copy number of archaeal 16S rRNA genes was 1.04×10(3)copies/g-sediment by digital PCR, whereas real-time PCR only resulted in 4.64×10(2)copies/g-sediment, which was most likely due to an inhibitory effect. The data from this study demonstrated that inhibitory substances had little effect on DNA quantification using microfluidics and digital PCR, and showed the great advantages of digital PCR in accurate quantifications of DNA extracted from various microbial habitats.     

3 次连续重复提取DNA 能较好反映土壤微生物丰度

【目的】研究同一个土壤需要反复提取几次才能在最大程度上反映土壤微生物的丰度,探讨风干土壤代替新鲜土壤用于微生物丰度研究的可行性。【方法】针对两种理化性质具有较大差异的旱地和稻田新鲜土壤及其风干土壤,分别对土壤微生物进行5次连续裂解提取DNA。通过实时荧光定量PCR技术分析连续反复提取对土壤古菌和细菌16S rRNA gene数量、氨氧化古菌和细菌功能基因amoA数量的影响。【结果】3次连续提取DNA占5次提取DNA总量的76%以上,氨氧化古菌、氨氧化细菌、古菌和细菌4类微生物的3次连续提取最低回收率为77.5%;与新鲜土壤相比,风干处理导致氨氧化古菌、氨氧化细菌、古菌、细菌的数量分别降低84.3%、81.2%、12.5%和90.3%,然而,2种土壤风干过程中主要微生物类群的数量变化规律基本一致,表明土壤微生物对风干处理的响应可能受土壤类型的影响较小。【结论】土壤微生物连续3次裂解能较好反映微生物丰度。与新鲜土壤相比,风干过程显著降低了土壤微生物丰度,然而,通过风干土壤中微生物丰度的变化趋势反映新鲜土壤中微生物数量变化规律具有一定的可行性。     

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.     

Simple absolute quantification method correcting for quantitative PCR efficiency variations for microbial community samples

Real-time quantitative PCR (qPCR) is a widely used technique in microbial community analysis, allowing the quantification of the number of target genes in a community sample. Currently, the standard-curve (SC) method of absolute quantification is widely employed for these kinds of analysis. However, the SC method assumes that the amplification efficiency (E) is the same for both the standard and the sample target template. We analyzed 19 bacterial strains and nine environmental samples in qPCR assays, targeting the nifH and 16S rRNA genes. The E values of the qPCRs differed significantly, depending on the template. This has major implications for the quantification. If the sample and standard differ in their E values, quantification errors of up to orders of magnitude are possible. To address this problem, we propose and test the one-point calibration (OPC) method for absolute quantification. The OPC method corrects for differences in E and was derived from the ΔΔC(T) method with correction for E, which is commonly used for relative quantification in gene expression studies. The SC and OPC methods were compared by quantifying artificial template mixtures from Geobacter sulfurreducens (DSM 12127) and Nostoc commune (Culture Collection of Algae and Protozoa [CCAP] 1453/33), which differ in their E values. While the SC method deviated from the expected nifH gene copy number by 3- to 5-fold, the OPC method quantified the template mixtures with high accuracy. Moreover, analyzing environmental samples, we show that even small differences in E between the standard and the sample can cause significant differences between the copy numbers calculated by the SC and the OPC methods.     

Incorporating 16S Gene Copy Number Information Improves Estimates of Microbial Diversity and Abundance

The abundance of different SSU rRNA (“16S”) gene sequences in environmental samples is widely used in studies of microbial ecology as a measure of microbial community structure and diversity. However, the genomic copy number of the 16S gene varies greatly – from one in many species to up to 15 in some bacteria and to hundreds in some microbial eukaryotes. As a result of this variation the relative abundance of 16S genes in environmental samples can be attributed both to variation in the relative abundance of different organisms, and to variation in genomic 16S copy number among those organisms. Despite this fact, many studies assume that the abundance of 16S gene sequences is a surrogate measure of the relative abundance of the organisms containing those sequences. Here we present a method that uses data on sequences and genomic copy number of 16S genes along with phylogenetic placement and ancestral state estimation to estimate organismal abundances from environmental DNA sequence data. We use theory and simulations to demonstrate that 16S genomic copy number can be accurately estimated from the short reads typically obtained from high-throughput environmental sequencing of the 16S gene, and that organismal abundances in microbial communities are more strongly correlated with estimated abundances obtained from our method than with gene abundances. We re-analyze several published empirical data sets and demonstrate that the use of gene abundance versus estimated organismal abundance can lead to different inferences about community diversity and structure and the identity of the dominant taxa in microbial communities. Our approach will allow microbial ecologists to make more accurate inferences about microbial diversity and abundance based on 16S sequence data.     

Enumeration of total bacteria and bacteria with genes for proteolytic activity in pure cultures and in environmental samples by quantitative PCR mediated amplification

Real-time quantitative PCR assays were developed for the absolute quantification of different groups of bacteria in pure cultures and in environmental samples. 16S rRNA genes were used as markers for eubacteria, and genes for extracellular peptidases were used as markers for potentially proteolytic bacteria. For the designed 16S rDNA TaqMan assay, specificity of the designed primer-probe combination for eubacteria, a high amplification efficiency over a wide range of starting copy numbers and a high reproducibility is demonstrated. Cell concentrations of Bacillus cereus, B. subtilis and Pseudomonas fluorescens in liquid culture were monitored by TaqMan-PCR using the 16S rDNA target sequence of Escherichia coli as external standard for quantification. Results agree with plate counts and microscopic counts of DAPI stained cells. The significance of 16S rRNA operon multiplicity to the quantification of bacteria is discussed.Furthermore, three sets of primer pair together with probe previously designed for targeting different classes of bacterial extracellular peptidases were tested for their suitability for TaqMan-PCR based quantification of proteolytic bacteria. Since high degeneracy of the probes did not allow accurate quantification, SybrGreen was used instead of molecular probes to visualize and quantify PCR products during PCR. The correlation between fluorescence and starting copy number was of the same high quality as for the 16S rDNA TaqMan assay for all the three peptidase gene classes. The detected amount of genes for neutral metallopeptidase of B. cereus, for subtilisin of B. subtilis and for alkaline metallopeptidase of P. fluorescens corresponded exactly to the numbers of bacteria investigated by the 16S rDNA targeting assay.The developed assays were applied for the quantification of bacteria in soil samples.     

Power analysis for real-time PCR quantification of genes in activated sludge and analysis of the variability introduced by DNA extraction

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.     

16S rRNA基因在微生物生态学中的应用

16S rRNA(Small subunit ribosomal RNA)基因是对原核微生物进行系统进化分类研究时最常用的分子标志物(Biomarker),广泛应用于微生物生态学研究中。近些年来随着高通量测序技术及数据分析方法等的不断进步,大量基于16S rRNA基因的研究使得微生物生态学得到了快速发展,然而使用16S rRNA基因作为分子标志物时也存在诸多问题,比如水平基因转移、多拷贝的异质性、基因扩增效率的差异、数据分析方法的选择等,这些问题影响了微生物群落组成和多样性分析时的准确性。对当前使用16S rRNA基因分析微生物群落组成和多样性的进展情况做一总结,重点讨论当前存在的主要问题以及各种分析方法的发展,尤其是与高通量测序技术有关的实验和数据处理问题。     

Assessment of bacterial community structure in the deep sub-seafloor biosphere by 16S rDNA-based techniques: a cautionary tale

Investigations into the deep marine environment have demonstrated the presence of a significant microbial biomass buried deep within sediments on a global scale. It is now believed that this deep biosphere plays a major role in the global cycling of elements and contains a large reservoir of organic carbon. This paper reports the development of a DNA extraction protocol that addresses the particular problems faced in applying molecular ecological techniques to samples containing very low biomass. Sediment samples were collected from different geographical locations within the Pacific Ocean and include the Ocean Drilling Program (ODP) Leg 190, Nankai Trough Accretionary Prism. Seven DNA extraction protocols were tested and a commercially available DNA extraction kit with modifications was shown to produce higher yields of polymerase chain reaction (PCR)-amplifiable DNA than standard laboratory methods. Denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA gene diversity revealed that template DNA from these extremely low biomass sediment samples was susceptible to PCR bias and random amplification. We propose that it is essential to screen 16S rRNA gene products for bacterial diversity by DGGE or other rapid fingerprinting methods, prior to their use in establishing a representative clone library of deep sub-seafloor bacteria. This represents a cautionary approach to analysis of microbial diversity in such sub-seafloor ecosystems.     

Semi-automated extraction of microbial DNA from feces for qPCR and phylogenetic microarray analysis

The human gastrointestinal tract (GI-tract) harbors a complex microbial ecosystem, largely composed of so far uncultured species, which can be detected only by using techniques such as PCR and by different hybridization techniques including phylogenetic microarrays. Manual DNA extraction from feces is laborious and is one of the bottlenecks holding up the application of microarray and other DNA-based techniques in large cohort studies. In order to enhance the DNA extraction step we combined mechanical disruption of microbial cells by repeated bead-beating (RBB) with two automated DNA extraction methods, KingFisher with InviMag Stool DNA kit (KF) and NucliSENS easyMAG (NeM). The semi-automated DNA extraction methods, RBB combined with either KF or NeM, were compared to the manual extraction method currently considered the most suited method for fecal DNA extraction by assessing the yield of 16S rRNA gene copies by qPCR and total microbiota composition by the HITChip, a phylogenetic microarray. Parallel DNA extractions from infant fecal samples by using the three methods showed that the KF and manual methods gave comparable yields of 16S rRNA gene copies as assessed by qPCR, whereas NeM showed a significantly lower yield. All three methods showed highly similar microbiota profiles in HITChip. Both KF and NeM were found to be suitable methods for DNA extraction from fecal samples after the mechanical disruption of microbial cells by bead-beating. The semi-automated methods could be performed in half of the time required for the manual protocol, while being comparable to the manual method in terms of reagent costs.     

Extraction of high molecular weight DNA from microbial mats

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction of high-quality, high molecular weight (HMW) community DNA. However, environmental mat samples often pose difficulties to obtaining large concentrations of high-quality, HMW DNA. Hypersaline microbial mats contain high amounts of extracellular polymeric substances (EPS)1 and salts that may inhibit downstream applications of extracted DNA. Direct and harsh methods are often used in DNA extraction from refractory samples. These methods are typically used because the EPS in mats, an adhesive matrix, binds DNA during direct lysis. As a result of harsher extraction methods, DNA becomes fragmented into small sizes. The DNA thus becomes inappropriate for large-insert vector cloning. In order to circumvent these limitations, we report an improved methodology to extract HMW DNA of good quality and quantity from hypersaline microbial mats. We employed an indirect method involving the separation of microbial cells from the background mat matrix through blending and differential centrifugation. A combination of mechanical and chemical procedures was used to extract and purify DNA from the extracted microbial cells. Our protocol yields approximately 2 μg of HMW DNA (35-50 kb) per gram of mat sample, with an A(260/280) ratio of 1.6. Furthermore, amplification of 16S rRNA genes suggests that the protocol is able to minimize or eliminate any inhibitory effects of contaminants. Our results provide an appropriate methodology for the extraction of HMW DNA from microbial mats for functional metagenomic studies and may be applicable to other environmental samples from which DNA extraction is challenging.     

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

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.     

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.     

Use of rpoB and 16S rRNA genes to analyse bacterial diversity of a tropical soil using PCR and DGGE

AIM: To evaluate the rpoB gene as a biomarker for PCR-DGGE microbial analyses using soil DNA from the Cerrado, Brazil. METHODS: DNA extraction from soil was followed by Polymerase Chain Reaction (PCR) amplification of rpoB and 16S rRNA genes. PCR products were compared by Denaturing Gradient Gel Electrophoresis (DGGE) to compare gene/community profiles. RESULTS: The rpoB DGGE profiles comprised fewer bands than the 16S rDNA profiles and were easier to delineate and therefore to analyse. Comparison of the community profiles revealed that the methods were complementary. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The gene for the beta subunit of the RNA polymerase, rpoB, is a single copy gene unlike 16S rDNA. Multiple copies of 16S rRNA genes in bacterial genomes complicate diversity assessments made from DGGE profiles. Using the rpoB gene offers a better alternative to the commonly used 16S rRNA gene for microbial community analyses based on DGGE.     

Agreement between amoA Gene-Specific Quantitative PCR and Fluorescence In Situ Hybridization in the Measurement of Ammonia-Oxidizing Bacteria in Activated Sludge

Microbial abundance is central to most investigations in microbial ecology, and its accurate measurement is a challenging task that has been significantly facilitated by the advent of molecular techniques over the last 20 years. Fluorescence in situ hybridization (FISH) is considered the gold standard of quantification techniques; however, it is expensive and offers low sample throughput, both of which limit its wider application. Quantitative PCR (qPCR) is an alternative that offers significantly higher throughput, and it is used extensively in molecular biology. The accuracy of qPCR can be compromised by biases in the DNA extraction and amplification steps. In this study, we compared the accuracy of these two established quantification techniques to measure the abundance of a key functional group in biological wastewater treatment systems, the ammonia-oxidizing bacteria (AOB), in samples from a time-series experiment monitoring a set of laboratory-scale reactors and a full-scale plant. For the qPCR analysis, we tested two different sets of AOB-specific primers, one targeting the 16SrRNA gene and one targeting the ammonia monooxygenase (amoA) gene. We found that there was a positive linear logarithmic relationship between FISH and the amoA gene-specific qPCR, where the data obtained from both techniques was equivalent at the order of magnitude level. The 16S rRNA gene-specific qPCR assay consistently underestimated AOB numbers.     

Development of a rapid quantitative PCR assay for direct detection and quantification of culturable and non-culturable Escherichia coli from agriculture watersheds

A real-time quantitative polymerase chain reaction (Q-PCR) assay was developed for detecting and quantifying Escherichia coli in water samples from agricultural watersheds. The assay included optimization of DNA extraction and purification from water samples, and Q-PCR amplification conditions using newly designed species-specific oligonucleotide primers derived from conserved flanking regions of the 16S rRNA gene, the internal transcribed spacer region (ITS) and the 23S rRNA gene. The assay was optimized using a pure culture of E. coli with known quantities spiked into autoclaved agricultural water samples. The optimized assay was capable of a minimum quantification limit of 10 cells/ml of E. coli in the spiked agricultural water samples. A total of 121 surface water samples from three agricultural watersheds across Canada were analyzed, and results were compared with conventional culture-based enumerations of E. coli. The Q-PCR assay revealed significantly higher numbers of E. coli in water samples than the culture-based assay in each agricultural watershed. The new Q-PCR assay can facilitate the quantification of E. coli in a single water sample in < 3 h, including melt curve analysis, across a range of agricultural water quality conditions.     

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 x 10(-1) and 1.14 x 10(4) 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 (> or =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.