Differential amplification of rRNA genes by polymerase chain reaction.

The polymerase chain reaction (PCR) is used widely to recover rRNA genes from naturally occurring communities for analysis of population constituents. We have found that this method can result in differential amplification of different rRNA genes. In particular, rDNAs of extremely thermophilic archaebacteria often cannot be amplified by the usual PCR methods. The addition of 5% (wt/vol) acetamide to a PCR mixture containing both archaebacterial and yeast DNA templates minimized nonspecific annealing of the primers and prevented preferential amplification of the yeast small-subunit rRNA genes.     

PCR-based dENATURING GRADIENT GEL ELECTROPHORETICtypification and identification of the microbial consortium present in Kefir grains

Kefir grains have a complex microbiological composition that makes it difficult to obtain an optimal and constant starter culture necessary for the production of a quality Kefir beverage. The microbes present in the grains have in the past been identified using traditional methods such as growth on selective media and morphological and physiological characteristics. The aim of this study was to typify and identify the complex microbial community present in mass cultured, traditionally cultured and Irish Kefir grains by PCR amplification of a variable part of the ribosomal RNA (rRNA) genes in Eubacteria and yeasts and resolving these PCR fragments by denaturing gradient gel electrophoresis (DGGE). Unique PCR-based DGGE fingerprints were obtained for the Eubacterial and yeast species present in the three different grain types. A part of the Eubacterial and yeast rRNA genes were sequenced and compared to sequences available on NCBI. The phylogenetic relatedness of the amplified and sequenced lactobacilli was determined. Different bands in the Eubacterial and the yeast DGGE profiles of the mass cultured grains were identified to species level. A DGGE marker was constructed providing a quick reference for the identification of the members of the Eubacterial microbial population in mass cultured Kefir grains.     

Comparison of 16S rRNA and protein-coding genes as molecular markers for assessing microbial diversity (Bacteria and Archaea) in ecosystems

PCR amplification of the rRNA gene is the most popular method for assessing microbial diversity. However, this molecular marker is often present in multiple copies in cells presenting, in addition, an intragenomic heterogeneity. In this context, housekeeping genes may be used as taxonomic markers for ecological studies. However, the efficiency of these protein-coding genes compared to 16S rRNA genes has not been tested on environmental data. For this purpose, five protein marker genes for which primer sets are available, were selected (rplB, pyrG, fusA, leuS and rpoB) and compared with 16S rRNA gene results from PCR amplification or metagenomic data from aquatic ecosystems. Analysis of the major groups found in these ecosystems, such as Actinobacteria, Bacteroides, Proteobacteria and Cyanobacteria, showed good agreement between the protein markers and the results given by 16S rRNA genes from metagenomic reads. However, with the markers it was possible to detect minor groups among the microbial assemblages, providing more details compared to 16S rRNA results from PCR amplification. In addition, the use of a set of protein markers made it possible to deduce a mean copy number of rRNA operons. This average estimate is essentially lower than the one estimated in sequenced genomes.     

Extrachromosomal circular ribosomal DNA in the yeast Saccharomyces carlsbergensis.

Purified ribosomal DNA from Saccharomyces carlsbergensis contains a small proportion of circular DNA molecules with a contour length of 3 micron or integral multiples thereof. Hybridization of yeast ribosomal DNA with 26 S rRNA, using the R-loop technique, reveals that these circular molecules contain sequences complementary to yeast ribosomal RNA. We suggest that these extrachromosomal rRNA genes may be intermediates in the amplification of rRNA genes in yeast.     

Rapid detection of yeast rRNA genes with primed in situ (PRINS) labeling

In yeast, rRNA genes can be detected with the FISH technique using rRNA gene probes. This technique yields reliable, reproducible and precise results, but is time-consuming. Here, the primed in situ DNA synthesis (PRINS) procedure has been optimized for rapid detection of yeast rRNA genes. PRINS, which is as sensitive as PCR and allows cytological localization of analyzed sequences, can be adapted for various screening tests requiring fast labeling of rRNA genes.     

实时定量PCR构建法夫酵母内参基因β-actin、 gpd、18S rRNA标准品质粒和标准曲线

为建立检测法夫酵母JMU-MVP14中虾青素合成相关基因在不同生长时期表达水平的实时定量PCR方法,构建法夫酵母JMU-MVP14的管家基因β-actin、gpd、18S rRNA的标准质粒,进行实时定量PCR,制作标准曲线及回归方程.β-actin基因标准曲线相关系数（R2）=0.9956,扩增效率（E） =96.93％;gpd基因标准曲线相关系数（R2） =0.9901,扩增效率（E） =93.78％;18S rRNA基因标准曲线相关系数（R2） =0.9981,扩增效率（E）=98.76％.3个基因片段的熔解曲线均呈单峰;扩增曲线呈典型的S型动力学曲线,指数期和平台期明显,为理想的熔解曲线和扩增曲线.用geNorm软件对三个管家基因的稳定性进行分析,三个基因的稳定性排序为β-actin＞ 18S rRNA＞ gpd,故β-actin和18S rRNA较适合作为研究法夫酵母JMU-MVP14定量实验的内参基因.     

Amplification and sequencing of variable regions in bacterial 23S ribosomal RNA genes with conserved primer sequences

Published bacterial 23S ribosomal RNA sequences were aligned, and universally conserved regions flanking highly variable regions were looked for. In strategically positioned conserved regions, six oligonucleotides suitable for polymerase chain reaction (PCR) and sequencing were designed, allowing fast sequencing of four of the most variable 23S rRNA regions. Two other primers were designed for PCR amplification of nearly complete 23S rRNA genes. All these primers successfully amplified fragments of 23S rRNA genes from seven unrelated bacteria. Four primers were used to determine 938 bp of sequence forCampylobacter jejuni subsp.jejuni. These results indicate that the oligonucleotide sequences presented here are useful for PCR amplification and sequence determination of variable 23S rRNA regions for a broad variety of eubacterial species.     

PCR-amplification of mixed 16S rRNA genes from an anaerobic, cyanide-degrading consortium

Abstract The microorganisms participating in the anaerobic biodegradation of cyanide were characterized using 16S rRNA genes as genetic markers of diversity. Segments of mixed population 16S rRNA genes were amplified using the polymerase chain reaction (PCR) and prokaryote-specific amplification primers. Restriction fragment length polymorphism (RFLPs) and screening with the 926f universal sequencing primer were used to categorized the cloned PCR products. Six unique prokaryote sequence were obtained, including four similar to methanogens and two similar to Gram-positive eubacteria.     

Frequency of formation of chimeric molecules as a consequence of PCR coamplification of 16S rRNA genes from mixed bacterial genomes.

PCR is routinely used in amplification and cloning of rRNA genes from environmental DNA samples for studies of microbial community structure and identification of novel organisms. There have been concerns about generation of chimeric sequences as a consequence of PCR coamplification of highly conserved genes, because such sequences may lead to reports of nonexistent organisms. To quantify the frequency of chimeric molecule formation, mixed genomic DNAs from eight actinomycete species whose 16S rRNA sequences had been determined were used for PCR coamplification of 16S rRNA genes. A large number of cloned 16S ribosomal DNAs were examined by sequence analysis, and chimeric molecules were identified by multiple-sequence alignment with reference species. Here, we report that the level of occurrence of chimeric sequences after 30 cycles of PCR amplification was 32%. We also show that PCR-induced chimeras were formed between different rRNA gene copies from the same organism. Because of the wide use of PCR for direct isolation of 16S rRNA sequences from environmental DNA to assess microbial diversity, the extent of chimeric molecule formation deserves serious attention.     

Design and Evaluation of Useful Bacterium-Specific PCR Primers That Amplify Genes Coding for Bacterial 16S rRNA

We report the design and evaluation of PCR primers 63f and 1387r for amplification of 16S rRNA genes from bacteria. Their specificity and efficacy were tested systematically with a variety of bacterial species and environmental samples. They were found to be more useful for 16S rRNA gene amplification in ecological and systematic studies than PCR amplimers that are currently more generally used.     

Molecular Identification of a Rare Subtype of Cryptosporidium hominis in Infants in China

Two Cryptosporidium isolates from separate infants suffering from diarrhea were obtained from a hospital in Zhengzhou, China and were genotyped by PCR amplification and sequence analysis of the small-subunit ribosomal RNA (rRNA) (SSU rRNA), 70-kDa heat shock protein (HSP70), and actin genes. Further subtyping was performed by PCR amplification and sequence analysis of the 60-kDa glycoprotein (gp60) gene. Both the isolates were identified as Cryptosporidium hominis subtype IdA21, a rare subtype previously found only in a human immunodeficiency virus-infected child in South Africa and another child in Jordan.     

Nonradioactive method to study genetic profiles of natural bacterial communities by PCR-single-strand-conformation polymorphism.

We describe a new method for studying the structure and diversity of bacterial communities in the natural ecosystem. Our approach is based on single-strand-conformation polymorphism (SSCP) analysis of PCR products of 16S rRNA genes from complex bacterial populations. A pair of eubacterial universal primers for amplification of the variable V3 region were designed from the 16S rRNA sequences of 1,262 bacterial strains. The PCR conditions were optimized by using genomic DNAs from five gram-positive and seven gram-negative strains. The SSCP analysis of the PCR products demonstrated that a bacterial strain generated its characteristic band pattern and that other strains generated other band patterns, so that the relative diversity in bacterial communities could be measured. In addition, this method was sensitive enough to detect a bacterial population that made up less than 1.5% of a bacterial community. The distinctive differences between bacterial populations were observed in an oligotrophic lake and a eutrophic pond in a field study. The method presented here, using combined PCR amplification and SSCP pattern analyses of 16S rRNA genes, provides a useful tool to study bacterial community structures in various ecosystems.     

5S ribosomal and U1 small nuclear RNA genes: a new linkage type in the genome of a crustacean that has three different tandemly repeated units containing 5S ribosomal DNA sequences.

We investigated the 5S ribosomal RNA (rRNA) genes of the isopod crustacean Asellus aquaticus. Using PCR amplification, three different tandemly repeated units containing 5S rDNA were identified. Two of the three sequences were cloned and sequenced. One of them was 1842 bp and presented a 5S rRNA gene and a U1 small nuclear RNA (snRNA) gene. This type of linkage had never been observed before. The other repeat consisted of 477 bp and contained only an incomplete 5S rRNA gene lacking the first eight nucleotides and a spacer sequence. The third sequence was 6553 bp long and contained a 5S rRNA gene and the four core histone genes. The PCR products were used as probes in fluorescent in situ hybridization (FISH) experiments to locate them on chromosomes of A. aquaticus. The possible evolutionary origin of the three repeated units is discussed.     

Critical evaluation of two primers commonly used for amplification of bacterial 16S rRNA genes

rRNA-based studies, which have become the most common method for assessing microbial communities, rely upon faithful amplification of the corresponding genes from the original DNA sample. We report here an analysis and reevaluation of commonly used primers for amplifying the DNA between positions 27 and 1492 of bacterial 16S rRNA genes (numbered according to the Escherichia coli rRNA). We propose a formulation for a forward primer (27f) that includes three sequences not usually present. We compare our proposed formulation to two common alternatives by using linear amplification-providing an assessment that is independent of a reverse primer-and in combination with the 1492 reverse primer (1492r) under the PCR conditions appropriate for making community rRNA gene clone libraries. For analyses of DNA from human vaginal samples, our formulation was better at maintaining the original rRNA gene ratio of Lactobacillus spp. to Gardnerella spp., particularly under stringent amplification conditions. Because our 27f formulation remains relatively simple, having seven distinct primer sequences, there is minimal loss of overall amplification efficiency and specificity.     

A novel multiplex PCR assay for the detection of Salmonella enterica serovar Enteritidis in human faeces

AIMS: To develop a multiplex PCR assay for the detection of Salmonella enterica serovar Enteritidis in human faeces. METHODS AND RESULTS: A total of 54 Salmonella strains representing 19 serovars and non-Salmonella strains representing 11 different genera were used. Five primer pairs were employed in the assay. Three of them targeted to the genes hilA, spvA and invA that encode virulence-associated factors. A fourth primer pair amplified a fragment of a unique sequence within S. enterica serovar Enteritidis genomes. An internal amplification control (a fragment of a conservative sequence within the 16S rRNA genes) was targeted by a fifth primer pair. The assay produced two or three amplicons from the invA, hilA and 16S rRNA genes for 19 Salmonella serovars. All Salmonella and non-Salmonella strains yielded a band of an internal amplification control. For S. enterica serovar Typhimurium, four products (the fourth from the spvA gene), and for S. enterica serovar Enteritidis five amplicons (the fifth from the sdf gene) were observed. S. enterica serovar Enteritidis was cultured from three of 71 rectal swabs from diarrhoeal patients. Five specific amplicons were generated with the multiplex PCR assay only from culture-positive faecal samples. CONCLUSION: The multiplex PCR assay specifically detects S. enterica serovar Enteritidis. SIGNIFICANCE AND IMPACT OF THE STUDY: This is a novel multiplex PCR assay, which contains an internal amplification control and enables concurrent survey for Salmonella virulence genes.     

A universal primer set for PCR amplification of nuclear histone H4 genes from all animal species

To control the quality of genomic DNA of samples from a wide variety of animals, a heminested PCR assay specifically targeting a nuclear gene has been developed. The histone H4 gene family comprises a small number of genes considered among the most conserved genes in living organisms. Tissue samples from necropsies and from cells belonging to 43 different species were studied, eight samples from invertebrates and 35 samples from vertebrates covering all classes. Ancient DNA samples from three Siberian woolly mammoths (Mammuthus primigenius) dating between 40,000 and 49,000 years before present were also tested for PCR amplification. Performance of HIST2H4 amplification were also compared with those of previously published universal PCRs (28S rRNA, 18S rRNA, and cytochrome b). Overall, 95% of species studied yielded an amplification product, including some old samples from gorilla and chimpanzees. The data indicate that the HIST2H4 amplimers are, thus, suitable for both DNA quality testing as well as species identification in the animal kingdom.     

Direct profiling of the yeast dynamics in wine fermentations

We present a method to directly characterize the yeast diversity present in wine fermentations by employing denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR)-amplified 26S ribosomal RNA (rRNA) genes. PCR-DGGE of a portion of the 26S rRNA gene was shown to distinguish most yeast genera associated with the production of wine. With this method the microbial dynamics in several model wine fermentations were profiled. PCR-DGGE provided a qualitative assessment of the yeast diversity in these fermentations accurately identifying populations as low as 1000 cells ml(-1). PCR-DGGE represents an attractive alternative to traditional plating schemes for analysis of the microbial successions inherent in the fermentation of wine.     

Selective and sensitive method for PCR amplification of Escherichia coli 16S rRNA genes in soil

A set of PCR primers targeting 16S rRNA gene sequences was designed, and PCR parameters were optimized to develop a robust and reliable protocol for selective amplification of Escherichia coli 16S rRNA genes. The method was capable of discriminating E. coli from other enteric bacteria, including its closest relative, Shigella. Selective amplification of E. coli occurred only when the annealing temperature in the PCR was elevated to 72 degrees C, which is 10 degrees C higher than the optimum for the primers. Sensitivity was retained by modifying the length of steps in the PCR, by increasing the number of cycles, and most importantly by optimizing the MgCl(2) concentration. The PCR protocol developed can be completed in less then 2 h and, by using Southern hybridization, has a detection limit of ca. 10 genomic equivalents per reaction. The method was demonstrated to be effective for detecting E. coli DNA in heterogeneous DNA samples, such as those extracted from soil.     

Identification and analysis of PCB dechlorinating anaerobic enrichments by amplification: accuracy of community structure based on restriction analysis and partial sequencing of 16S rRNA genes

The composition of polychlorinated biphenyl (PCB) dechlorinating mixed communities was analysed by restriction fragment length polymorphism of PCR amplified rDNAs (ARDRA) and partial sequencing of 16S rRNA genes amplified from PCB degrading enrichments. Restriction analysis confirms that the 16S rRNA genes amplified from PCB dechlorinating communities vary depending on the PCB congener dechlorinated. Comparison of 16S rRNA sequences to published ribosomal databases indicates that the two most abundant Operational Taxonomic Units (OTUs) appear to be species of the genus Clostridium . The amount that the amplification procedure contributed to this result was determined by varying the amplification procedure and by creating an artificial template mixture. Varying the amount of template by sixfold in the amplification did not affect the distribution of OTUs but the number of OTUs observed decreased with decreasing template concentration. Comparison of products amplified from mixtures of 16S rDNA clones indicates that the more abundant Clostridium OTU did not amplify more efficiently than those of less abundant OTUs. Hybridization to a probe designed to detect the most abundant OTUs indicates that two other OTUs are closely related to this Clostridium species.