Detection of human papillomaviruses by polymerase chain reaction and ligation reaction on universal microarray

Sensitive and specific detection of human papillomaviruses (HPV) in cervical samples is a useful tool for the early diagnosis of epithelial neoplasia and anogenital lesions. Recent studies support the feasibility of HPV DNA testing instead of cytology (Pap smear) as a primary test in population screening for cervical cancer. This is likely to be an option in the near future in many countries, and it would increase the efficiency of screening for cervical abnormalities. We present here a microarray test for the detection and typing of 15 most important high-risk HPV types and two low risk types. The method is based on type specific multiplex PCR amplification of the L1 viral genomic region followed by ligation detection reaction where two specific ssDNA probes, one containing a fluorescent label and the other a flanking ZipCode sequence, are joined by enzymatic ligation in the presence of the correct HPV PCR product. Human beta-globin is amplified in the same reaction to control for sample quality and adequacy. The genotyping capacity of our approach was evaluated against Linear Array test using cervical samples collected in transport medium. Altogether 14 out of 15 valid samples (93%) gave concordant results between our test and Linear Array. One sample was HPV56 positive in our test and high-risk positive in Hybrid Capture 2 but remained negative in Linear Array. The preliminary results suggest that our test has accurate multiple HPV genotyping capability with the additional advantages of generic detection format, and potential for high-throughput screening.     

Development of a 16S rRNA gene-based prototype microarray for the detection of selected actinomycetes genera

Actinomycetes are known for their secondary metabolites, which have been successfully used as drugs in human and veterinary medicines. However, information on the distribution of this group of Gram-positive bacteria in diverse ecosystems and a comprehension of their activities in ecosystem processes are still scarce. We have developed a 16S rRNA-based taxonomic microarray that targets key actinomycetes at the genus level. In total, 113 actinomycete 16S rRNA probes, corresponding to 55 of the 202 described genera, were designed. The microarray accuracy was evaluated by comparing signal intensities with probe/target-weighted mismatch values and the Gibbs energy of the probe/target duplex formation by hybridizing 17 non-actinomycete and 29 actinomycete strains/clones with the probe set. The validation proved that the probe set was specific, with only 1.3% of false results. The incomplete coverage of actinomycetes by a genus-specific probe was caused by the limited number of 16S rRNA gene sequences in databases or insufficient 16S rRNA gene polymorphism. The microarray enabled discrimination between actinomycete communities from three forest soil samples collected at one site. Cloning and sequencing of 16S rRNA genes from one of the soil samples confirmed the microarray results. We propose that this newly constructed microarray will be a valuable tool for genus-level comparisons of actinomycete communities in various ecological conditions.     

基于16S rRNA基因测序分析微生物群落多样性

微生物群落多样性的研究对于挖掘微生物资源,探索微生物群落功能,阐明微生物群落与生境间的关系具有重要意义。随着宏基因组概念的提出以及测序技术的快速发展,16S rRNA基因测序在微生物群落多样性的研究中已被广泛应用。文中系统地介绍了16S rRNA基因测序分析流程中的四个重要环节,包括测序平台与扩增区的选择、测序数据预处理以及多样性分析方法,就其面临的问题与挑战进行了探讨并对未来的研究方向进行了展望,以期为微生物群落多样性相关研究提供参考。     

Polymerase chain reaction detection of bacterial 16S rRNA gene in human blood

Bacterial 16S ribosomal RNA genes (rDNA) were detected in blood samples from two healthy individuals by PCR under conditions involving 30 cycles that did not produce any visible products from negative control saline. Even from control samples, PCR involving 35-40 cycles yielded visible bands. Major clones detected in the blood samples, but not in control, were the Aquabacterium subgroup, Stenotrophomonas subgroup, Budvicia subgroup, Serratia subgroup, Bacillus subgroup and Flavobacteria subgroup. No clone was located within the bacteroides-clostridium-lactobacillus cluster, which is indigenous to gastrointestinal flora.     

Development and validation of a prototype 16S rRNA-based taxonomic microarray for Alphaproteobacteria

The microarray approach has been proposed for high throughput analysis of the microbial community by providing snapshots of the microbial diversity under different environmental conditions. For this purpose, a prototype of a 16S rRNA-based taxonomic microarray was developed and evaluated for assessing bacterial community diversity. The prototype microarray is composed of 122 probes that target bacteria at various taxonomic levels from phyla to species (mostly Alphaproteobacteria). The prototype microarray was first validated using bacteria in pure culture. Differences in the sequences of probes and potential target DNAs were quantified as weighted mismatches (WMM) in order to evaluate hybridization reliability. As a general feature, probes having a WMM > 2 with target DNA displayed only 2.8% false positives. The prototype microarray was subsequently tested with an environmental sample, which consisted of an Agrobacterium-related polymerase chain reaction amplicon from a maize rhizosphere bacterial community. Microarray results were compared to results obtained by cloning-sequencing with the same DNA. Microarray analysis enabled the detection of all 16S rRNA gene sequences found by cloning-sequencing. Sequences representing only 1.7% of the clone library were detected. In conclusion, this prototype 16S rRNA-based taxonomic microarray appears to be a promising tool for the analysis of Alphaproteobacteria in complex ecosystems.     

Development of a 60-mer oligonucleotide microarray on the basis of benzene monooxygenase gene diversity

We constructed a 60-mer oligonucleotide microarray on the basis of benzene monooxygenase gene diversity to develop a new technology for simultaneous detection of the functional gene diversity in environmental samples. The diversity of the monooxygenase genes associated with benzene degradation was characterized. A new polymerase chain reaction (PCR) primer set was designed using conserved regions of benzene monooxygenase gene (BO12 primer) and used for PCR-clone library analysis along with a previously designed RDEG primer which targeted the different types of benzene monooxygenase gene. We obtained 20 types of amino acid sequences with the BO12 primer and 40 with the RDEG primer. Phylogenetic analysis of the sequences obtained suggested the large diversity of the benzene monooxygenase genes. A total of 87 60-mer probes specific for each operational taxonomical unit were designed and spotted on a microarray. When genomic DNAs of single strains were used in microarray hybridization assays, corresponding sequences were successfully detected by the microarray without any false-negative signals. Hybridization with soil DNA samples showed that the microarray was able to detect sequences that were not detected in clone libraries. Constructed microarray can be a useful tool for characterizing monooxygenase gene diversity in benzene degradation. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Electrochemical detection of point mutation based on surface ligation reaction and biometallization

A highly sensitive electrochemical method for point mutation detection based on surface enzymatic ligation reaction and biometallization is demonstrated. In this method the surface-immobilized allele-specific probe, complementary to the mutant target, undergoes allele-specific ligation with the 5'-phosphorylated ligation probe in the presence of the mutant oligonucleotide target and E. coli DNA ligase. If there is an allele mismatch, no ligation takes place. After thermal treatment at 90 degrees C, the formed duplex melts apart, which merely allows the ligation product to remain on the electrode surface. Then, biotinylated detection probes hybridize with the ligation product. With the binding of streptavidin-alkaline phosphatase (SA-ALP) to the biotinylated probes, a non-reductive substrate of alkaline phosphatase, ascorbic acid 2-phosphate (AA-P), can be converted into ascorbic acid (AA) at the electrode surface. Silver ions in solution are then reduced by AA, resulting in the deposition of silver metal onto the electrode surface. Linear sweep voltammetry (LSV) is used to detect the amount of deposited silver. The proposed approach has been successfully implemented for the identification of single base mutation in codon 12 of K-ras oncogene target with a detection limit of 80fM, demonstrating that this method provides a highly specific, sensitive and cost-efficient approach for point mutation detection.     

Development of a 16S rRNA gene primer and PCR-restriction fragment length polymorphism method for rapid detection of members of the genus Megasphaera and species-level identification

The genus Megasphaera is relevant to the environment, human health and food, and renewable energy for the future. In this study, a primer set was designed for PCR-restriction fragment length polymorphism (RFLP) analyses to detect and identify the members of Megasphaera. Direct detection and identification were achieved for environmental samples and isolates.     

Multiplex polymerase chain reaction and ligation detection reaction/universal array technology for the traceability of genetically modified organisms in foods

A multiplex polymerase chain reaction (PCR) system was developed for the simultaneous detection of target sequences in genetically modified soybean (Roundup Ready) and maize (MON810, Bt176, Bt11, and GA21). Primer pairs were designed to amplify the junction regions of the transgenic constructs analyzed and the endogenous genes of soybean (lectin) and maize (zein) were included as internal control targets to assess the efficiency of all reactions. This multiplex PCR has constituted the basis for an efficient platform for genetically modified organism traceability based on microarray technology. In particular, the ligation detection reaction combined to a universal array approach, using the multiplex PCR as target, was applied. High specificity and sensitivity were obtained.     

Advantage of using inosine at the 3' termini of 16S rRNA gene universal primers for the study of microbial diversity

To overcome the shortcomings of universal 16S rRNA gene primers 8F and 907R when studying the diversity of complex microbial communities, the 3' termini of both primers were replaced with inosine. A comparison of the clone libraries derived using both primer sets showed seven bacterial phyla amplified by the altered primer set (8F-I/907R-I) whereas the original set amplified sequences belonging almost exclusively to Proteobacteria (95.8%). Sequences belonging to Firmicutes (42.6%) and Thermotogae (9.3%) were more abundant in a library obtained by using 8F-I/907R-I at a PCR annealing temperature of 54 degrees C, while Proteobacteria sequences were more frequent (62.7%) in a library obtained at 50 degrees C, somewhat resembling the result obtained using the original primer set. The increased diversity revealed by using primers 8F-I/907R-I confirms the usefulness of primers with inosine at the 3' termini in studying the microbial diversity of environmental samples.     

Sequence diversity of a fragment of the 16S RNA gene from Helicobacter pylori

Helicobacter pylori is one of the most common bacterial pathogens. It is the main cause of gastric and duodenal ulcers and has been associated with other diseases. The organism seems to be more genetically diverse than other bacterial pathogens, and the source of these differences awaits explanation. The sequence of a fragment of the 16S rRNA gene was determined for ten strains of H. pylori to examine the contribution of point mutation within a conserved gene. There were few differences between the sequences from the various strains and it was concluded that such differences were not the most important source of diversity.     

Genetic diversity of vaginal lactobacilli from women in different countries based on 16S rRNA gene sequences

AIMS: Lactobacilli are widely distributed in food and the environment, and some colonize the human body as commensal bacteria. The aim of this study was to determine the species of lactobacilli that colonize the vagina and compare them with those found in food and the environment. METHODS AND RESULTS: Thirty-five Lactobacillus strains from women from seven countries were isolated, and sequences from 16S rRNA genes were determined and compared with existing data in GenBank. A phylogenetic tree was achieved using the Neighbour-Joining method based on the analysis of 1465 nucleotides. The results showed that most vaginal isolates were L. crispatus, L. jensenii and L. gasseri. Some were L. vaginalis, L. fermentum, L. mucosae, L. paracasei and L. rhamnosus. Two isolates from a native American woman displayed distinct branches, indicating novel phylotypes. Few vaginal isolates matched food or environmental Lactobacillus species. CONCLUSIONS: Most women worldwide were colonized by three common Lactobacillus species: L. crispatus, L. jensenii and L. gasseri. SIGNIFICANCE AND IMPACT OF THE STUDY: Knowledge of vaginal Lactobacillus species richness and distribution in women worldwide may lead to the design of better probiotic products as bacterial replacement therapy.     

Development of a high-throughput DNA microarray for drug-resistant gene detection and its preliminary application

Most bacteria are resistant to a wide variety of antibiotics and other drugs, which decrease the effectiveness of clinical drug therapies. The present study developed a high-throughput DNA microarray for drug-resistant gene detection. A total of 115 specific oligonuclieotide probes with lengths of 42 nt to 45 nt and comparable Tm values were selected from 17 categories of drug-resistant genes in the National Center for Biotechnology Information database and were chemically synthesized. The entire bacterial DNA was extracted, randomly amplified, and labeled using Cy3-dCTP. The hybridization conditions of the microarray test were optimized to improve sensitivity and specificity. The drug-resistant genes were detected and genotyped using microarray analysis after hydration at 42°C for 4h with 2× hybridization solution. The microarray test sensitivity was 20ng/μL DNA. The performance of the microarray was validated using reference strains and clinical isolates. The results were consistent with direct DNA sequence analysis and drug susceptibility tests. The developed DNA microarray could be used to detect and screen drug-resistant bacteria rapidly and simultaneously. Thus, the present study could be helpful in effectively using antibiotics and controlling infectious diseases.     

Phylogeny of hagfish based on the mitochondrial 16S rRNA gene

The phylogenetic relationships among the species belonging to the family Myxinidae are still debatable. The mitochondrial DNA sequences from the large ribosomal RNA gene may be of great value for systematic and phylogenetic studies within families. Partial sequences of the 16S rRNA gene were obtained for comparisons among the following hagfish species, Paramyxine nelsoni, Paramyxine sheni, Paramyxine taiwanae, Paramyxine yangi, Paramyxine cheni, Eptatretus burgeri, Eptatretus stouii, Eptatretus cirrhatus, Myxine glutinosa, Myxine formosana, Myxine circifrons, Myxine sp1, and Myxine sp2. The boundary of four Paramyxine species (P. sheni, P. taiwanae, P. nelsoni, and P. yangi) from 16S rRNA sequences is ambiguous, however, they are valid based on our unpublished isozyme data as well as the gill aperture arrangement pattern. Both NJ and MP trees constructed from the present molecular data indicate that the genus Paramyxine is diphyletic and Eptatretus paraphyletic. The complexity of Eptatretus and Paramyxine in the clade would not be solved until the farther departed P. cheni is included to form a new clade under the genus Eptatretus. The other clade of Myxininae contains but single genus Myxine.     

Simultaneous analysis of seven 16S rRNA hypervariable gene regions increases efficiency in marine bacterial diversity detection

Environmental DNA sequencing is the gold standard to reveal microbial community structures. In most applications, a one-fragment PCR approach is applied to amplify a taxonomic marker gene, usually a hypervariable region of the 16S rRNA gene. We used a new reverse complement (RC)-PCR-based assay that amplifies seven out of the nine hypervariable regions of the 16S rRNA gene, to interrogate bacterial communities in sediment samples collected from different coastal marine sites with an impact gradient. In parallel, we employed a traditional one-fragment analysis of the hypervariable V3–V4 region to investigate whether the RC-PCR reveals more of the ‘unseen’ diversity obtained by the one-fragment approach. As a benchmark for the full deck of diversity, we subjected the samples to PCR-free metagenomic sequencing. None of the two PCR-based approaches recorded the full taxonomic repertoire obtained from the metagenomics datasets. However, the RC-PCR approach detected 2.8 times more bacterial genera compared to the near-saturation sequenced V3–V4 samples. RC-PCR is an ideal compromise between the standard one-fragment approach and metagenomics sequencing and may guide future environmental sequencing studies, in which bacterial diversity is a central subject.     

Development of a 16S rRNA primer for the detection of Brevibacterium spp

AIM: To develop a PCR method for the rapid identification of the genus Brevibacterium. METHODS AND RESULTS: Genus-specific primers were designed by aligning and comparing the 16S sequence of all Brevibacterium spp. with closely related genera. The primer set was tested with all validly described Brevibacterium spp. and their closest neighbours. SIGNIFICANCE: Until today brevibacteria could only be identified with laborious and time-consuming phenotypic characterization. The primer from this study offers a rapid alternative to the detection of Brevibacterium spp. Brevibacteria have been isolated from food, blood, ear discharge, from a wound and from an intravascular catheter.     

The 16S rRNA and mcrA gene based comparative diversity of methanogens in cattle fed on high fibre based diet

In the present study, the diversity of rumen methanogens in crossbred Karan Fries cattle was determined by constructing 16S rRNA and mcrA (methyl coenzyme-M reductase α subunit) gene libraries using specific primers. All thirteen OTUs or phylotypes from 16S rRNA library clustered with order Methanobacteriales, twelve of which aligned with Methanobrevibacter spp., whereas one OTU resemble with Methanosphaera stadtmanae. Out of eighteen OTUs identified from mcrA gene library, fifteen clustered with order Methanobacteriales, two resemble with Methanomicrobiales and remaining one grouped with Methanosarcinales. These results revealed that Methanobrevibacter phylotype was predominantly present in Karan Fries crossbred cattle fed on high fibrous diet containing wheat straw. Compared to 16S rRNA gene, mcrA gene OTUs clustered in three orders providing better insights of rumen methanogens diversity in cattle.     

应用16S rRNA基因文库技术分析土壤细菌群落的多样性

[目的]土壤微生物在菜田生态系统中具有重要的生态功能,通过16S rRNA基因克隆文库技术分析典型菜田土壤细菌群落结构的组成情况,为揭示典型的菜田土壤微生物的多样性以及土地利用变化与生态环境效应之间的关系奠定基础.[方法]采用未培养技术直接从北京和山东两地典型菜田土壤样品中提取微生物总的DNA,分别构建基于通用引物PCR扩增的土壤细菌16S rRNA基因克隆文库,通过Hinf Ⅰ和Hae Ⅲ限制性内切酶对两地土壤细菌16s rRNA基因文库中的克隆进行ARDRA(Amplified Ribosomal DNA Rstriction Analysis)分析,将所有阳性克隆分为若干个可操作分类单元(OTU).[目的]通过构建两地细菌克隆文库的系统发育树,并分析主要种群的组成表明:北京和山东菜田土壤细菌克隆文库的优势种群均为γ、β、α变形细菌亚群.两地的细菌种类组成分别包括124个OTUs和92个OTUs.[结论]北京地区和山东地区典型蔬菜地土壤细菌种群中优势种群均为变形细菌,但是土壤细菌多样性降低,这可能与典型菜田的多年连作,种植蔬菜种类单一直接相关.同时,也可能是造成菜田土壤病害普遍发生,土壤退化的一个重要原因.     

Assessing the genetic diversity and population structure of Culter alburnus in China based on mitochondrial 16S rRNA and COI gene sequences

The genetic diversity and population genetic structure of Culter alburnus were investigated using mitochondrial cytochrome c oxidase subunit I (COI) and ribosomal 16S subunit (16S rRNA) gene sequences. A total of 89 individuals from four localities were included in the analysis. Overall, 12 polymorphic sites were observed and 10 haplotypes were defined. The C. alburnus populations were characterized by high haplotype diversity (0.587 ± 0.047) and low nucleotide diversity (0.00197 ± 0.00073). Pairwise fixation index (F_ST) analysis indicated significant genetic differentiation among different populations. The hierarchical analysis of molecular variance (AMOVA) analysis also showed significant genetic divergence (φ_ST = 0.3792, P < 0.01) among these populations. The present results suggest that subdivisions exist among four C. alburnus populations, and should be considered as different management unit for effective conservation and management purposes. This study provides new information for genetic assessment and will be crucial for establishing fisheries management and strategies for this species.