Diversity of Crenarchaeota in terrestrial hot springs in Tengchong, China

Diversity of Crenarchaeota was investigated in eight terrestrial hot springs (pH 2.8–7.7; temperature 44–96°C) located in Tengchong, China, using 16S rRNA gene phylogenetic analysis. A total of 826 crenarchaeotal clones were sequenced and a total of 47 operational taxonomic units (OTUs) were identified. Most (93%) of the identified OTUs were closely related (89–99%) to those retrieved from hot springs and other thermal environments. Our data showed that temperature may predominate over pH in affecting crenarchaeotal diversity in Tengchong hot springs. Crenarchaeotal diversity in moderate-temperature (59–77°C) hot springs was the highest, indicating that the moderately hot-temperature springs may provide optimal conditions for speciation of Crenarchaeota.     

Diversity and abundance of nematode-trapping fungi from decaying litter in terrestrial, freshwater and mangrove habitats

Nematode-trapping fungi are ubiquitous in terrestrial habitats in dung, soils, litter and woody debris and they also occur in freshwater, but only one species has been found in marine habitats. The purpose of this study was therefore to investigate whether nematode-trapping fungi occurred in mangrove habitats. To achieve this we assessed the diversity of nematode-trapping fungi on decaying litter from mangroves, freshwater and terrestrial habitats (22 sites) in Hong Kong. Composite samples (n = 1,320) of decaying litter (wood and leaves) were examined and a total of 31 species of nematode-trapping fungi belonging to four genera, Arthrobotrys, Monacrosporium, and Dactylella were recorded. Twenty-nine species reported in this study are new records for Hong Kong and 16 species are new records from mangrove habitats worldwide. Nematode trapping fungi are therefore present in marine environments. Commonly encountered taxa were Arthrobotrys oligospora and Monacrosporium thaumasium which are abundant in all habitats. A. oligospora, M. thaumasium and Arthrobotrys musiformis were frequent (F > 10%). Twenty-six species were rare (0.16–9.32%). Species richness and diversity was higher in terrestrial than in freshwater and mangrove habitats (ANOVA, P < 0.001). A higher mean diversity was observed on decaying leaves as compared to decaying wood in all habitats (P < 0.001). Based on Shannon diversity index, it was also observed that taxa characterized by adhesive nets were more frequent in all habitats. This can be explained by the fact that these taxa may have a better competitive saprotrophic ability which would allow them to compete favourably in nutrient limited environments. Abiotic factors that could be linked to differences in species diversity between decaying wood and leaves are also discussed.     

俄罗斯堪察加地区热泉及其周边生境的泉古菌多样性

【目的】泉古菌为陆地热泉系统的主要古菌类群,可能在自然界生源元素的地球化学循环中发挥着重要作用。本研究旨在揭示俄罗斯堪察加地区热泉以及热泉周边区域的泉古菌多样性,同时基于之前已获得的我国云南地区热泉数据,比较两地区泉古菌群落差异。【方法】通过构建16S rRNA基因片段克隆文库获得序列信息和丰度,随后进行物种多样性、系统发育和群落结构差异分析。【结果】高温热泉Burlyashi Liza(BSL,89℃)中的泉古菌全部属于热变形菌纲(Thermoprotei)内的物种。中温热泉TF Vent 2(TFV,49℃)的群落结构主要由不确定的热变形菌纲类群、不确定的泉古菌类群、高温水环境泉古菌类群Ⅱ(HWCG-Ⅱ)和奇古菌下的Group1.1b类群组成。热泉周边常温环境的主要物种与热泉环境的代表性克隆pJP41一起聚成一个较大的遗传分枝。Jackknife聚类树和主坐标分析(Principal coordinates analysis,PCoA)的结果显示:温度相似的样点,其泉古菌群落结构相对来说更为相似。【结论】俄罗斯堪察加地区与我国云南地区热泉中的泉古菌存在着一定程度上的不同。陆地热泉系统影响着其周边环境的泉古菌类群。热泉中泉古菌群落结构受温度的明显影响。     

A new real time PCR (TaqMan PCR) system for detection of the16S rDNA gene associated with fecal bacteria

A recent PCR detection technique (TaqMan) based on a 3'-Minor Groove Binder (MGB) probe was applied to the detection of fecal-dominant bacteria to assess fecal contamination in environmental samples. Primers and probes used bacterial 16S ribosomal DNA (16S rDNA) as a gene marker and accurately defined with specificity a cluster of phylotypes within the Gram-positive low GC division. This cluster of phylotypes, called Fec1, corresponds to around 5% of human fecal microflora. Fec1 clustered 16S rDNA and strains (Eubacterium rectale) of fecal origin. A range of samples made up of feces and intestinal samples from humans and animals tested positive whereas other microbial ecosystems (soils, laboratory reactor, subsurface water) were negative. In order to circumvent problems related to DNA extraction efficiency, quantitative results took the form of the ratio between Fec1 16S rDNA and total bacterial 16S rDNA. The threshold of detection, defined as the ratio between Fec1 and total 16S rDNA, was measured as 0.006%.     

RNA microarray for estimating relative abundance of 16S rRNA in microbial communities

We developed an RNA microarray protocol in which total RNA from a microbial community was attached to a slide glass, and rRNA was detected by fluorescently labeled oligonucleotide probes. The RNA microarray requires only 4 h for hybridization and enables double staining and estimating relative abundance of rRNA.     

Detection of Leishmania infantum in animals and their ectoparasites by conventional PCR and real time PCR

Visceral leishmaniosis (VL) is a parasitic disease caused by Leishmania infantum, which is primarily transmitted by phlebotomine sandflies. However, there has been much speculation on the role of other arthropods in the transmission of VL. Thus, the aim of this study was to assess the presence of L. infantum in cats, dogs and their ectoparasites in a VL-endemic area in northeastern Brazil. DNA was extracted from blood samples and ectoparasites, tested by conventional PCR (cPCR) and quantitative real time PCR (qPCR) targeting the L. infantum kinetoplast DNA. A total of 280 blood samples (from five cats and 275 dogs) and 117 ectoparasites from dogs were collected. Animals were apparently healthy and not previously tested by serological or molecular diagnostic methods. Overall, 213 (76.1 %) animals and 51 (43.6 %) ectoparasites were positive to L. infantum, with mean parasite loads of 795.2, 31.9 and 9.1 fg in dogs, cats and ectoparasites, respectively. Concerning the positivity between dogs and their ectoparasites, 32 (15.3 %) positive dogs were parasitized by positive ectoparasites. The overall concordance between the PCR protocols used was 59.2 %, with qPCR being more efficient than cPCR; 34.1 % of all positive samples were exclusively positive by qPCR. The high number of positive animals and ectoparasites also indicates that they could serve as sentinels or indicators of the circulation of L. infantum in risk areas.     

Diversity and abundance of polyadenylated RNA from Achlya ambisexualis

The diversity, abundance, and DNA sequence representation of poly(adenylic acid) containing RNA derived from cells of Achlya ambisexualis cultured in defined and undefined media have been determined. The kinetics of hybridization of polyadenylated RNA with complementary DNA were the same for both culture conditions and revealed the presence of three frequency classes containing 29, 220, and 3000 different sequences of an average length of 1150 nucleotides. Complexity estimates derived from experiments in which polyadenylated RNA was hybridized to unique sequence DNA were in good agreement with these results. The kinetics of hybridization of complementary DNA with an excess of nuclear DNA indicate that approximately 10% of the RNA is transcribed from reiterated DNA sequences while the remainder is transcribed from single copy sequences.     

Diversity in methane enrichments from agricultural soil revealed by DGGE separation of PCR amplified 16S rDNA fragments

Denaturing gradient gel electrophoresis (DGGE) profiles of PCR amplified V3 regions of 16S rRNA genes were used to assess the diversity in enrichment cultures with methane as the only carbon and energy source. The enrichments originated from two agricultural soils. One was a sandy soil with low (10%) organic content, the other an organic soil with approximately 50% organic content. DGGE provided a fast evaluation of the distribution of amplifiable sequence types indicating that specific bacterial populations had been enriched from each soil. The DGGE profiles revealed a broader range of amplified V3 fragments in the community derived from organic soil than from sandy soil. Fragments from 19 individual DGGE bands were sequenced and compared with 27 previously published 16S rRNA gene sequences. The sequences confirmed the high diversity with the presence of different methylotrophic populations in each enrichment. No affiliation was found with type I methanotrophs, instead type II methanotroph sequences were found in the enrichments from both soil types. Some of the fragments from the organic soil enrichment were not affiliated with methylotrophs. Most of the sequences clustered distantly on a branch within the α-Proteobacteria. These facts suggested that previously undescribed methylotrophs are abundant in methane enrichments from agricultural soil.     

Overestimation of the abundance of sulfate-reducing bacteria in human feces by quantitative PCR targeting the Desulfovibrio 16S rRNA gene

The dominant genus of sulfate-reducing bacteria (SRB) in humans is Desulfovibrio, and quantitative PCR (QPCR) targeting the 16S rRNA gene is often used in assays. We show that the 16S rRNA gene assay overestimated SRB abundance in feces from 24 adults compared to QPCR assays using primers targeting two genes involved in SRB energy metabolism.     

Archaeal diversity in a terrestrial acidic spring field revealed by a novel PCR primer targeting archaeal 16S rRNA genes

The phylogenetic diversity of archaeal 16S rRNA genes in a thermoacidic spring field of Ohwakudani, Hakone, Japan, was investigated by PCR-based analysis using a novel Archaea-specific primer designed in the present study. Clone libraries of archaeal 16S rRNA genes were constructed from hot water (78 °C) and mud (28 °C) samples by PCR using a newly designed forward primer and a previously reported forward primer with reverse primers. Most phylotypes found in the libraries from the hot water sample were related to cultured (hyper)thermophiles. The phylotypes and their detection frequencies from the hot water sample were similar for the libraries amplified with the two different primer sets. In contrast, phylotypes having a low similarity (<95%) to cultured Archaea were found in the libraries from the mud sample. Some of the phylotypes were relatively close to members of Thermoplasmata (80-93% similarity) and the others were not clearly affiliated with Crenarchaeota and Euryarchaeota, but related to Thaumarchaeota and Korarchaeota. The phylotypes and their detection frequencies were significantly different between the two libraries of the mud sample. Our results from the PCR-based analysis using the redesigned primer suggest that more diverse, uncultured Archaea are present in acidic environments at a low temperature than previously recognized.     

Detection of pathogenic Vibrio parahaemolyticus in oyster enrichments by real time PCR

A real time polymerase chain reaction (PCR) assay was developed and evaluated to detect the presence of the thermostable direct hemolysin gene (tdh), a current marker of pathogenicity in Vibrio parahaemolyticus. The real time PCR fluorogenic probe and primer set was tested against a panel of numerous strains from 13 different bacterial species. Only V. parahaemolyticus strains possessing the tdh gene generated a fluorescent signal, and no cross-reaction was observed with tdh negative Vibrio or non-Vibrio spp. The assay detected a single colony forming unit (CFU) per reaction of a pure culture template. This sensitivity was achieved when the same template amount per reaction was tested in the presence of 2.5 microl of a tdh negative oyster:APW enrichment (oyster homogenate enriched in alkaline peptone water overnight at 35 degrees C). This real time technique was used to test 131 oyster:APW enrichments from an environmental survey of Alabama oysters collected between March 1999 and September 2000. The results were compared to those previously obtained using a streak plate procedure for culture isolation from the oyster:APW enrichment combined with use of a non-radioactive DNA probe for detection of the tdh gene. Real time PCR detected tdh in 61 samples, whereas the streak plate/probe method detected tdh in 15 samples. Only 24 h was required for detection of pathogenic V. parahaemolyticus in oyster:APW enrichments by real time PCR, whereas the streak plate/probe method required 3 days and was more resource intensive. This study demonstrated that real time PCR is a rapid and reliable technique for detecting V. parahaemolyticus possessing the tdh gene in pure cultures and in oyster enrichments.     

Diversity and geographical distribution of members of the Streptomyces violaceusniger 16S rRNA gene clade detected by clade-specific PCR primers

The Streptomyces violaceusniger 16S rRNA gene clade contains organisms that are of ecological interest and a rich source of novel bioactive metabolites. Improvements in the classification of members of the S. violaceusniger clade made it possible to design, evaluate and use an oligonucleotide primer set to gain an insight into the presence, distribution and taxonomic diversity of members of this taxon in environmental samples. In silico testing showed that the primers had a perfect match with representatives of the S. violaceusniger clade. The primers, designated S-S-Svio-66-a-S-20 and S-S-Svio-1274-a-A-20, amplified an approximately 1190-bp stretch of 16S rRNA gene from authenticated members of the S. violaceusniger clade, but not from representatives of other actinomycete taxa. Following amplification of DNA extracted from sediment and soil samples, the sequences of cloned PCR products confirmed the specific amplification of target sequences in 87% of the clones; the use of 16S rRNA gene fragment similarity correlations indicated that the clones represented new species. The primers can be used to facilitate the isolation of novel members of the S. violaceusniger 16S rRNA gene clade by allowing prescreening of environmental samples and the subsequent detection and retrieval of targetted strains through the use of selective isolation procedures.     

PCR and real time PCR for the detection of Cryptosporidium parvum oocyst DNA

Three DNA extraction kits were used, all without preliminary procedures, then DNA extraction was preceded with freeze/thaw cycles in three versions. A lack of desired effect resulted in the application of liquid nitrogen/water bath cycles before the use of the extractions in further experiments. The effectiveness of DNA extraction was measured by PCR signal and C(T) values of real time PCR. A comparison of the efficiency of various Cryptosporidium parvum undiluted oocyst treatments prior to DNA extraction with the use of three kits has shown that the best results were obtained after extraction of DNA with the QIAamp DNA Tissue Mini Kit (T kit), preceded by triple liquid nitrogen/water bath in 100 degrees C for 2 minutes and with overnight proteinase K digestion. After extraction with the T kit, the detection limit was 50 oocysts per 200 microl when effectiveness was evaluated with PCR and 10 oocysts in the case of real time PCR.     

腐皮镰刀菌SYBR Green实时荧光定量PCR快速检测方法的建立

目的建立一种能够快速、灵敏、特异的鉴定腐皮镰刀菌的SYBR Green实时荧光定量PCR。方法运用SYBR Green实时荧光定量PCR反应体系检测腐皮镰刀菌,并对此方法的特异性、灵敏度和稳定性进行评价。结果通过对45例样品的检测,结果显示SYBR Green实时荧光定量PCR特异性好,其检出率高于普通PCR;灵敏度高,对重组质粒标准品的检测灵敏度为1.0×10~2copies/μL;稳定性好,对质粒为1.0×10~7copies/μL、1.0×10~5copies/μL、1.0×10~3copies/μL的标准品重复检测10次,结果显示扩增反应Ct值的变异系数为0.96%~1.68%。结论SYBR Green实时荧光定量PCR检测腐皮镰刀菌,不仅特异性好,灵敏度高,稳定性好,而且简便、快速、易操作。