TaqMan-MGB探针实时荧光定量PCR联合检测难辨梭菌菌属基因及毒素基因方法学的建立

目的：建立一种简便、快速难辨梭菌菌属鉴定及其毒素基因筛查的荧光定量PCR检测方法。方法采用TaqMan-MGB探针，通过real-time PCR分析系统，同时检测难辨梭菌菌属基因磷酸丙糖异构酶（Tpi）、A毒素（TcdA）、B毒素（TcdB）及缺失部分基因的A毒素（TcdAT），从特异度、灵敏度及其抗干扰性等方面评价该方法，并联合全自动酶联荧光免疫系统（VIDAS）检测50例临床不明原因腹泻病例粪便标本探讨其应用价值。结果难辨梭菌非产毒株Tpi基因（tpi）的检测下限是6×10-2 CFU/μl，产毒株tpi、tcdA、tcdB、tcdAT的检测下限为6×10-1 CFU/μl；难辨梭菌非产毒株tpi检测下限批内、批间变异率分别为2.1%和2.3%；产毒株tpi、tcdA、tcdB、tcdAT的检测下限批内、批间变异率依次为3.0%和3.4%、2.9%和3.2%、5.3%和5.7%、2.7%和2.8%。临床常见分离菌株及梭菌属其他细菌对检测无干扰。50例不明原因腹泻病例粪便标本中，采用TaqMan-MGB探针实时荧光PCR与VIDAS酶标免疫检测39例阴性标本其符合率为100%；6例两方法检测均为阳性；3例VIDAS酶标免疫检测为可疑及2例为阴性，经TaqMan-MGB探针实时荧光PCR检测为A-/B＋菌株。结论建立的TaqMan-MGB探针实时荧光定量PCR具有高通量、高灵敏度和重复性好的特性，且可筛查携带缺失部分基因的TcdA难辨梭菌菌株。

沙门菌属stn基因LAMP快速检测方法的建立及应用

目的本研究以沙门菌属肠毒素基因(stn)为靶基因,运用环介导恒温扩增技术(LAMP),建立LAMP快速检测方法。方法对沙门菌属stn基因的6个特征区域设计4条引物,利用具有链置换活性的Bst DNA聚合酶在恒温条件下催化新链合成,使目的基因高效扩增;对LAMP反应体系和反应条件进行优化,用沙门菌标准株和非沙门菌标准株检测方法的特异性和敏感性。与显色培养基法同时检测肉、蛋、奶、等样品共计100份,检测方法的实用性。结果以沙门菌属stn基因为靶基因建立的LAMP快速检测方法,沙门菌株阳性检出率100%,非沙门菌株检测均为阴性。100份检验样品检测结果与显色培养基检测结果对比,一致率100%。结论沙门菌属stn基因LAMP检测技术敏感性、特异性高,简便快速,是一种适合口岸及基层检验、检疫部门使用的沙门菌快速检测方法。

糖多孢红霉菌bldD基因提高活跃链霉菌诺西肽产量的研究

目的将糖多孢红霉菌bldD基因转入活跃链霉菌(Streptomyces actuosus)N-H,探讨其对诺西肽产量的影响。方法将bldD基因连接到链霉菌整合型表达载体pZMW上构建pZMW-bldD质粒,利用PEG介导的原生质体转化方法将重组质粒pZMW-bldD及对照质粒pZMW-vgb分别导入活跃链霉菌,通过对枯草芽孢杆菌抑菌试验和分光光度计法测定发酵液中诺西肽产量。结果成功构建了活跃链霉菌N-H/pZMW-bldD工程菌株及对照菌株N-H/pZMW-vgb。与出发菌株N-H相比,N-H/pZMW-bldD菌株诺西肽产量提高了68%;与对照菌株N-H/pZMW-vgb相比,N-H/pZMW-bldD菌株诺西肽产量提高了19%。结论在活跃链霉菌中导入糖多孢红霉菌bldD基因可以提高诺西肽产量,其作用效果较vgb基因更为明显,说明bldD基因对抗生素产量的提高具有广谱性。

Cloning and Efficient Expression of Bacillus sp. BH072 tas A Gene in Escherichia coli

The Bacillus strain BH072 isolated from a honey sample showed strong antifungal activity against phytopathogen. Gene cloning test demonstrated that the strain had a tasA gene encoding an antifungal TasA protein. Although the wild strain simultaneously produced various antifungal substances, only the physicochemical property and antifungal activity of TasA protein were unclear due to the difficulty in extraction. In this study, tasA gene encoding the protein from Bacillus sp. BH072 was amplified by using the polymerase chain reaction （PCR） method and cloned into pET 28a （＋） vector, and then expressed in host cells Escherichia coli BL21 （DE3）. The expressed proteins were collected by centrifugation and ultrasonic treatment, and then purified by using nickel-nitrilotriacetic acid （Ni-NTA） metal affinity column and dialysis methods. The result of sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE） test showed that an expected protein band appeared with a size of 31 kDa. The expressed products possessed antifungal activity against the phytopathogenic indicator strain Botrytis cinerea. A genetically engineered strain tasA orE, coli was established in this study which can efficiently express Tas A protein.

Comparison of three PCR methods for detection of Helicobacter pylori DNA and detection of cagA gene in gastric biopsy specimens

AIM:To comparatively evaluate PCR and other diagnostic methods (the rapid urease test and/or culture) in order to determine which of the three PCR methods (ureA,glmM and 26-kDa,SSA gene) was most appropriate in the diagnosis of Helicobacterpylori(Hpylori) infection and also to evaluate the detection of a putative virulence marker of H pylori,the cage,gene,by PCR in biopsy specimens. METHODS:One hundred and eighty-nine biopsy specimens were collected from 63 patients (three biopsies each) undergoing upper gastroduodenal endoscopy for various dyspeptic symptoms.The PCR methods used to detect H pylori DNA directly from biopsies were the glmM,26-kDa, ureA and then cagA was used to compare the culture technique and CLO for urease with the culture technique being used as the gold standard. RESULTS:Thirty-five percent of the biopsies were positive for H pylori DNA using the 3 PCR methods,while 68% of these were positive for the cagA gene.Twenty-four percent of the biopsies were negative for H pylori DNA in all PCR methods screened.The remaining 41% were either positive for ureA gene only,glmM only,26-kDa only,or ureA+glmM, ureA+26-kDa,glmM+26-kDa.Out of the 35% positive biopsies,41% and 82% were positive by culture and CLO respectively,while all negative biopsies were also negative by culture and cagA.Cag A+ infection was also predominantly found in H pylori DNA of the biopsies irrespective of the clinical diagnosis. CONCLUSION:This method is useful for correctly identifying infections caused by H pylori and can be easily applied in our laboratory for diagnostic purposes.

Functional genomic approach to the study of biodiversitywithin Trichoderma

Trichoderma is a fungal genus of great and demonstrable biotechnological value, but its genome is poorly surveyed compared with other model microorganisms. Due to their ubiquity and rapid substrate colonization, Trichoderma species have been widely used as biocontrol organisms for agriculture, and their enzyme systems are widely used in industry. Therefore, there is a clear interest to explore beyond the phenotype to exploit the underlying genetic systems using functional genomics tools. The great diversity of species within the Trichoderma genus, the absence of optimized systems for its exploration, and the great variety of genes expressed under a wide range of ambient conditions are the main challenges to consider when starting a comprehensive functional genomics study. An initial project started by three Spanish groups has been extended into the project TRICHOEST, funded by the EU (FP5, QLRT-2001-02032) to target the transcriptome analysis of selected Trichoderma strains with biocontrol potential, in conditions related to antagonism, nutrient stress and plant interactions. Once specific conditions were defined, cDNA libraries were produced and used for EST sequencing. Nine strains from seven Trichoderma species have been considered in this study and an important amount of gene sequence data has been generated, analyzed and used to compare the gene expression in different strains. In parallel to sequencing, genomic expression studies were carried out by means of macro-arrays to identify genes expressed in specific conditions. In silico analysis of DNA sequencing data together with macro-array expression results have lead to a selection based on the potential use of the gene sequences. The selected clone sequences were completed and cloned in appropriate vectors to initiate functional analysis by means of expression studies in homologous and heterologous systems.

Regulatory puzzle of xyn1 gene (xylanase1) expression in Trichoderma reesei

Xylanase 1 (Xyn1) is one of the two major representatives of the xylanase system of T. reesei; the mechanisms governing its expression were analysed throughout this study. All factors and regulatory motifs responsible for transcriptional regulation and the model of their interplay in induction and repression will be presented. Using in vivo foot printing analysis of xylan-induced and glucose repressed mycelia, we detected three adjacent nucleotide sequences contacted by DNA-binding proteins. Protection within the inverted repeat of the Cre1 (SYGGRG) consensus sequence on the non coding strand under repressing conditions is in perfect agreement with the previously reported Cre1 dependent glucose repression of xyn1. Constitutive protein binding could be observed to a CCAAT-box and an inverted repeat of a 5′ GGCTAA 3′ sequence. EMSA with crude extracts from induced and repressed mycelia revealed that the latter motifs are sufficient for formation of the basal transcriptional complex under all conditions. The inverted repeat of GGCTAA closely resembles the consensus sequences of the cellulase and xylanase regulators Ace1, Ace2 and, Xyr1 (encoded by xyr1, cloned and characterised in this study) EMSA with heterologously expressed components of each factor and of the T. reesei Hap2/3/5 protein complex revealed that the basal transcriptional complex is formed by Xyr1 and the Hap2/3/5. Additionally to the Cre1 mediated carbon catabolite repression a yet unknown mechanism antagonizing induction of xyn1 expression could be elucidated. Latter occurs through competition of the repressor Ace1 and Xyr1 for the GGCTAA motif. In vivo proof for the relevance of identified motifs could be given through analysis of T. reesei transformants containing correspondingly mutated versions of the xyn1 promoter fused to the A. niger goxA gene. The results indicated that the basal as well as the induction level of xyn1 gene transcription is dependent on an interaction of Xyr1 with the GGCTAA motif while formation of the CCAAT-Hap2/3/5 complex slightly reduces induction. It can be concluded that mutations impairing protein binding in vitro lead to a loss of distinct regulatory functions in xyn1 gene expression in vivo. A respective model of gene regulation will be presented.

Neither gastric topological distribution nor principle virulence genes of Helicobacter pylori contributes to clinical outcomes

AIM: Studies on Helicobacter pylori (H pylon) and gastroduodenal diseases have focused mainly on the distal sites of the stomach, but relationship with the gastric cardia is lacking. The aim of this study is to determine if the gastric topology and genotypic distribution of Hpyloriwere associated with different upper gastrointestinal pathologies in a multiethnic Asian population. METHODS: Gastric biopsies from the cardia, body/corpus and antrum were endoscoped from a total of 155 patients with dyspepsia and/or reflux symptoms, with informed consent. H pylori isolates obtained were tested for the presence of 26kDa, ureC, cagA, vacA, iceA1, iceA2 and babA2 genes using PCR while DNA fingerprints were generated using random amplification polymorphic DNA (RAPD). RESULTS: Hpyloriwas present in 51/155 (33%) of patients studied. Of these, 16, 15 and 20 were isolated from patients with peptic ulcer diseases, gastroesophageal reflux diseases and non-ulcer dyspepsia, respectively. Of the Hpyloripositive patients, 75% (38/51) had Hpyloriin all three gastric sites. The prevalence of various genes in the H pylori isolates was shown to be similar irrespective of their colonization sites as well as among the same site of different patients. The RAPD profiles of H pylori isolates from different gastric sites were highly similar among intra-patients but varied greatly between different patients. CONCLUSION: Topographic colonization of H pylori and the virulence genes harboured by these isolates have no direct bearing to the clinical state of the patients. In multiethnic Singapore, the stomach of each patient is colonized by a predominant strain of H pylori，irrespective of the clinical diagnosis.

Identification and characterization of the Vibrio anguillarum prtV gene encoding a new metalloprotease

We cloned and sequenced a prtV-like gene from Vibrio anguillarum M3 strain.This prtV gene encodes a putative protein of 918 amino acids,and is highly homologous to the V.cholerae prtV gene.We found that a prtV insertion mutant strain displayed lower gelatinase activity on gelatin agar,lower protease activity against azocasein,and lower activity for four glycosidases.This prtV mutant strain also had increased activity for two esterases in its extracellular products,as analyzed by the API ZYM system.In addition,the prtV mutant strain exhibited decreased growth in turbot intestinal mucus and reduced hemolytic activity on turbot erythrocytes.Infection experiments showed that the LD50 of the prtV mutant strain increased by at least 1 log compared to the wild-type in turbot fish.We propose that prtV plays an important role in the pathogenesis of V.anguillarum.

Numerical Classification of Brevibacterium and Related Genera Using Linocin M18 Bacteriocin

Fifty bacterial strains isolated from dairy product, skin and blood from cancer and kidney failure dialysis patients were identified to 22 species and the following genera： Brevibacterium, Corynebacterium, Arthrobacter, Actinomyces, Exiguobacterium, Kocuria, Micrococcus, Rothia, Rhodococcus and classified numerically using a set of 52 phenetic characteristics, using simple matching coefficient （Ssm） and clustering method of unweighted average linkage between groups by SPSS program. They were also grouped to 7 main clusters and 29 sub-clusters in the hierarchical tree. Twelve isolates of the different species from the genera Brevibacterium, Arthrobacter, Corynebacterium, Kocuria, Rhodococcus, Rothia were selected from the taxonomic clusters and probed for lin gene by peR. One species Kocuria rhizophila which inhibited most of the test organism did not have lin gene in the chromosome while the species Corynebacterium glucuronolyticum, Arthrobacter comminsii, Arthrobacter oxydans have the lin gene. Our results establish a wide distribution of the structural gene encoding this Iinocin M 18 within coryneform bacteria and also in the genus Kocuria.

DNA barcoding of the fungal genus Neonectria and the discovery of two new species

To determine a suitable DNA barcode for the genus Neonectria,the internal transcribed spacer rDNA,β-tubulin,EF-1α,and RPB2 genes were selected as candidate markers.A total of 205 sequences from 19 species of the genus were analyzed.Intra-and inter-specific divergences and the ease of nucleotide sequence acquisition were treated as criteria to evaluate the feasibility of a DNA barcode.Our results indicated that any single gene among the candidate markers failed to serve as a successful barcode,while the combination of the partial EF-1α and RPB2 genes recognized all species tested.We tentatively propose the combined partial EF-1α and RPB2 genes as a DNA barcode for the genus.During this study,two cryptic species were discovered,based on the combined data of morphology and DNA barcode information.We described and named these two new species N.ditissimopsis and N.microconidia.