通用PCR反向斑点杂交快速检测及鉴定致病性酵母菌

建立PCR结合寡核苷酸探针反向斑点杂交技术,快速检测及鉴定致病性酵母菌.将待检酵母菌种特异性寡核苷酸探针固定在尼龙膜上,然后用生物素标记的真菌通用引物扩增的各真菌DNA片段,与固定在膜上的探针杂交.结果表明所用的真菌通用引物可扩增临床常见的真菌DNA,9种特异性探针具有高度的特异性.该方法检测35例临床分离菌株,结果与常规鉴定方法一致.该技术检测时间短、操作简单、不需要特殊设备,能部分满足临床检测的通量要求.具有很好的临床应用前景.     

Rapid Screening for Freshwater Bacterial Groups by Using Reverse Line Blot Hybridization

The identification of phylogenetic clusters of bacteria that are common in freshwater has provided a basis for probe design to target important freshwater groups. We present a set of 16S ribosomal RNA gene-based oligonucleotide probes specific for 15 of these freshwater clusters. The probes were applied in reverse line blot hybridization, a simple method that enables the rapid screening of PCR products from many samples against an array of probes. The optimized assay was made stringent to discriminate at approximately the single-mismatch level. This made 10 of the probes highly specific, with at least two mismatches to the closest noncluster member in the global database. Screening of PCR products from bacterioplankton of 81 diverse lakes from Belgium, The Netherlands, Denmark, Sweden, and Norway showed that the respective probes were reactive against 5 to 100% of the lake samples. Positive reactivity of six highly specific probes showed that bacteria from actinobacterial clusters ACK-M1 and Sta2-30 and from verrucomicrobial cluster CLO-14 occurred in at least 90% of the investigated lakes. Furthermore, bacteria from alpha-proteobacterial cluster LD12 (closely related to the marine SAR11 cluster), beta-proteobacterial cluster LD28 and cyanobacterial cluster Synechococcus 6b occurred in more than 70% of the lakes. Reverse line blot hybridization is a new tool in microbial ecology that will facilitate research on distribution and habitat specificity of target species at relatively low costs.     

Reverse Line Blot Macroarray for Simultaneous Detection and Characterization of Four Biological Warfare Agents

The need for a rapid detection and characterization of biowarfare (BW) agents cannot be over emphasized. With diverse array of potential BW pathogen available presently, rapid identification of the pathogen is crucial, so that specific therapy and control measures can be initiated. We have developed a multiplex polymerase chain reaction based reverse line blot macroarray to simultaneously detect four pathogens of BW importance viz. Bacillus anthracis, Yersinia pestis, Brucella melitensis and Burkholderia pseudomallei. The multiplex PCR utilizes 14 pairs of primers targeting 18 specific markers. These markers include genes which are genus specific, species-specific chromosomal sequences and virulence markers of plasmid origin. The assay was evaluated on various human, environment and animal isolates. The assay w successful in simultaneous detection and characterization of isolates of the four pathogens on as a single platform with sensitivity ranging from 0.3 pg to 0.3 ng of genomic DNA. The assay was able to detect 5 × 10² cfu/ml for B. anthracis, 8 × 10² cfu/ml for Yersinia sp., 1.4 × 10² cfu/ml for B. melitensis and 4 × 10² cfu/ml for B. pseudomallei.     

诺如病毒RT-PCR-反向斑点杂交检测方法的建立

旨在建立诺如病毒RT-PCR-反向斑点杂交检测方法。选取诺如病毒较为保守的RdRp基因作为扩增对象,经RT-PCR扩增后将目的片段克隆到pGEM-T载体中。以重组质粒为模版,选择合成寡核苷酸探针及生物素标记引物。生物素标记引物的扩增产物经热变性后与固定在硝酸纤维素膜上的探针进行杂交反应,经显色后判定结果。出现明显的蓝紫色斑点为诺如病毒阳性,如无斑点则为阴性。对5份临床样品进行检测,并以RT-PCR对比验证。结果显示,利用反向斑点杂交法对重组质粒的检测限为100拷贝/μL,在5例实际样品检测中有1例为阳性,与RT-PCR判定结果一致。建立了诺如病毒的RT-PCR-反向斑点杂交检测方法,该方法特异性好,灵敏度高,操作简便,具有重要的应用价值。     

Patterns of Limnohabitans Microdiversity across a Large Set of Freshwater Habitats as Revealed by Reverse Line Blot Hybridization

Among abundant freshwater Betaproteobacteria, only few groups are considered to be of central ecological importance. One of them is the well-studied genus Limnohabitans and mainly its R-BT subcluster, investigated previously mainly by fluorescence in situ hybridization methods. We designed, based on sequences from a large Limnohabitans culture collection, 18 RLBH (Reverse Line Blot Hybridization) probes specific for different groups within the genus Limnohabitans by targeting diagnostic sequences on their 16 S–23 S rRNA ITS regions. The developed probes covered in sum 92% of the available isolates. This set of probes was applied to environmental DNA originating from 161 different European standing freshwater habitats to reveal the microdiversity (intra-genus) patterns of the Limnohabitans genus along a pH gradient. Investigated habitats differed in various physicochemical parameters, and represented a very broad range of standing freshwater habitats. The Limnohabitans microdiversity, assessed as number of RLBH-defined groups detected, increased significantly along the gradient of rising pH of habitats. 14 out of 18 probes returned detection signals that allowed predictions on the distribution of distinct Limnohabitans groups. Most probe-defined Limnohabitans groups showed preferences for alkaline habitats, one for acidic, and some seemed to lack preferences. Complete niche-separation was indicated for some of the probe-targeted groups. Moreover, bimodal distributions observed for some groups of Limnohabitans, suggested further niche separation between genotypes within the same probe-defined group. Statistical analyses suggested that different environmental parameters such as pH, conductivity, oxygen and altitude influenced the distribution of distinct groups. The results of our study do not support the hypothesis that the wide ecological distribution of Limnohabitans bacteria in standing freshwater habitats results from generalist adaptations of these bacteria. Instead, our observations suggest that the genus Limnohabitans, as well as its R-BT subgroup, represent ecologically heterogeneous taxa, which underwent pronounced ecological diversification.     

用反向斑点杂交方法检测猪常见致病菌

目的:建立检测猪常见致病菌的反向斑点杂交方法。方法:将23S rRNA基因芯片用的针对12种细菌的25～30 mer探针加长到30～38 mer,2对通用引物序列不变。用地高辛标记下游引物,以尼龙膜为载体制备膜芯片,检验探针/膜杂交的特异性和敏感性;另外设计1条大肠杆菌K88基因探针、一段带K88探针的报告基因和1对报告基因的反向PCR引物,在PCR体系中增加封口的K88报告基因和反向引物对,被检样品扩增后进行膜杂交。结果:修改的13条探针与参考目标菌株在膜上成特异性杂交,对52个参考菌株和野外分离株的检测准确率为92%;膜杂交的敏感性与玻片芯片接近,最小检出量为100 fg DNA;在尼龙膜上增加K88探针,与3重PCR产物杂交,可以检测到大肠杆菌K88毒力基因。结论:建立的反向斑点杂交方法简便快速,检测成本低,可用于仪器设备不足的实验室,同时可以加入检测如大肠杆菌K88等致病基因,提高基于保守基因的芯片的诊断能力。     

Hybridization and Polyploidization of Saccharomyces cerevisiae Strains by Transformation-Associated Cell Fusion

Hybrid or polyploid clones of Saccharomyces cerevisiae produced by protoplast fusion were easily isolated by selecting transformants with the plasmid phenotype because the transformation was directly associated with cell fusion. When haploid cells were used as the original strain, the transformants were mostly diploids with a significant fraction of polyploids (triploids or tetraploids). Repeated transformation after curing the plasmid gave rise to clones with higher ploidy, but the frequency of cell fusion was severely reduced as ploidy increased.     

Genomic Comparison of Plant Pathogenic and Nonpathogenic Serratia marcescens Strains by Suppressive Subtractive Hybridization

Cucurbit yellow vine disease (CYVD) is caused by disease-associated Serratia marcescens strains that have phenotypes significantly different from those of nonphytopathogenic strains. To identify the genetic differences responsible for pathogenicity-related phenotypes, we used a suppressive subtractive hybridization (SSH) strategy. S. marcescens strain Z01-A, isolated from CYVD-affected zucchini, was used as the tester, whereas rice endophytic S. marcescens strain R02-A (IRBG 502) was used as the driver. SSH revealed 48 sequences, ranging from 200 to 700 bp, that were present in Z01-A but absent in R02-A. Sequence analysis showed that a large proportion of these sequences resembled genes involved in synthesis of surface structures. By construction of a fosmid library, followed by colony hybridization, selection, and DNA sequencing, a phage gene cluster and a genome island containing a fimbrial-gene cluster were identified. Arrayed dot hybridization showed that the conservation of subtracted sequences among CYVD pathogenic and nonpathogenic S. marcescens strains varied. Thirty-four sequences were present only in pathogenic strains. Primers were designed based on one Z01-A-specific sequence, A79, and used in a multiplex PCR to discriminate between S. marcescens strains causing CYVD and those from other ecological niches.     

Rapid Detection of Potato Spindle Tuber Viroid (PSTV) by Dot Blot Hybridization

A PSTV probe of 350 bases, prepared in our laboratory, was used to detect picogram quantities of viroids by a simplified, improved, dot blot, hybridization technique in crude potato and tomato extracts. Optimum conditions for certification of PSTV-free potato plants were established, involving formamide concentration, washing stringencies, exposure time during autoradiography and method of probe radiolabelling. Ten pg of purified PSTV in water and 50 pg of purified PSTV added to healthy plant extract were detected. Also hybridization signals could be detected from as little as 0.075 mg of infected plant tissue.     

Identification of Frankia Strains by Direct DNA Hybridization of Crushed Nodules

A hybridization procedure was developed to identify Frankia strains inside actinorhizae by direct probing of crushed root nodules. The probe consisted of an indigenous cryptic plasmid. This well-conserved, 8-kilobase plasmid was detected in Frankia isolates that were very close taxonomically (they possessed a very high DNA sequence homology). The probe did not hybridize to the DNA of Frankia isolates which did not carry the plasmid. Endophyte DNA was extracted by a modification of a technique originally developed for the detection of plasmids in Frankia isolates. The hybridization procedure applied to nodules collected in a stand of alder permitted determination of a distribution map of the plasmid-bearing Frankia strains.     

Detection and Identification by PCR of a Highly Virulent Phylogenetic Subgroup among Extraintestinal Pathogenic Escherichia coli B2 Strains

Closely related Escherichia coli B2 strains O1:K1, O2:K1, O18:K1, and O45:K1 constitute a major subgroup causing extraintestinal infections. A DNA pathoarray analysis was used to develop a PCR specific for this subgroup that was included in the multiplex phylogenetic-grouping PCR method. Our PCR may serve to identify this virulent subgroup among different ecological niches.     

Specific Detection of the Gene for the Extracellular Neutral Protease of Bacillus cereus by PCR and Blot Hybridization

A pair of primers and a gene probe for the amplification and detection of the Bacillus cereus neutral protease gene (NPRC) were developed. Specificity for the npr genes of the B. cereus group members B. cereus, B. mycoides, and B. thuringiensis was shown. Restriction polymorphism patterns of the PCR products confirmed the presence of the NPRC gene in all three species.     

Simultaneous Detection and Identification of Enteric Viruses by PCR-Mass Assay

Simultaneous detection of enteric viruses that cause similar symptoms (e.g. hand, foot and mouth disease) is essential to the prevention of outbreaks and control of infections. In this study, a novel PCR-Mass assay combining multiplex polymerase chain reaction (PCR) with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was developed and used for simultaneous detection of eight distinct human enteric viruses. Enteric viral isolates and standard viral RNAs were examined to determine the sensitivity and specificity of the PCR-Mass assay. Clinical performance was evaluated with a total of 101 clinical specimens from patients suspected of having hand, foot and mouth disease (HFMD). The results were compared to those of previous analyses using real-time RT-PCR. The identification of specific viruses and clinical specimens shows that the PCR-Mass assay performed as well as or better than standard methods with respect to indicating the presence of multiplex pathogens in a single specimen.     

Simultaneous Identification of Mutations by Dual-Parameter Multiplex Hybridization in Peptide Nucleic Acid-Containing Virtual Arrays

The physical entrapment of peptide nucleic acids (PNA) in electrophoresis media provides a system for performing real-time hybridization. DNA strands fully complementary to the target PNA are retarded compared to single-nucleotide mismatched strands. A second parameter, that of amplicon length, has been introduced to perform multiplex analyses on several mutations simultaneously. Size fractionation creates a virtual array of PCR products that can hybridize to one of a set of mutation-specific PNAs present within the matrix. Each targeted mutation can be identified by the size of its corresponding amplicon. Its genotype is characterized by its interaction with a specific PNA that gives a visually resolved distinction between wildtype and mutant allele. In contrast to conventional hybridization, heterozygotes are readily distinguished from homozygotes. Using a capillary electrophoresis-based DNA sequencer, this approach has been used to automate the identification of the H63D, S65C, and C282Y mutations in the hereditary hemochromatosis gene.     

Identification of DNA Sequences Specific for Vibrio vulnificus Biotype 2 Strains by Suppression Subtractive Hybridization

Vibrio vulnificus can be divided into three biotypes, and only biotype 2, which is further divided into serovars, contains eel-virulent strains. We compared the genomic DNA of a biotype 2 serovar E isolate (tester) with the genomic DNAs of three biotype 1 strains by suppression subtractive hybridization and then tested the distribution of the tester-specific DNA sequences in a wide collection of bacterial strains. In this way we identified three plasmid-borne DNA sequences that were specific for biotype 2 strains irrespective of the serovar and three chromosomal DNA sequences that were specific for serovar E biotype 2 strains. These sequences have potential for use in the diagnosis of eel vibriosis caused by V. vulnificus and in the detection of biotype 2 serovar E strains.     

Specific Detection of Arcobacter and Campylobacter Strains in Water and Sewage by PCR and Fluorescent In Situ Hybridization

The aim of this study was to evaluate PCR and fluorescent in situ hybridization (FISH) techniques for detecting Arcobacter and Campylobacter strains in river water and wastewater samples. Both 16S and 23S rRNA sequence data were used to design specific primers and oligonucleotide probes for PCR and FISH analyses, respectively. In order to assess the suitability of the methods, the assays were performed on naturally and artificially contaminated samples and compared with the isolation of cells on selective media. The detection range of PCR and FISH assays varied between 1 cell/ml (after enrichment) to 10³ cells/ml (without enrichment). According to our results, both rRNA-based techniques have the potential to be used as quick and sensitive methods for detection of campylobacters in environmental samples.     

Identification of Differences in Genome Content among phlD-Positive Pseudomonas fluorescens Strains by Using PCR-Based Subtractive Hybridization

Certain 2,4-diacetylphloroglucinol-producing strains of Pseudomonas fluorescens colonize roots and suppress soilborne diseases more effectively than others from which they are otherwise phenotypically almost indistinguishable. We recovered DNA fragments present in the superior colonizer P. fluorescens Q8r1-96 but not in the less rhizosphere-competent strain Q2-87. Of the open reading frames in 32 independent Q8r1-96-specific clones, 1 was similar to colicin M from Escherichia coli, 3 resembled known regulatory proteins, and 28 had no significant match with sequences of known function. Seven clones hybridized preferentially to DNA from strains with superior rhizosphere competence, and sequences in two others were highly expressed in vitro and in the rhizosphere.     

Species-Specific Detection of Vibrio anguillarum in Marine Aquaculture Environments by Selective Culture and DNA Hybridization

Methods for specific detection of Vibrio anguillarum in complex microbial communities within diverse marine aquaculture environments were evaluated. A system for the detection of culturable cells based on the combined use of a selective medium and a nonradioactively labeled oligodeoxynucleotide complementary to 16S rRNA was developed. Four hundred fourteen bacterial cultures were evaluated in order to assess the specificity of the method. When both the selective medium and the specific probe gave positive results, the cultures were always identified as V. anguillarum. The selectivity for colony hybridization was 1 V. anguillarum cell in 10,000 total bacterial cells in environmental samples. The utility of the method was also compared with detection by dot blot hybridization of either raw DNA purified from environmental samples or PCR-amplified DNA of 16S rRNA genes, using universal eubacterial primers. The post-PCR hybridization was more sensitive (8 x 10(sup2) cells) than direct hybridization of the whole purified DNA (10(sup6) cells). However, the selective medium-probe combined method was as sensitive as post-PCR hybridization, albeit more specific.     

环介导等温扩增技术原理及其在检测诊断病原微生物中的应用

环介导等温扩增技术(loop-mediated isothermal amplification,LAMP)是近年来发展出来的一种核酸扩增技术.通过设计识别目的片段6个序列的4个特异引物,整个反应可以在恒温(60～65℃)条件下1 h内完成,由于采用4个引物,与传统的核酸扩增技术相比,它具有敏感、特异的特点;且产物中有大量的副产物--白色焦磷酸镁沉淀,扩增产物可通过肉眼观察或浊度计即可判定结果;反应不需要精密控温设备和高级复杂的分析仪器,对操作人员的熟练度和专业水平要求不高,因此该方法特别适合基层或者实验条件较差的试验室进行病原微生物的快速检测.就其原理及其在病原微生物的检测中的应用做一综述.     

扣除杂交法筛选与非小细胞性肺癌转移相关的基因

非小细胞性肺癌病人的术后死亡率很高 ,其原因是该病易发生转移。收集了肺癌早期患者的样品 ,并根据患者资料分成转移型 (n =4)和非转移型 (n =5 ) ,采用扣除杂交法筛选与非小细胞性肺癌转移相关的基因。扣除后的cDNA文库中得到了 2 2 5个有效克隆。对这些克隆进行了测序 ,在基因文库中比较了核苷酸同源性 ,初步确定了这些克隆对应的基因 ,并根据基因可能涉及的功能加以分类。通过实时 (realtime)RT PCR鉴定 ,发现 10种基因在用于扣除试验的转移病人样品中的平均表达量比非转移病人高。进一步对 70位患非小细胞性肺癌病人 (I至IIIA期 )的样品进行了检测。根据统计分析 ,在I和II期病人中 ,2种基因MALA1和EIF4A1在发生转移的病人样品中的表达与在非转移的病人中有显著差异性。这些结果将有助于分析非小细胞性肺癌病变发生转移的可能性