反相斑点杂交法对解脲脲原体分型的研究

目的研究以聚合酶链反应为基础的快速检测与鉴定解脲脲原体基因型的方法。方法选择2003年11月至2005年11月在中山大学附属第二医院门诊就诊的有外阴阴道炎症状和体征的患者601例,设为病例组,同期无自觉症状的正常体检人群306例,设为对照组,分别取宫颈分泌物待检测。将解脲脲原体不同基因型的特异探针固定在硝酸纤维素膜上,临床标本按常规方法提取解脲脲原体DNA,采用生物素标记的解脲脲原体特异通用引物PCR扩增DNA,然后分别与解脲脲原体不同基因型特异探针杂交、显色。结果病例组解脲脲原体阳性421例占70.0%,对照组解脲脲原体阳性126例占41.2%。病例组中单型别感染的U.parvum占65.4%,其中1型、3型、6型和14型分别占28.8%、43.3%、26.0%和1.9%,U.urealyticum占18.4%;对照组中单型别感染的U.parvum占79.3%,其中1型、3型、6型和14型分别占63.2%、21.1%、15.7%和0.0%,U.urealyticum占13.8%。18例阳性标本随机DNA测序鉴定,均为相应的解脲脲原体基因型。结论U.parvum群,尤其是其中的1、3、6型别是正常人群携带的可能性较大,U.urealyticum则有可能和1型起协同作用或独自导致疾病。用反相斑点杂交进行解脲脲原体基因分型,方法简单、实用,适用于临床。     

Development of a selective and sensitive polymerase chain reaction assay for the detection of Mycoplasma pirum

Abstract A new assay using the polymerase chain reaction to amplify a 173-nucleotide DNA fragment within the 16S ribosomal RNA gene of Mycoplasma pirum has been developed. The assay selectively amplified DNA from all strains of M. pirum tested with a high level of sensitivity, even in a context of human DNA. DNA from other mollicute species, including those closely related to M. pirum , from bacteria phylogenetically close to mollicutes ( Clostridium innocuum, C. ramosum and Bacillus subtilis ), from Escherichia coli and from human peripheral blood mononuclear cells, did not produce the amplified DNA product specific for M. pirum .     

拉米夫定联合阿德福韦酯治疗乙型肝炎的疗效及对HBV耐药基因突变的影响

目的探讨拉米夫定联合阿德福韦酯治疗慢性乙型肝炎的疗效,并利用反向点杂交技术检测其对HBV基因耐药突变的影响。方法156例慢性乙型肝炎患者随机分为2组：对照组70例采用拉米夫定治疗,治疗组86例采用拉米夫定联合阿德福韦酯治疗。采用实时荧光定量PCR和ELISA检测2组治疗前和治疗后48周的HBV-DNA载量和HBeAg并采用PCR-反向点杂交技术（PCR-RDB）检测2组治疗48周后的HBV耐药基因突变情况。结果对照组及治疗组在经过48周治疗后HBV-DNA载量较治疗前都明显下降（P 〈0. 05）,治疗组HBV-DNA载量明显低于对照组（P〈0.05）。治疗组经过48周治疗后HBeAg的阴转率为54.9%,明显高于对照组15.0% （P 〈0.05）。对照组44例未出现耐药突变,25例拉米夫定耐药突变中rtL180M突变6例,rtM204V/I突变11例,rtL180M ＋ rtM204V/I混合突变8例;阿德福韦酯HN236T耐药突变1例。治疗组77例未出现耐药突变;5例拉米夫定耐药突变中rtL180M突变1例,rtM204V/I突变2例,rtL180M ＋ rtM204V/I混合突变2例;阿德福韦酯耐药突变中rtN236T突变1例;拉米夫定和阿德福韦酯交叉耐药rtN236T ＋ rtM204V/I混合突变3例。对照组耐药突变率为37. 1%（26/70）明显髙于治疗组的10.5%（9/86）（P〈0.05）。结论拉米夫定联合阿德福韦酯对治疗慢性乙型肝炎方面有效并在减少HBV耐药基因突变方面具有一定的作用。     

Development of a PCR assay combined with a short enrichment culture for detection of Campylobacter jejuni in estuarine surface waters

Abstract Two extraction procedures were examined, and it was found that DNA recovered from Campylobacter jejuni lysed by the cetyltrimethylammonium bromide (CTAB) method was more suitable for use as a PCR template than DNA released by the boiling method. The region targeted for PCR amplification was a 1.73-kb portion of the flagellin A gene of C. jejuni . The detection limit was lower than 30 cells per 100 ml in artificially contaminated waters. PCR assay and conventional culturing method had the same sensitivity, but results of the PCR technique were available within 48 h and so shortened the time necessary for detection by 48 h.     

A self-probing primer PCR method for detection of very short DNA fragments

A novel self-probing primer method that based on the fluorescence resonance energy transfer principle is designed to detect DNA fragments of approximately 40 bp. Four self-probing primer reaction systems were developed to target a maize endogenous reference gene (HMG), a soybean endogenous reference gene (Lectin), a rapeseed endogenous reference gene (CruA) and an exogenous gene 5-enolpyruvylshikimate-3-phosphate synthase (ctp2-cp4epsps). These four primer systems were confirmed to have a high level of inter-species specificity and good intra-species stability. The limit of detection was estimated to be 10 copies of haploid genomes for all four assays. The validation results demonstrated that the self-probing primer methods are able to quantify the DNA amount in the different samples with good sensitivity and precision. When highly processed food products were assayed, the self-probing primer method produced better results than the TaqMan probe method. Overall, the self-probing primer method is suitable for qualitative and quantitative detection of very short DNA targets in samples of different sources.     

Development of molecular techniques for detection of lymphocystis disease virus in different marine fish species

AIMS: The development and evaluation of a protocol based on polymerase chain reaction (PCR) and nucleic acid hybridization techniques for the specific detection of lymphocystis disease virus (LCDV) in several marine fish species. METHODS AND RESULTS: The pair of primers for PCR, OBL3 and OBL4, was designed based on published nucleotide sequence (LCDV-1) and amplifies a fragment within the major capsid protein. The sensitivity was evaluated using DNA from purified viral particles, as well as from cells inoculated with several viral concentrations. The PCR combined with slot blot was the most sensitive methodology, detecting 2.5 ng of viral DNA. Using this methodology LCDV was detected at 5 days postinoculation from SAF-1 cells initially inoculated with 10(-5) TCID(50) ml(-1). The combination of PCR with membrane hybridization has also been proved to be adequate to detect LCDV from apparently healthy carriers by means of caudal fin sample analysis. This asymptomatic infection was also demonstrated by classical virological methods (cell culture and immunoblot). CONCLUSIONS: The protocol described in this study allows the specific detection of LCDV, both in cell cultures and in fin homogenates from asymptomatic fish. SIGNIFICANCE AND IMPACT OF THE STUDY: The detection of asymptomatic carriers by a rapid molecular method using caudal fin sampling, which does not imply animal killing, could be an important tool to control epizootics caused by LCDV, as fish could be analysed before their introduction and/or mobilization in farm facilities.     

高灵敏可视化核酸试纸条法快速检测HBV DNA

目的：本研究旨在建立一种基于试纸条的快速、灵敏及可视化检测乙型肝炎病毒核酸的方法。方法：利用聚合酶链反应扩增乙肝病毒的保守区,其中上、下游引物的5＇端分别修饰异硫氰酸荧光素和生物素。核酸试纸条上的胶体金以及检测线处分别标记有链霉亲和素以及抗荧光素抗体。将扩增产物与展开液混合后点样,10 min后即可用肉眼判读结果。在优化了展开液成分、上样体积以及上样浓度之后,对该方法的灵敏度进行了评价。最后收集15例阴性样本及33例HBsAg阳性样本,按血清标志物结果进行分类后使用核酸试纸条进行检测,并与实时荧光PCR的结果进行了比较。结果：试纸条检测乙肝病毒核酸的灵敏度为250copies/mL。在临床样本的测定中,该方法与实时荧光定量PCR的特异性均为100%。且两种方法检测不同血清标志物类型的阳性检出率无差异。结论：核酸试纸条技术能够用于乙肝病毒核酸的可视化检测,与实时荧光PCR相比检测速度快,具有较好的灵敏度和特异性,适合流行病学调查以及在基层医院体检使用。     

Use of a thin film biosensor for rapid visual detection of PCR products in a multiplex format

Rapid, sensitive assays for nucleic acid amplification products have utility for the identification of bacterial or viral infections. We have developed a nucleic acid hybridization assay utilizing thin film technology that permits visual detection of hybrids. The silicon-based biosensor detects the presence of target sequences by enzymatically transducing the formation of nucleic acid hybrids into molecular thin films. These films alter the interference pattern of light on the biosensor surface, producing a perceived color change. We have applied this technology to the development of a chip containing capture probes specific for human respiratory virus sequences including respiratory syncytial virus, influenza virus A and B, parainfluenza virus types 1 and 3, and rhinovirus. In a ten-minute assay, the biosensor permits unambiguous identification of viral-specific RT/PCR products from infected cell lysates.     

基于聚合酶螺旋反应技术快速检测铜绿假单胞菌

【背景】铜绿假单胞菌是一种重要的水源和食源性致病菌,可引起急性肠道炎、脑膜炎、败血症和皮肤炎症等疾病。加强铜绿假单胞菌的快速检测,对保障食品安全具有重要的意义。【目的】建立聚合酶螺旋反应(Polymerasespiralreaction,PSR)方法快速检测铜绿假单胞菌。【方法】针对铜绿假单胞菌外毒素A调控基因——ETA基因(toxA)设计引物,通过引入加速引物、优化反应条件和筛选颜色指示剂,建立快速检测铜绿假单胞菌的PSR方法,并研究方法的特异性、敏感性和可靠性。【结果】建立的方法在等温65°C条件下,40 min内可完成PSR反应,且可通过钙黄绿素和羟基萘酚蓝直接判读结果。方法特异性强、灵敏度高,最低检出限分别为20 CFU/mL细菌和1.011 5 pg/μL基因组DNA。可视化PSR方法检测包装饮用水来源的分离菌株与传统生化方法检测结果一致。【结论】研究建立的可视化PSR方法为铜绿假单胞菌DNA快速检测提供了一种可行的有效手段。     

Recombinant DNA probes and polymerase chain reaction for detection of Mycoplasma gallisepticum strains

Abstract Two recombinant DNA clones, pMG286.2 and pMG301.1, were isolated from the partial genomic library of Mycoplasma gallisepticum strain S6. Recombinant M. gallisepticum specific fragments were used as probes in Southern hybridisation with 10 M. gallisepticum strains whose DNA was digested by Eco RI, Hin dIII, Bgl II, Rsa I and Bam HI. The 1.5 kb fragment pMG301.1 did not show polymorphism in hybridisation patterns with M. gallisepticum strains, while the 3.5 kb fragment pMG286.2 enabled differentiation of M. gallisepticum strains into clusters. The DNA sequence of pMG301.1 was used to design a pair of 27-mer oligonucleotides flanking a 1.3 kb genomic region. These two primers directed specific in vitro amplification of all M. gallisepticum strains assayed giving an expected 1.3 kb product. Digestion of polymerase chain reaction products by Dde I enabled simple differentiation between clusters of M. gallisepticum strains and may be useful for improved epizootiological studies of M. gallisepticum infections in poultry.     

Development of a genus specific primer set for detection of Leishmania parasites by polymerase chain reaction

Abstract We have compared the sequences of a major class of kinetoplast DNA (kDNA) minicircle (pLURkE3) of Leishmania strain UR6 with other minicircle sequences from different Leishmania species. Alignment of these sequences allowed the selection of a pair of oligonucleotides suitable as primers in polymerase chain reaction (PCR) which is specific for Leishmania parasites. PCR with this genus-specific primer set is capable of detecting 1 femtogram of kDNA. These primers have been tested with kDNAs from both old world and new world Leishmania species. The results indicate that the primers may be suitable for detection of any kind of leishmaniasis.     

Development and evaluation of a reverse dot blot assay for the simultaneous detection of common toxic microalgae along the Chinese coast

Toxic microalgae currently pose a great threat to human health, ecosystem, fishery, tourism, and aquaculture along the Chinese coast. The detection of toxic microalgae by routinely monitoring natural waters is necessary to provide timely mitigation. Therefore, an effective, simultaneous detection protocol should be established for the simple, rapid, and accurate identification of causative algae. This study developed and evaluated a reverse dot blot assay (RDBA) combined with a low-density membrane-based DNA array for the rapid and simultaneous detection of toxic microalgae that are commonly distributed along the Chinese coast. The large subunit rDNA D1–D2 regions of the target species were first sequenced to design taxonomic probes. Probe specificity was validated by performing a cross-reactivity test with dot blot hybridization. The tailed probes were immobilized onto a nylon membrane to prepare a low-density DNA array for RDBA. The established detection procedure involved DNA extraction, biotin (Bio)-labeling of objective sequences by multiple polymerase chain reaction (M-PCR), RDBA, coloration, and judgment of hybridization by the naked eye. Bio-labeled primer-based labeling proved to be an economical and effective method to prepare Bio-labeled PCR products for RDBA. The detection limits of RDBA using the M-PCR-labeling products from DNA templates prepared by different methods were also compared, and a kit-based DNA extraction method displayed the lowest detection limit of 0.5 cells. Simulation results showed that RDBA can recover all target species and was not affected by background DNA. RDBA was proven effective, specific, and sensitive for the simultaneous detection of toxic microalgae in the field samples. Therefore, this method may be used in the field monitoring of natural samples.     

解脲脲原体生物群聚合酶链反应-毛细管电泳检测方法的建立及应用

目的 建立一种检测解脲脲原体生物群1、2的聚合酶链反应-毛细管电泳(PCR-CE)方法,研究解脲脲原体生物群与男性非淋菌性尿道炎(NGU)的关系。方法 合成解脲脲原体生物群1、2的通用引物,进行PCR,然后使用CE检测解脲脲原体生物群1、2的扩增产物。对该方法进行敏感性、特异性的评价。使用该方法检测不同男性人群尿样中解脲脲原体2个生物群。结果 PCR-CE法的敏感性为10拷贝/50μl。该方法仅特异扩增解脲脲原体生物群1、2。解脲脲原体生物群2在NGU组、非沙眼衣原体引起的NGU(NCNGU)组中的检出率高于对照组(P<0.05),解脲脲原体生物群1在NGU及NCNGU组中的检出率和对照组无差异(P>0.05)。结论 建立了一种敏感性高、特异性强、分辨率高的检测解脲脲原体2个生物群的PCR-CE方法。解脲脲原体生物群2与男性NGU有一定的关系。解脲脲原体生物群1不能引起男性NGU。     

Design and development of a DNA array for rapid detection and identification of multiple tomato vascular wilt pathogens

Fusarium wilt, caused by Fusarium oxysporum f. sp. lycopersici, and Verticillium wilt, caused by either Verticillium albo-atrum or Verticillium dahliae, are devastating diseases of tomato (Lycopersicon esculentum) found worldwide. Monitoring is the cornerstone of integrated pest management of any disease. The lack of rapid, accurate, and reliable means by which plant pathogens can be detected and identified is one of the main limitations in integrated disease management. In this paper, we describe the development of a molecular detection system, based on DNA array technology, for rapid and efficient detection of these vascular wilt pathogens. We show the utility of this array for the sensitive detection of these pathogens from complex substrates like soil, plant tissues and irrigation water, and samples that are collected by tomato growers in their greenhouses.     

Ingested foreign (phage M13) DNA survives transiently in the gastrointestinal tract and enters the bloodstream of mice

Is the epithelial lining of the mammalian gastrointestinal (GI) tract a tight barrier against the uptake of ingested foreign DNA or can such foreign DNA penetrate into the organism? We approached this question by pipette-feeding circular or linearized double-stranded phage M13 DNA to mice or by adding M13 DNA to the food of mice whose fecal excretions had previously been shown to be devoid of this DNA. At various post-prandial times, the feces of the animals was tested for M 13 DNA sequences by Southern or dot blot hybridization or by the polymerase chain reaction (PCR). On Southern blot hybridization, the majority of M13 DNA fragments were found in the size range between < 200 and 400 by (base pairs). For the PCR analysis, synthetic oligodeoxyribonucleotide primers were spaced on the M13 DNA molecule such that the sizes of the persisting M13 DNA fragments could be determined. We also extracted DNA from whole blood or from sedimented blood cells of the animals at different times after feeding M t3 DNA and examined these DNA preparations for the presence of M13 DNA by dot blot hybridization or by PCR. M13 DNA fragments were found between 1 and 7 h postprandially in the feces of mice. By PCR analysis, fragments of 712, 976, and 1692 by in length were detected. In DNA from blood, M13 DNA fragments of up to 472 by were found by PCR between 2 and 6 h after feeding. Dot blot or Southern blot hybridization revealed M13 DNA at 2 and 4 h, but not at 1, 8 or 24 h after feeding. This DNA was shown to be DNase sensitive. M13 DNA was found both in blood cells and in the serum. A segment of about 400 by of the DNA amplified by PCR from feces or blood was analyzed for its nucleotide sequence which was found to be identical to that of authentic M13 DNA, except for a few deviations. M13 DNA could not be detected in the feces or in the blood of the animals prior to feeding or prior to 1 h and later than 7 h after feeding. These controls attest to the validity of the results and also argue against the possibility that the murine GI tract had been colonized by phage M13. Moreover, M13 DNA-positive bacterial colonies were never isolated from the feces of animals that had ingested M13 DNA. The results of reconstitution experiments suggested that 2 to 4% of the orally administered M13 DNA could be detected in the GI tract of mice. A proportion of about 0.01% to 0.1% of the M13 DNA fed could be retrieved from the blood.     

Development of a generic PCR detection of deoxynivalenol- and nivalenol-chemotypes of Fusarium graminearum

Based on the intergenic sequences of Tri5-Tri6 genes involved in the mycotoxin pathways of Fusarium species, a generic PCR assay was developed to detect a 300 bp fragment of deoxynivalenol (DON)-chemotypes and a 360 bp sequence of nivalenol (NIV)- chemotypes of Fusarium graminearum. Mycotoxin chemotypes identified by the PCR assays were confirmed by the chemical analyses of HPLC or GC/MS. Further analysis of 364 F. graminearum isolates from 12 provinces of China showed that 310 were DON-chemotypes and 54 were NIV-chemotypes. Sequence analyses revealed that DON-chemotypes display more variations than NIV-chemotypes. This PCR assay could be used to detect mycotoxin-producing Fusarium-species and may thus help to develop strategies to avoid or reduce mycotoxin contamination of cereals. Also this assay may provide useful alternatives to antibody-based mycotoxin tests.     

Development of a Low-cost Polymerase Chain Reaction-based Method for Studying Differentially Expressed Genes in Developing Rice Leaves

Gene expression studies are important for revealing gene functions putatively involved in biological processes. We were interested in identifying differentially expressed genes during leaf development in rice. We combined the RNA arbitrarily primed-polymerase chain reaction （RAP.PCR） and dot blot hybridization methods to screen a rice leaf primordium cDNA library. Three developmental stages during vegetative growth were examined. The cDNA clones showing different hybridization patterns were further analyzed and verified. Here we demonstrate that the combination of RAP-PCR and dot blot hybridization could provide an efficient and relatively low-cost cDNA library screening approach to discover genes not previously known to be associated with leaf development in rice, We believe that the findings described here will help to elucidate the molecular mechanism（s） underlying the developmental processes of rice leaf     

Direct quantification of polymerase chain reaction fragments using field-amplified sample injection in capillary zone electrophoresis for gene dosage estimation

To assess gene dosages for clinical application, especially for prognostication of cancer, we developed a direct quantification method for polymerase chain reaction products. We report on an application of field amplified sample injection (FASI) to capillary zone electrophoresis which allows the quantification of PCR products without sample preparation. Using an external standard and UV detection for the quantification of DNA, a low coefficient of variation has been obtained. Overall, the described method provides a fast and easy tool for PCR product quantification in clinical laboratories.     

Development of a Low-cost Polymerase Chain Reaction-based Method for Studying Differentially Expressed Genes in Developing Rice Leaves

Gene expression studies are important for revealing gene functions putatively involved in biological processes. We were interested in identifying differentially expressed genes during leaf development in rice, We combined the RNA arbitrarily primed-polymerase chain reaction (RAP-PCR) and dot blot hybridization methods to screen a rice leaf primordium cDNA library. Three developmental stages during vegetative growth were examined. The cDNA clones showing different hybridization patterns were further analyzed and verified. Here we demonstrate that the combination of RAP-PCR and dot blot hybridization could provide an efficient and relatively low-cost cDNA library screening approach to discover genes not previously known to be associated with leaf development in rice. We believe that the findings described here will help to elucidate the molecular mechanism(s) underlying the developmental processes of rice leaf.