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 .     

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.     

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 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。     

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 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.     

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.     

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.     

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。     

Development of a PCR-based technique for detection of Helicobacter pylori

Abstract A primer-set was designed for specific detection of genes that encode for 16S rRNA of Helicobacter pylori , using direct polymerase chain reaction (PCR). The primers were selected in the hypervariable regions, derived from a complete small subunit 16S rRNA sequence of the reference strain H. pylori CCUG 17874. The primer-set amplified a 537 base pair (bp) sequence specifically from chromosomal H. pylori DNA. Amplification of purified chromosomal H. pylori DNA was achieved at concentrations as low as 1 femto gram (fg), equivalent to 5 bacteria. Furthermore, as few as 1 lysed H. pylori cell was detected by this PCR technique. The specificity of the primers was 100%, since purified chromosomal DNA was detected from all 32 various H. pylori isolates, whereas no other bacteria species were detected, whether related to Helicobacter or not. The 16S rDNA primers successfully detected H. pylori in antral biopsy specimens collected from infected patients.     

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     

基于PCR-LDR平台的近交系小鼠遗传质量快速检测方法

目的建立基于PCR-LDR平台的近交系小鼠SNP快速分型方法,用于检测实验小鼠的遗传质量与品系纯度。方法利用可移植性极高的PCR-LDR技术,以常见近交系小鼠为研究对象,选取了21条染色体上的45个SNP位点,分别设计引物和探针,经过筛选和验证,建立了多重PCR-LDR(polymerase chain reaction and ligase detection reaction,PCR-LDR)分型方案。结果四组多重PCR-LDR可实现45个SNP位点的基因分型,其中43个、44个与45个SNP在样本中的检出率分别为100%、90.9%与36.4%。所有样本经分型确定为纯合体,并得到了常见近交系小鼠SNP位点信息。结论实现了常见近交系小鼠快速、高通量的基因分型,可用于遗传质量检测和品系鉴定。     

Identification of specific gene sequences in preimplantation embryos by genomic amplification: detection of a transgene

Endogenous and foreign DNA sequences can be detected in an extremely small number of cells via sequence amplification in vitro. The polymerase chain reaction (PCR) technique applied in multiple cycles allows the amplification of specific short regions of the genome to levels that can be detected by DNA blotting techniques. Cow and mouse blastocysts were analyzed by PCR for the presence of an endogenous singlecopy gene or an integrated foreign gene. The endogenous single-copy gene encoding the beta chain of bovine luteinizing hormone was detectable in cow blastocysts and in purified bovine genomic DNA representing as few as 25 cells. To determine whether exogenous genes (transgenes) can be detected in preimplantation embryos, transgenic male mice hemizygous for the prokaryotic gene encoding neomycin resistance were bred to nontransgenic females, and the resulting blastocysts were analyzed. The neo gene was detected in approximately half of the embryos. The capability to identify specific gene sequences in a limited number of embryonic cells affords investigators the opportunity to study genetics in early development.     

Species-specific detection of Legionella using polymerase chain reaction and reverse dot-blotting

Abstract Legionella pneumophila and some other Legionella species are capable of causing Legionnaire's disease, a potentially fatal pneumonia. The identification of legionellae by standard laboratory techniques such as culture is difficult and time-consuming. In the present work, the DNA sequence of the 23S-5S spacer region was determined for 43 Legionella isolates, and the sequence information was used to develop a species-specific detection system using PCR and reverse dot-blotting which employs just one PCR amplicon to perform genus- and species-specific detection. L. pneumophila serogroups 1–16 as well as 21 non- pneumophila isolates could be identified and differentiated at the species level using this system.     

Simplified procedures for detection of amplified DNA using fluorescent label incorporation and reverse probing

Conventional methods of detecting polymerase chain reaction (PCR) products require equipment and expertise which may not be available in diagnostic bacteriology laboratories, especially in developing countries. To this end we have examined other methods of product detection, including fluorescein-12-dUTP incorporation during PCR amplification, and reverse probing, where the PCR product is used as the probe in a scaled down hybridization with a fixed capture probe consisting of a fragment entirely internal to the sequence of the PCR product. These techniques have shown sensitivities of 20 fg of purified mycobacterial DNA, which corresponds to approximately five cells.