金黄色葡萄球菌反向线性杂交探针检测方法的建立

目的建立一种检测金黄色葡萄球菌的简单、快速、灵敏、准确的方法。方法根据金黄色葡萄球菌的耐热核酸酶nuc基因,设计一对通用引物及两条特异性探针,用生物素标记通用引物的5＇端,将两条特异性探针固定于硝酸纤维膜上,使PCR产物与探针杂交。结果建立的反向线性杂交探针方法,其检测限为2 ng/μL,检测特异性和准确性均为100%。结论建立的反向线性杂交检测方法具有较高的敏感性和特异性,可用于实验动物金黄色葡萄球菌的快速检测。     

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.     

反向斑点杂交法快速检测HBV基因型反应条件的优化和建立*

利用反向斑点杂交技术,设计出特异性基因分型探针,并将探针固定在带正电荷的尼龙膜上,与PCR扩增带有地高辛标记的临床血清样本进行杂交。通过优化杂交反应条件,建立起简单、快速、特异地检测HBV基因型的方法。利用该方法对重庆地区临床样本进行分型检测,并与直接测序结果比较。结果表明新建的HBV基因分型方法可对拷贝数在103以上的血清样本准确分型,特异性达到96.67%。重庆地区感染HBV主要以B型为主。     

应用膜反向斑点杂交技术快速检测结核分支杆菌耐乙胺丁醇基因型的研究

应用膜反向斑点杂交技术快速检测结核分支杆菌对乙胺丁醇（EMB）耐药性。设计与合成用于检测结核分支杆菌耐EMB基因embB的寡核苷酸探针,点于硝酸纤维素膜上,与结核分支杆菌临床分离株生物素标记的聚合酶链反应（PCR）产物进行反向斑点杂交,并与PCR单链构象多态性（PCRSSCP）和PCR直接测序（PCRDS）结果比较。对81株结核分支杆菌临床分离株进行分析,31株EMB敏感株中,26株embB基因的SSCP图谱、膜反向斑点杂交结果与标准株(H37Rv)完全相同;其余5株SSCP图谱出现泳动变位,其中3株E1b杂交阳性,PCRDS分析为embB基因306位密码子ATG→GTG突变;2株E1d杂交阳性,PCRDS分析为embB基因306位密码子ATG→ATA突变。50株耐EMB菌株中,24株PCRSSCP 图谱与标准菌株相同,E1杂交阳性;26株PCRSSCP图谱出现泳动变位,其中18株E1b杂交阳性,2株E1c杂交阳性,5株E1d杂交阳性,1株E1e杂交阳性,未发现E1f杂交阳性,与PCRSSCP、PCRDS分析结果一致。突变检出率为52%。 膜反向斑点杂交技术可能成为检测部分结核分支杆菌乙胺丁醇耐药基因型简便、快速的方法。     

克隆化反向杂交探针的制备

反向斑点杂交是将寡核苷酸探针固定在膜上,用标记的靶序列与固定在膜上的探针进行杂交．与正向杂交相比,它通过一次杂交即可确定多种基因型,是一种快速筛查DNA点突变的诊断方法.我们选择β－地中海贫血基因-28 (A→ G), CD17 (A→T) and CD41～42 (-TTCT) 三种点突变为模型采用PCR扩增产生串联多拷贝序列探针并将其克隆化．将克隆化探针固定于尼龙膜上,与同位素标记的β－珠蛋白基因PCR片段进行杂交,检测β－地贫患者的基因型．     

反向斑点杂交检测泰泽病原体

目的建立一种简洁稳定、特异灵敏的检测泰泽病原体(Tyzzer’s organism)的反向斑点杂交方法(reverse dot blot,RDB)。方法根据泰泽病原体16S rDNA保守基因组序列设计引物和特异性探针,上游引物用生物素标记,进行PCR扩增,建立反向斑点杂交方法,用此法进行了特异性和灵敏度实验,同时,用RDB、ELISA和IFA对41只小鼠、38只大鼠进行了检测。结果 RDB特异性强,最低检测限为4.5 ng/μL。对79例实验动物的检测中,与ELISA检测结果一致性为100%,阳性率是7.59%(6/79);与IFA一致性为92.4%(73/79),IFA阳性率是0%。结论建立了PCR扩增与分子杂交相结合的准确、灵敏、特异的泰泽病原体反向斑点杂交检测方法。     

β-地中海贫血基因检测膜条制备及其临床评价

根据GenBank报道的人β-珠蛋白序列及中国人β-地中海贫血基因特点,设计并合成30条探针用于检测在中国人中发现的17个β-地中海贫血基因突变位点,然后将探针点至醋酸纤维膜上,制成反向杂交膜条,通过检测950份标本,对其临床检测效果进行评价.共制备了含30条寡核苷酸探针的反向杂交膜条,临床验证结果表明,以对照膜条为标准,制备膜条阳性符合率为99.76%(421/422),特异性为99.05%(523/528),总符合率为99.37%.对6份检测结果不符的标本进行测序验证,结果为TATAbox-28/Int双重杂合子(1份)、TATAbox--32杂合子(1份)、IVS-1-5杂合子(3份)、-30杂合子(1份),均为少见突变位点,与制备膜条检测结果一致;采用等位基因特异性PCR法检测5个常见β-地中海贫血基因位点(CD41/42,IVS-2-654,CD17,TATAbox--28,CD71/72),检测结果与制备膜条完全相符.结果表明,研制的β-地中海贫血基因检测反向杂交膜条敏感度高,特异性好,不仅能准确检测中国人常见的β-地中海贫血基因类型,而且还能检测少见基因突变类型.     

反向线性探针杂交在检测实验动物沙门菌中的研究

目的建立简便、快速、准确、灵敏、特异的沙门菌检测方法。方法根据沙门菌argT基因序列设计通用引物和3'、5'均加有polyC的特异性探针。上游引物5'标记生物素,将探针线性固定在硝酸纤维素膜上,使沙门菌PCR扩增产物与探针进行杂交,通过优化杂交条件,建立反向线性探针杂交检测方法。利用该方法对重庆地区74只实验动物进行检测,同时与传统分离培养方法比较。结果反向线性探针杂交方法灵敏度高,对沙门菌PCR扩增产物在3ng/μL以上可有效检测。从细菌分离培养及DNA提取到PCR扩增及反向杂交结束仅需27h。该检测方法特异性高,对6种非沙门菌的检测中,其特异性为100%。应用传统分离培养方法和反向线性探针杂交方法分别检测42只KM小鼠和32只SD大鼠,两种方法检测结果一致性为100%。结论反向线性探针杂交检测方法,具有快速、可靠、敏感和特异的特点,可用于沙门菌感染时的检测,适合应用于实验动物沙门菌的监测。     

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。     

一种基于寡核苷酸微阵列芯片的多重可扩增探针杂交技术

多重可扩增探针杂交技术(multiplex amplifiable probe hybridization,MAPH)是近年来发展起来的一种用于基因组中DNA拷贝数检测的新技术。并发展了一种基于寡核苷酸微阵列芯片的MAPH技术。该方法根据所检测的DNA序列,制备若干具有通用引物的FCR产物作为可扩增探针组,与固定在尼龙膜上待测的基因组DNA杂交。用磁珠回收特异性杂交的探针,经生物素标记的通用引物扩增后,与相应的寡核苷酸微阵列芯片杂交。该特异性的寡核苷酸微阵列芯片包括10个抗肌营养不良基因的外显子探针和阴性、阳性探针。杂交清冼后,链霉亲和素-Cy3染色用芯片扫描仪得到杂交的荧光图像。分析荧光信号的强度差异给出特定基因片段拷贝数的变化。该方法用微阵列技术代替MAPH中的电泳检测技术,可大幅度增加检测的通量。选择了一个正常男性、一个正常女性和一个肌营养不良症患者的基因组DNA来进行验证。结果表明,该方法能够同时给出抗肌营养不良基因多个外显子中的基因片段拷贝数差异信息。     

产气荚膜梭菌实时荧光PCR方法的建立

目的:利用荧光定量PCR技术,建立快速敏感特异的检测产气荚膜梭菌的方法。方法:以产气荚膜梭菌基因为靶序列设计引物和探针,以自产气荚膜梭菌菌株中提取的DNA为模板,优化引物和探针的浓度比,同时验证方法的特异性、敏感性。结果:建立的反应体系在上游引物浓度为0.45μmol/L、下游引物浓度为0.15μmol/L、探针浓度为0.3μmol/L时,具有良好的特异性和敏感性,与创伤弧菌等12种相关细菌均无交叉反应;对纯菌检测的灵敏度低于10 CFU/反应体系。结论:建立的实时荧光PCR方法特异、灵敏、快速,能对战时气性坏疽做出快速准确的报告,实现对这种战时高发疾病的安全、快速和定量检测。     

Haplosporidiosis in the Pacific oyster Crassostrea gigas from the French Atlantic coast

Two cases of haplosporidian infection occurred during 1993 in Pacific oysters Crassostrea gigas from the French Atlantic coast. The localization and ultrastructure of the plasmodia are described. In situ hybridization of infected tissue sections was conducted with DNA probes for oyster-infecting haplosporidians. The Haplosporidium nelsoni-specific DNA probe MSX1347 hybridized with the C. gigas parasite, and the H. costale-specific probe SSO1318 did not hybridize. Total genomic DNA was extracted from the infected tissue sections for polymerase chain reaction (PCR) amplification of the haplosporidian. PCR amplifications with H. nelsoni-specific primers and with 'universal' actin primers did not yield the expected products of 573 and 700 bp, respectively. A series of primers was designed to amplify short regions of small subunit ribosomal DNA (SSU rDNA) from most haplosporidians. The primers encompass a highly variable region of the SSU rDNA and did not amplify oyster DNA. PCR amplification of the infected C. gigas genomic DNA with these primers yielded the expected-sized product from the primer pair targeting the shortest region (94 bp). This PCR product was sequenced and it was identical to the corresponding SSU rDNA region of H. nelsoni.     

Design and application of an oligonucleotide microarray for the investigation of compost microbial communities

A microarray consisting of oligonucleotide probes targeting variable regions of the 16S rRNA gene was designed and tested for the investigation of microbial communities in compost. Probes were designed for microorganisms that have been previously reported in the composting process and for plant, animal and human pathogens. The oligonucleotide probes were between 17 and 25 bp in length and included mostly species-specific sequences. Validation of probe specificity and optimization of hybridization conditions were conducted using fluorescently labeled 16S rRNA gene PCR products of pure culture strains. A labeling method employing a Cy3 or Cy5-labeled forward primer together with a phosphate-conjugated reverse primer for the production of single stranded DNA after a digestion step was optimised and used to label target DNA. A combination of two different DNA extraction methods using both physical and chemical lysis was found to give the best DNA yields. Increased hybridization signal intensities were obtained for probes modified with a 12 mer T-spacer. The microarray was found to have a detection limit of 10(3) cells, although in compost spiking experiments, the detection limit was reduced to 10(5) cells. The application of the microarray to compost samples indicated the presence of Streptococcus, Acinetobacter lwoffii, and Clostridium tetani in various compost samples. The presence of A. lwoffii in those compost samples was confirmed by PCR using primers specific for the organism. The aim of this study was to develop a molecular tool that would allow screening for the presence or absence of different microorganisms within compost samples.     

Locked nucleic acids for optimizing displacement probes for quantitative real-time PCR

Displacement probes have recently been described as a novel probe-based detection system for use in both quantitative real-time polymerase chain reaction (PCR) and single nucleotide polymorphism genotyping analysis. Previous reports have shown that shorter probes (23 mer) had improved detection sensitivity relative to longer probes (29 mer), with the likely reason for this effect being the improved hybridization kinetics of shorter probes. Sterically modified locked nucleic acids (LNAs) have been used to improve the design of a range of real-time PCR probes by raising the melting temperature (Tm) of the probe and enabling shorter probe designs to be considered. A displacement probe for gapdh was designed and tested successfully, and this probe was then redesigned with LNAs to an 11 mer probe. This probe showed increased detection sensitivity compared with the original 26 mer probe. To detect the widest range of displacement probe designs at maximum sensitivity, we have also developed a novel fluorescence capture two-step PCR protocol. This method produces enhanced probe quenching with a single standardized protocol ideal for high-throughput applications. The displacement probes tested produced sensitive and efficient quantitative analyses of template serial dilutions when compared with a range of commercially available predesigned real-time PCR detection systems, including TaqMan MGB probes, QuantiTect MGB probes, and LUX primers.     

Molecular diagnostics,field validation,and phylogenetic analysis of Quahog Parasite Unknown (QPX), a pathogen of the hard clam Mercenaria mercenaria

Quahog Parasite Unknown (QPX) is a protistan parasite that causes disease and mortality in the hard clam Mercenaria mercenaria. PCR primers and DNA oligonucleotide probes were designed and evaluated for sensitivity and specificity for the QPX organism specifically and for the phylum Labyrinthulomycota in general. The best performing QPX-specific primer pair amplified a 665 bp region of the QPX small-subunit ribosomal DNA (SSU rDNA) and detected as little as 1 fg cloned QPX SSU rDNA and 20 fg QPX genomic DNA. The primers did not amplify DNA of uninfected hard clams M. mercenaria or of the thraustochytrids Schizochytrium aggregatum, Thraustochytrium aureum, and T. striatum. The general labyrinthulomycete primers, which were designed to offer broader specificity than the QPX primers, amplified a 435 bp region of SSU rDNA from QPX, and a 436 to 437 bp region of SSU rDNA from S. aggregatum, T. aureum, and T. striatum, but did not amplify that of the clam M. mercenaria. Field validation of the QPX-specific primer pair, through comparative sampling of 224 clams collected over a 16 mo period from a QPX endemic site in Virginia, USA, indicated that the PCR assay is equivalent to histological diagnosis if initially negative PCR products are reamplified. Oligonucleotide DNA probes specific for QPX and the phylum Labyrinthulomycota were evaluated for in situ hybridization assays of cell smears or paraffin-embedded tissues. Two DNA probes for QPX offered limited sensitivity when used independently; however, when used together as a probe cocktail, sensitivity was greatly enhanced. The probe cocktail hybridized to putative QPX organisms in tissues of hard clams collected from Virginia, New Jersey, Massachusetts and Canada, suggesting that the QPX organisms in these areas are either very closely related or the same species. The QPX probe cocktail did not hybridize with clam tissue or with the thraustochytrids S. aggregatum, T. aureum, and T. striatum. The labyrinthulomycete DNA probe hybridized with QPX and the 3 thraustochytrids, with no background hybridization to clam tissue. SSU rDNA sequences were obtained for the putative QPX organisms from geographically distinct sites. Phylogenetic analyses based on the QPX and Labyrinthulomycota sequences confirmed earlier reports that QPX is a member of this phylum, but could not definitively demonstrate that all of the QPX organisms were the same species.     

环介导间接PCR检测方法的建立

建立环介导间接PCR检测体系,为分子诊断提供一种新的检测工具。以质粒pUC18的核苷酸序列为模板,设计两条特异性探针,采用常规PCR技术将特异性探针标记于大豆Lectin基因的左右两端充当报告基因,此标记的报告基因与待检的pUC18质粒经杂交和缺口补平后形成一环状DNA分子,然后采用反向PCR技术扩增报告基因,建立针对pUC18质粒的环介导间接PCR检测方法。结果表明,该检测方法的检测底限为0.32 pg/μL,与常规PCR相当,并且与其他质粒和动物DNA检测无交叉反应,是一种简单、快速、灵敏、特异的PCR检测方法。     

RTPrimerDB: the real-time PCR primer and probe database

The real-time polymerase chain reaction (PCR) methodology has become increasingly popular for nucleic acids detection and/or quantification. As primer/probe design and experimental evaluation is time-consuming, we developed a public database application for the storage and retrieval of validated real-time PCR primer and probe sequence records. The integrity and accuracy of the data are maintained by linking to and querying other reference databases. RTPrimerDB provides free public access through the Web to perform queries and submit user based information. Primer/probe records can be searched for by official gene symbol, nucleotide sequence, type of application, detection chemistry, LocusLink or Single Nucleotide Polymorphism (SNP) identifier, and submitter's name. Each record is directly linked to LocusLink, dbSNP and/or PubMed to retrieve additional information on the gene/SNP for which the primers/probes are designed. Currently, the database contains primer/probe records for human, mouse, rat, fruit fly and zebrafish, and all current detection chemistries such as intercalating dyes (SYBR Green I), hydrolysis probes (Taqman), adjacent hybridizations probes and molecular beacons. Real-time PCR primer/probe records are available at http://www.realtimeprimerdatabase.ht.st.     

Rapid simultaneous detection and identification of six species Candida using polymerase chain reaction and reverse line hybridization assay

The aim of this study was to develop and evaluate PCR based reverse line blot (RLB) hybridization assay for rapid detection of the most common Candida isolates from clinical specimens. A pair of universal primers targeting the ITS2 region of the gene from 28S rRNA to 5.8S rRNA was designed for PCR amplification of DNA from 6 Candida species (C. albicans, C. tropicalis, C. krusei, C. glabrata, C. parapsilosis, C. dubliniensis), the reverse primer was biotin labeled. PCR products, which were 302-441 bp length, were hybridized with 6 specific oligonucleotides probes immobilized on a nylon membrane. These 6 probes proved specific (they hybridized with only their target molecules). The assay was shown to be sensitive in detecting yeast to a concentration of 10 CFU/ml. This method was used to test 100 isolates and 200 vaginal swabs. The results agreed with those of culture for all but 3 of 100 isolates. Sequencing was performed on these 3 samples and confirmed that the culture results were inaccurate. Our results show the PCR-RLB positive rate (49%) is higher than culture (39%) and smear microscopic screening (27%) (P<0.05). In conclusion, the PCR/RLB developed in this study is specific and offers increased sensitivity compared to culture for the detection of Candida species in swab specimens. Moreover, the improved detection of cases of polycandidal candidiasis is advantageous.     

Rapid Detection and Quantification of Members of the Archaeal Community by Quantitative PCR Using Fluorogenic Probes

We describe a rapid, reproducible, and sensitive method for detection and quantification of archaea in naturally occurring microbial communities. A domain-specific PCR primer set and a domain-specific fluorogenic probe having strong and weak selectivity, respectively, for archaeal rRNA genes (rDNAs) were designed. A universal PCR primer set and a universal fluorogenic probe for both bacterial and archaeal rDNAs were also designed. Using these primers and probes, we demonstrated that detection and quantification of archaeal rDNAs in controlled microbial rDNA assemblages can be successfully achieved. The system which we designed was also able to detect and quantify archaeal rDNAs in DNA samples obtained not only from environments in which thermophilic archaea are abundant but also from environments in which methanogenic archaea are abundant. Our findings indicate that this method is applicable to culture-independent molecular analysis of microbial communities in various environments.