PCR结合反向膜杂交快速鉴定临床常见念珠菌方法学的建立

目的建立PCR结合反向膜杂交技术快速检测临床常见致病念珠菌的方法。方法根据真菌rDNA高度可变区设计白色念珠菌、热带念珠菌,近平滑念珠菌,都柏林念珠菌,光滑念珠菌、克柔念珠菌的种特异性探针,加尾后固定在尼龙膜上,生物素标记的通用引物扩增转录间隔区ITS1,利用反向膜杂交技术快速检测念珠菌种群。结果以上六种念珠菌均能扩增出大小为218 bp的条带,每张膜上只有与靶序列对应的探针点有阳性结果,其他探针点均为阴性。正常人类基因组DNA及临床常见的几种细菌DNA扩增结果均为阴性。结论 PCR结合反向膜杂交技术方法学的建立,可以快速检测临床常见致病的念珠菌。该方法特异性高,耗费时间短。可以快速、准确的为临床抗真菌药物的使用提供指导依据。     

应用DNA探针检测甲氧西林耐药葡萄球菌

目的建立一种快速检测葡萄球菌和甲氧西林耐药葡萄球菌的斑点杂交技术。方法设计金黄色葡萄球菌nuc基因、甲氧西林耐药mecA基因、葡萄球菌tuf基因的特异引物,用聚合酶链反应合成其特异DNA探针,并用生物素标记,分别与固定在硝酸纤维素膜上的标准菌株和临床分离株模板DNA杂交,观察其敏感性和特异性。结果3对引物分别扩增出270bp、310bp、370bp3种DNA探针,均具有高度特异性。50株金黄色葡萄球菌tuf、nuc基因均为阳性：mecA基因阳性者22株。30株表皮葡萄球菌tuf基因均为阳性,nuc基因均为阴性,mecA基因阳性者9株。而其他非葡萄球菌与3种DNA探针杂交结果均为阴性。该方法可检测出1 ng细菌DNA。结论斑点杂交技术检测耐甲氧西林葡萄球菌快速、有效,具有较高的应用价值。     

分子杂交化学发光法检测血清中乙型肝炎病毒DNA

目的：用非放射性的异羟基洋地黄毒甙（地高辛，Dig)标记探针与靶DNA杂交，结合化学发光检测系统检测乙肝患者血清中HBV DNA。方法：应用灵曼公司生产的Dig DNA标记检测试验盒。将Dig标记的HBV DNA探针与点印迹在尼龙膜或硝酸纤维素膜上的靶DNA杂交，杂交后的膜与Dig－AP抗体保温，然后用化学发光底物CSPD与之作用，导致在波长477nm处发光，感光于X光底片上。结果：待检的129份血清标本，用此法检测HBV DNA阳性率达58．4％，敏感性2.0pg-0.5pg。为了比较，329份血清标本用显色法测定，阳性率45．0％，敏感性5.0pg-1.0pg。对照组呈阴性结果。结论：Dig标记探针分子杂交化学发光法具有较高特异性，敏感性和简便等优点，可用于血清中HBV DNA的检测。     

云南石屏汉族与彝族患者外阴阴道念珠菌病的菌种构成

目的探讨云南省石屏县汉族和彝族患者外阴阴道念珠菌病(VVC)的菌种构成情况。方法收集来源于云南省石屏地区念珠菌病患者的阴道分泌物143份,其中汉族84份,彝族59份;根据科玛嘉(CHROMagar)显色培养基人工鉴定结果,同时经PCR扩增ITS1-ITS4区段,用测序法进行菌种鉴定,用DNAStar软件对测序结果进行分析。将科玛嘉显色培养基法与测序结果进行对比。结果科玛嘉显色培养基法检出4种菌种,分别是光滑念珠菌,克柔念珠菌,白色念珠菌和热带念珠菌。测序法在VVC阴道分泌物中检测出5种菌种,分别是白色念珠菌113株、光滑念珠菌25株、近平滑念珠菌3株、热带念珠菌和季也蒙念珠菌各1株;汉族患者未检测到季也蒙念珠菌,彝族患者未检测到热带念珠菌。汉族患者的白色念珠菌、光滑念珠菌、近平滑念珠菌和热带念珠菌的携带率分别为78.57%(66株)、17.86%(15株)、2.38%(2株)和1.19%(1株);彝族患者的白色念珠菌、光滑念珠菌、近平滑念珠菌和季也蒙念珠菌的携带率分别为79.66%(47株)、16.95%(10株)、1.69%(1株)和1.69%(1株)。云南省石屏县汉族VVC患者的念珠菌菌种构成比与彝族患者相比较,差异无统计学意义(P0.05)。结论 PCR测序法优于科玛嘉显色培养基法鉴定念珠菌的菌种,云南省石屏县汉族与彝族VVC患者的病原菌以白色念珠菌为主。     

中国汉族人群中白色念珠菌的25S rDNA基因分型及耐药性分析

目的调查白色念珠菌25S核蛋白体脱氧核糖核酸(rDNA)基因型分布,并分析白色念珠菌各基因型与耐药性之间的相关性。方法以经API 20C AUX真菌鉴定板和科玛嘉念珠菌显色培养基鉴定到种的76株白色念珠菌为研究对象,转沙保罗氏培养基纯培养24 h,收集菌液,裂解法提取质粒DNA,用特异性引物扩增白色念珠菌25S rDNA基因,根据聚合酶链反应(PCR)产物电泳图谱进行基因分型;采用微量肉汤稀释法测定各基因型白色念珠菌对4种常用抗真菌药物5-氟胞嘧啶(5-FC)、两性霉素B(AMB)、氟康唑(FCZ)、伊曲康唑(ICZ)的最低抑菌浓度(MIC)。结果 76例白色念珠菌分为A、B、C 3个基因型(A型48例,B型8例,C型20例),按照有无长度为379 bp的Ⅰ型内含子的插入,将76例白色念珠菌分为有内含子组和无内含子组,分别进行4种抗真菌药物耐药率的比较,结果发现2组间对FCZ耐药率差异有统计学意义(P0.01),而对5-FC、AMB和ICZ耐药率差异无统计学意义(P值均大于0.05)。结论白色念珠菌25S rDNA基因内含子(379 bp)的缺失或者插入可能与其对FCZ的耐药性有关。     

两种酵母样真菌培养鉴定方法评价

目的 选择和评价实用性强、快速、准确酵母样真菌培养鉴定方法。为临床诊断和治疗真菌感染性疾病提供可靠的病原学依据。方法 将API酵母样真菌鉴定试条鉴定出的白色念珠菌（48株），热带念珠菌（15株），克柔氏念珠菌（19株）。光滑念珠菌（6株）转种科玛嘉酵母样真菌显色平板，经35℃，24—48小时培养，分析科玛嘉酵母样真菌显色平板鉴定结果正确率。结果 科玛嘉显色平板鉴定出白色念珠菌43株，热带念珠菌13株，克柔氏念珠菌8株，光滑念珠菌5株。科玛嘉显色平板法与酵母样真菌鉴定符合率为80％-90％．结论 科玛嘉显色平板法操作简单、快速。价格低廉，可作为酵母样真菌鉴定的初筛方法。     

聚合酶链反应反向斑点杂交快速检测及鉴定念珠菌

目的　研究以非培养为基础的快速检测与鉴定念珠菌的方法。方法　将白色念珠菌、热带念珠菌、光滑念珠菌种特异探针加尾后固定在硝酸纤维素膜上;用氯化苄法提取念珠菌ＤＮＡ,采用真菌特异通用引物ＰＣＲ 扩增ＤＮＡ,在扩增过程中用Ｂｉｏ１１ｄＵＴＰ 标记扩增产物,然后分别与白色念珠菌、热带念珠菌、光滑念珠菌特异探针杂交。结果　对１４ 种念珠菌扩增均为阳性,对３ 种常见细菌扩增均为阴性。３ 种念珠菌只与其对应探针杂交。结论　该方法简便、快速、准确,适于临床实验室快速检测与鉴定念珠菌。     

基因芯片技术检测临床常见致病性念珠菌和新型隐球菌及ERG11基因点突变的实验分析

目的建立基因芯片技术检测临床常见致病性念珠菌和新型隐球菌及氟康唑耐药白念珠菌相关ERG11基因点突变的试验方法。方法针对临床常见的5种致病性念珠菌和新型隐球菌5.8S r DNA与28S r DNA间的内转录间区2(ITS-2)基因设计、合成一系列寡核苷酸探针,制备寡核苷酸芯片,以鉴定5种致病性念珠菌和新型隐球菌;设计、合成能够特异性扩增ERG11基因点突变的引物,并采用不对称荧光聚合酶链反应(PCR)扩增ERG11基因,将PCR产物与芯片进行杂交。结果采用特异性引物和不对称荧光PCR从12株临床分离耐药株中成功扩增出ERG11基因4个突变点;采用基因芯片杂交技术成功鉴定5种临床常见致病性念珠菌和新型隐球菌。结论制备的寡核苷酸基因芯片,可以用于鉴定临床常见的致病性念珠菌和新型隐球菌。     

科玛嘉念珠菌显色培养基在快速检测念珠菌的应用

目的 评价科玛嘉（CHROMagar)念珠菌显色培养基在快速检测念珠菌的应用价值。方法 将标本同时接种于沙保弱培养基（SDA）和CHROMagar念珠菌显色培养基上进行培养,SDA生长的酵母菌经ATB仪鉴定菌株,CHROMagar念珠菌显色培养基根据菌落颜色直接判读。结果 1874份标本经SDA培养出白色念珠菌412株,热带念珠菌167株,克柔念珠菌31株,光滑念珠菌154株;CHROMagar念珠菌显色培养基鉴定符合率为：白色念珠菌100％,热带念珠菌97．6％,克柔念珠菌96．7％,光滑念珠菌97．4％。69份标本为混合念珠菌感染,其中41例（59．4％）在SDA上很容易漏检,而CHROMagar念珠菌显色培养基一目了然。结论 CHROMagar念珠菌显色培养基能快速、简便、准确地分离鉴定临床常见念珠菌,明显促进对混合念珠菌感染的识别。     

8种深部真菌DNA微阵列鉴定方法的建立及应用

目的 建立一种能快速鉴定临床常见8种深部真菌的DNA微阵列.方法 选取临床常见的8种真菌,包括白色假丝酵母、光滑假丝酵母、热带假丝酵母、近平滑假丝酵母、土曲霉、黄曲霉、烟曲霉和米根霉.利用通用引物扩增真菌ITS区,生物素标记目的基因.针对扩增靶序列的可变区段设计探针,以尼龙膜为载体,建立DNA微阵列.采用所建立的DNA微阵列对45株临床真菌分离株进行检测.结果 应用DNA微阵列技术成功地对8种临床常见真菌菌种进行了检测,检测时间可缩短至8 h.白色假丝酵母、光滑假丝酵母、热带假丝酵母、近平滑假丝酵母、米根霉、黄曲霉以及土曲霉的探针的检测特异度高,无非特异性反应;烟曲霉的检测探针与黄曲霉之间有微弱非特异性反应,但是不影响鉴定结果.45例临床真菌分离株的DNA微阵列鉴定结果与临床检出结果完全一致.结论 该研究建立的DNA微阵列技术可在8 h内快速、准确地检测临床常见深部感染真菌菌种,具备应用于临床辅助诊断的前景.     

Evaluation of a DNA probe of plasmid origin for the detection of Chlamydia trachomatis in cultures and clinical specimens

This study evaluates five cryptic plasmid-derived DNA probes in a 4-h slot-blot hybridization assay for the detection of Chlamydia trachomatis in cultures and clinical specimens. The probes, consisting of either the entire cloned 7.5 kbp cryptic plasmid pSE8 or one of four Hin dIII/Eco RI fragments measuring 710, 1055, 710, and 500 bp, respectively, were labelled with Photoprobe biotin. The probe was detected using a streptavidin-alkaline phosphatase conjugate followed by addition of BCIP and NBT. The sensitivity of the assay, using 25 ng of probe DNA, ranged from 50 pg (with the entire plasmid as probe) to 5 ng (with the 500 bp fragment as probe). A total of 103 reference strains of Chlamydia trachomatis and other bacteria were tested for reactivity with the probes. All 18 reference strains of C. trachomatis hybridized with the probes. None of the DNA from the heterologous organisms tested was found to hybridize with any of the probes. A total of 174 samples taken from patients visiting the STD clinic at the University Hospital, University of Seville were used in the study. The overall sensitivity of the assay, using the 710 bp biotinylated probe was 94.5% compared to culture while the specificity was 97.5%. Positive and negative predictive values were 96.5% and 97.5%, respectively. It appears that the plasmid-derived probes used in this study could serve as useful tools for the rapid and specific detection of Chlamydia trachomatis in cell cultures and clinical specimens.     

常用消毒剂对念珠菌最小杀菌浓度的测定

在实验室内测定了８种常用消毒剂对８种２５株念珠菌的最小杀菌浓度。结果表明，不同消毒剂对同种念珠菌的杀灭作用不一致，同一消毒剂对不同种念珠菌的杀灭作用亦相异，但对同种内不同株者无差别。在各种念珠菌中，以白色念珠菌与热带念珠菌的抗力较强，其它种念珠菌较弱，且基本相似。ｐＨ与作用时间变化可影响消毒效果。     

Construction and use of PCR primers from a 65 kDa mannoprotein gene for identification of C. albicans

A method for detection of Candida albicans in biological samples (blood, serum, urine) was developed by the use of polymerase chain reaction (PCR) amplification of a DNA fragment of a gene coding for a 65 kDa mannoprotein of C. albicans (CaMP65). The PCR amplifies a 220 bp fragments whose specificity for C. albicans was demonstrated by Southern blot with a non-radioactive probe, leading to the differentiation from all other yeast species or human and bacterial DNA. The sensitivity of this assay was 5-10 C. albicans cells per milliliter of biological sample.     

Evaluation of a DNA probe of plasmid origin for the detection of Neisseria gonorrhoeae in cultures and clinical specimens

This study evaluates a cryptic plasmid-derived DNA probe in a dot-blot hybridization assay of 4-h duration, using both known bacterial isolates and clinical specimens. The probe, consisting of a 237 bp segment of the plasmid-encoded gene cppB, sequences of which are also found in the chromosome, was labelled with digoxigenin-11-dUTP. The sensitivity of the probe was approximately 25 pg of DNA or 500 cfu of Neisseria gonorrhoeae. A total of 170 reference strains of Neisseria gonorrhoeae and other related bacteria were tested for reactivity with the probe. All N. gonorrhoeae strains, including three plasmid-free strains, hybridized with the probe. Among the heterologous bacterial cultures, only one strain of N. cinerea reacted with the probe when the cell concentration was 5 x 10(6) cfu. The probe was also evaluated in a clinical study. A total of 201 patients visiting the STD clinic at the University Hospital, University of Seville, participated in the study. The sensitivity of the assay was 95% while the specificity was 98%. Positive and negative predictive values were 97% and 98%, respectively. It appears that the plasmid-derived probe used in this study could serve as a useful tool in the rapid and specific detection of Neisseria gonorrhoeae in clinical specimens.     

Detection of hepatitis B virus DNA in serum with nucleic acid probes labelled with 32P, biotin, alkaline phosphatase or sulphone.

To facilitate the clinical application of dot-blot hybridization for assaying hepatitis B virus (HBV) DNA, we compared the ability of nucleic acid probes labelled with 32P or with various non-radioactive markers to detect HBV DNA in patient serum. Cloned HBV DNA was hybridized with (1) 32P-labelled HBV DNA cloned in M13, (2) the 32P-labelled HBV RNA probe included in the HepProbe kit, (3) an alkaline phosphatase-labelled synthetic oligonucleotide of HBV, (4) biotin-labelled HBV DNA, and (5) sulphonated HBV DNA. Detection was either by autoradiography or an enzymatic colour reaction. The lowest level of detection of cloned HBV DNA was achieved with the 32P-labelled HBV RNA probe (0.3 pg HBV DNA, corresponding to 3 x 10(4) genomes in 50 microliters), followed by the 32P-labelled DNA probe (0.3-2 pg), sulphonated DNA (1-2 pg), biotin-labelled DNA (4 pg), and an alkaline phosphatase-labelled synthetic oligonucleotide (30 pg). Subsequently, sera from 159 patients with various constellations of HBsAg, HBeAg, and anti-HBe were tested with the most sensitive radioactive method (HepProbe) and the corresponding nonradioactive method (sulphonation). The overall concordance rate was 71% (r = 0.42). Compared with HepProbe results, sulphonation showed a sensitivity of 80% and a specificity of 67%. We conclude that radiolabelling (in particular 32P-labelling of HBV RNA) still allows the most sensitive and reliable detection of HBV DNA in patient serum using conventional dot-blot hybridization.     

In Vitro Activity of 5-Fluorocytosine Against Candida and Torulopsis Species

One hundred yeasts were studied. Tests included detailed identification and determination of 24- and 48-hr minimal inhibitory concentrations and minimal fungicidal concentrations of 5-fluorocytosine (5-FC). Final identifications included 57 isolates of Candida albicans, 15 isolates of C. tropicalis, 13 isolates of C. parapsilosis, 7 isolates of Torulopsis glabrata, 3 isolates of C. guilliermondii, 2 isolates each of C. stellatoidea and Cryptococcus neoformans, and 1 isolate of Candida krusei. Twenty-three original identifications were in error; these involved mostly C. albicans, C. tropicalis, C. parapsilosis, and T. glabrata. Inhibitory end point readings based on 24 hr of incubation were misleading. Whereas 79 of 91 isolates of Candida appeared to be inhibited at 24 hr by 12.5 mug or less of 5-FC/ml, only 52 were inhibited at 48 hr; whereas only 12 isolates appeared to be resistant to 100 mug/ml after 24 hr, 37 were resistant after 48 hr. Susceptibility varied amount the different species. C. tropicalis was the most susceptible, with 10 of 15 isolates (66.7%) being inhibited by 12.5 mug or less/ml and 7 (46.7%) being killed by 100 mug or less/ml. C. albicans was similarily susceptible; 33 of 57 isolates (57.9%) were inhibited by 12.5 mug or less/ml and 25 (43.9%) were killed by 100 mug or less/ml. C. parapsilosis was quite resistant, as only 4 of 13 isolates (30.8%) were inhibited by 12.5 mug or less/ml and 3 (23.1%) were killed by 100 mug or less/ml.     

两种DNA探针在肺炎链球菌DNA检测中的应用价值

目的研究寡核苷酸探针和长链DNA探针在肺炎链球菌检测中的诊断价值。方法合成肺炎链球菌特异的21bp寡核苷酸探针和263bp长链DNA探针并用生物素标记,两种探针分别与细菌全染色体DNA进行斑点杂交,比较两种探针检测痰液标本的敏感性和特异性,并与痰培养法比较。结果寡核苷酸探针和263bp探针检测肺炎链球菌基因组DNA的敏感性分别是100ng和1ng。培养法和杂交法同时检测100份痰标本,培养法、21bp探针和263bp探针的阳性检出率分别是7%、6%和17%。结论263bp长链DNA探针是检测肺炎链球菌DNA的较佳选择。     

Development of a rapid method for determining the susceptibility of Mycobacterium tuberculosis to isoniazid using the Gen-Probe DNA Hybridization System.

A rapid test was developed for determining the susceptibility of Mycobacterium tuberculosis to isoniazid by using nucleic acid hybridization. The method was based on quantification of total mycobacterial rRNA hybridized to a 125I-labeled DNA probe in the absence and presence of various concentrations of isoniazid. The radioactive hybridized complex was isolated by adsorption to hydroxyapatite crystals and measured in a gamma counter. The susceptibilities of four reference strains and 20 clinical isolates were compared by the Gen-Probe DNA Hybridization System and the critical concentration method. Overall agreement between the two methods was excellent. Results were obtained with the DNA probe after 3 to 5 days of incubation instead of the 14 to 21 days required for the critical concentration method. These findings indicate that susceptibility testing of M. tuberculosis by nucleic acid hybridization has merit for the clinical laboratory. Additional studies are needed to determine the efficacy of the DNA probe method for determining the susceptibility of M. tuberculosis to other antimycobacterial agents and its correlation with clinically significant levels of resistance.     

血液细菌16S rRNA基因检测的诊断价值

目的探索快速可靠的检测细菌感染的新方法。方法用PCR技术扩增实验室保留株10株的16SrRNA基因,以乙型肝炎病毒-DNA、白假丝酵母菌和人类基因组DNA为对照,检测该方法的特异性;采用10倍比稀释法进行该方法的灵敏度检测。结果对所测细菌株均获得475bp扩增产物,而与乙型肝炎病毒-DNA、白假丝酵母菌和人基因组DNA无交叉反应;PCR最低能检测1.5×104/L大肠埃希氏菌。结论16SrRN基因PCR检测细菌感染的方法具有特异性、快速性和敏感性高等特点。     

阴道感染三种病原菌多重PCR检测方法的建立及其应用研究

目的 探讨阴道感染病原体多重PCR快速检测白念珠菌、加德纳菌和阴道滴虫的方法建立及其临床应用。方法 根据白念珠菌EO3基因、加德纳菌ITS-23srRNA基因及阴道滴虫TVK3～TVK7基因设计特异性PCR引物,采用加热煮沸裂解法制备DNA模板,进行多重PCR扩增及琼脂糖凝胶电泳检测分析,在同一体系内完成三种阴道常见病原体的快速检测。结果 经特异性试验证明所建立的方法能特异地检测出白念珠菌125 bp,加德纳菌433 bp和阴道滴虫300 bp的目的片段,而其它非目的菌株均不能扩增出相应片段; 敏感性试验证实白念珠菌为10 cfu/ml,加德纳菌为10 cfu/ml,阴道滴虫为20 cfu/ml; 对30份已知临床标本和10份模拟混合标本进行检测均可检测出相应的病原体。结论 实验证明该研究所建立的白念珠菌、加德纳菌和阴道滴虫的多重PCR体系,具有灵敏度高、特异度强,可一步同时检测阴道分泌物中三种病原体,用于阴道感染的早期快速诊断。