聚合酶链反应快速检出耐甲氧西林金黄色葡萄球菌的研究

目的 建立耐甲氧西林金黄色葡萄球菌的快速检出方法。方法 利用聚合酶锭反应（ＰＣＲ）快速检出耐甲氧西林金黄色葡萄球菌（金葡菌），建立一种从葡萄球菌中快速提取ＤＮＡ方法，以粗提ＤＮＡ作为ＰＣＲ模板，检测编码耐甲氧西林金葡菌青霉素结合蛋白２（ＰＢＰ２ａ）的ｍｅｃＡ基因。结果 １８４金葡菌有ＰＣＲ方法及药敏法比较，药敏法鉴定为耐甲氧西林金葡菌（ＭＲＳＡ）５８株，仅一株ＰＣＲ扩增ｍｅｃＡ基因阴性，１２６株甲     

应用聚合酶链反应快速检测临床标本中的肺炎支原体

应用聚合酶链反应（ＰＣＲ）技术检测临床标本中的肺炎支原体（ＭＰ）。在１４０例非细菌性肺炎患者的标本中，ＰＣＲ试验阳性３０例。其中间接血凝试验阴性面ＰＣＲ试验阳性者２１例。ＰＣＲ阳性标本经ＭＰ５－４寡核苷酸探针检测验证，均为阳性结果。     

Tropheryma whippelii endocarditis confirmed by polymerase chain reaction

This report concerns a patient with cardiac manifestation ofWhipple's disease. For the first time, the gene that encodesthe 16s rRNA of Tropheryma whippelii was identified in a nativeaortic valve by means of a polymerase chain reaction technique.DNA amplification gives evidence of Tropheryma whippelii asa causative organism in infective endocarditis.     

聚合酶链反应快速检出耐甲氧西林凝固酶阴性葡萄球菌的研究

目的快速检出耐甲氧西林凝固酶阴性葡萄球菌。方法利用聚合酶链反应（ＰＣＲ）检测耐甲氧西林凝固酶阴性葡萄球菌，建立一种从葡萄球菌中快速提取ＤＮＡ方法，用粗提物为模板，检测编码耐甲氧西林葡萄球菌青霉素结合蛋白的ｍｅｃＡ基因，扩增产物为５３３ｂｐ大小。结果药敏法鉴定为耐甲氧西林凝固酶阴性葡萄球菌２１株，１００％ｍｅｃＡ基因阳性，２３株对甲氧西林敏感的凝固酶阴性葡萄球菌中有９株用ＰＣＲ扩增出ｍｅｃＡ基因片段。利用ｒ－３２Ｐ标记寡核苷酸作为探针，Ｓｏｕｔｈ－ｅｒｎ杂交结果与ＰＣＲ检测结果完全一致，证实５３３ｂｐ片段为耐甲氧西林凝固酶阴性葡萄球菌ｍｅｃＡ基因特异性片段。结论ＰＣＲ方法具有快速、敏感的优点，并能特异地检出临界（ＭＩＣ＝０．５～２ｍｇ／Ｌ）耐甲氧西林凝固酶阴性葡萄球菌株。     

Molecular basis and hematological characterization of Hb H disease in Southeast Asia

We molecularly characterized sixty-seven cases of Hb H disease by the polymerase chain reaction. The strategy depends on amplifying the α-thalassemia-1 (α-thal-1) gene by prlmers flanking the breakpoint and sequence differences of the 3′ end of the α-globin gene and the nonhomologous elements I, II, and III among different types of α-thala-2. In the 67 cases studied, all involved α-thal-1 of the Southeast Asia type (SEA) In combination with deletional or nondeletional α-thal-2. Thirty-two cases were of the deletion form and 35 cases were of the nondeletion form. In 32 cases of the deletion form, 29 cases were rightward deletion (-α^3.7), and three cases were leftward deletion (-α^4.2). We found that all of the nondeletion forms were α-thal-1 of SEA type with Hb CS. After the subtyping of Hb H with -α^3.7, 26 out of 29 were type I deletion and 3 out of 29 were type II deletion. Comparlsons of clinical data of deletion forms and the nondeletion form showed that there were earlier occurrence of anemic symptoms and a larger erythrocyte volume in the nondeletion form group (P < 0.005). © 1994 Wiley-Liss, Inc.     

聚合酶链反应诊断军团菌感染的实验研究

目的早期诊断军团菌感染。方法采用聚合酶链反应（ＰＣＲ）检测嗜肺军团菌（Ｌｐ）ｍｉｐ基因的特异性片段，并应用于豚鼠米克戴德军团菌（Ｌｍ）感染后的组织标本检测。结果可以扩增出ＬｐＩ型和Ｌｍ６３０ｂｐ的特异性片段，其他临床分离株不被测得，检测灵敏度为１５０ｆｇ军团菌基因组ＤＮＡ（相当于１５～３０个军团菌）。对２２份Ｌｍ感染后不同时间获取的豚鼠组织标本，应用该法与培养法检测结果比较显示，在感染后３．５天培养法阳性率为８８．９％，ＰＣＲ法１００％，在感染后７．５天培养法阳性率为０，而ＰＣＲ法仍有２２．２％的阳性率。结论本方法敏感、特异，既能检出嗜肺军团菌，又能测得Ｌｍ，大大提高了军团病的诊断率     

Rare occurrence of Hb Lepore-Baltimore in African Americans: molecular characteristics and variations of Hb Lepores

Hb Lepore is the hybrid hemoglobin (Hb) composed of two α-globin chains and two δβ hybrid chains and is associated with the clinical findings of thalassemia minor in its heterozygous form. Hb Lepore can be found in many ethnic groups, commonly in southern European countries, but rarely in African Americans. The first Hb Lepore case in an African-American individual was named Hb Lepore-The Bronx (Hb Lepore-Boston). Hb Lepore-Washington-Boston and Hb Lepore-Baltimore with a breakpoint of (δ50Ser/β86Ala) were later reported. In this paper, we describe an Hb Lepore-Baltimore (δ68Leu/β84Thr) δβ-fusion gene with a different breakpoint detected for the first time in an African-American female. We have used state-of-the-art technology, combining protein- and DNA-based methods, in the analysis of the hybrid hemoglobin and discuss its molecular characteristics.     

多重PCR检测方法在鼠疫监测中的应用

目的验证在鼠疫疫源地调查中应用多重PCR方法快速检测鼠疫菌的实用性。方法在14个省的17个鼠疫监测点采集标本2524份。选用针对鼠疫菌特异序列的引物,并加入内部对照模板,直接从鼠肝、脾等脏器标本中进行鼠疫核酸检测,与细菌分离培养结果相比较并进行统计学分析。结果两种检测方法的符合率为92.67%。PCR方法阳性检出率为10.38%,较细菌培养阳性检出率(4.48%)高(x~2=682.25,P<0.01)。结论多重PCR方法可应用于鼠疫监测中,比传统方法迅速、灵敏,但还有待于优化。     

PCR快速检测腹泻患者粪中痢疾杆菌致病基因ipaH和ial

目的: 建立一种快速、特异、敏感的分子生物学诊断方法对腹泻患者粪中痢疾杆菌致病基因ipaH及ial进行检测.方法:分别应用常规培养生化血清学鉴定方法、普通PCR、巢氏PCR(nested-PCR)方法, 对71例腹泻患者粪标本进行检测. 痢疾杆菌致病基因ipaH及ial的扩增产物经电泳分离.结果: 在71例腹泻患者粪标本中, 常规培养生化血清学鉴定方法分离出福氏痢疾杆菌24例, 宋内痢疾杆菌23例, 志贺痢疾杆菌1例, 沙门菌23例. 采用普通PCR法检测痢疾杆菌粪标本48例, ipaH基因均阳性(100%);ial基因阳性34例(70.8%), 余14例为阴性. 对该14例粪标本进而采用巢氏PCR法进行ial基因扩增, 其中12份标本阳性. 48例痢疾杆菌粪标本中46例ipaH和ial基因为双阳性(46/48). 沙门菌粪标本23例中ipaH及ial基因表达均为阴性. 以上两种方法的诊断一致性检验Kappa = 0.937, 差异有统计学意义(P<0.05). 71例腹泻患者粪标本采用PCR和巢氏PCR法扩增得到的ipaH和ial基因特异片段与基因库中发表的标准菌株序列比较, 一致性为100%.结论:建立了快速诊断腹泻患者粪中痢疾杆菌致病基因ipaH和ial的PCR检测方法, 具有简便、快速、特异和敏感的特点.     

聚合酶链技术检测慢性前列腺炎病原体的评价(附2071例报告)

目的探索慢性前列腺炎的病因，提高诊治水平。方法应用聚合酶链技术(PCR)对门诊疑诊慢性前列腺炎患者的前列腺液行淋球菌(NG)、沙眼衣原体(CT)及解脲支原体(UU)检测。结果检出单纯NG感染33例(9．09％)，CT感染32例(5．15％)，UU感染250例(23％)，CT与UU合并感染13例，NG合并UU感染5例，NG合并CT感染4例，并应用PCR监测治疗效果。结论PCR检测慢性前列腺炎病原体具有快速、敏感性高、特异性强等优点，值得临床推广应用。     

原位逆转录PCR检测胃癌及癌前组织中端粒酶RNA及其临床意义

目的　研究胃癌及癌前病变胃粘膜端粒酶RNA的检测及其临床意义。方法　选取经病理组织学证实的胃粘膜活检标本 15 0例 ,包括慢性浅表性胃炎 3 2例、肠上皮化生 3 6例、不典型增生 3 4例、胃癌 48例 ,采用原位逆转录PCR、端粒重复序列扩增 (TRAP)法检测上述胃粘膜端粒酶RNA与端粒酶活性。结果　原位逆转录PCR技术检测胃粘膜活检标本端粒酶RNA阳性率为 5 5 .3 % (83 / 15 0 ) ,显著高于TRAP法检测端粒酶活性阳性率 (4 0 .0 % ,60 / 15 0 ) ,P <0 .0 5。端粒酶RNA在胃癌及癌前病变 (包括肠上皮化生与异型增生 )中检出率显著高于浅表性胃炎 (P <0 .0 5 ) ,胃癌中端粒酶RNA检出率亦显著高于肠上皮化生及不典型增生 (P <0 .0 5 ) ,但后两者比较差异无显著性 (P >0 .0 5 )。端粒酶RNA主要分布于胃粘膜癌细胞及癌前病变上皮细胞的胞核内。结论　端粒酶RNA与胃癌的发生密切相关。原位逆转录PCR技术检测胃粘膜端粒酶RNA对胃粘膜癌变的早期诊断和预测有重要价值 ,而且可能是较端粒酶活性更灵敏的生物学指标     

Molecular Basis of Hb H Disease in Southwest Iran

Although α⁰-thalassemia (thal) defects are not very frequent in the Iranian population, Hb H disease does occur in the country. We have analyzed the α gene cluster of 13 patients showing the presence of Hb H to establish the molecular background of this disease in southwest Iran (Shiraz and Hormozgan provinces). Using gap-polymerase chain reaction (gap-PCR) and direct DNA sequencing we have found the ? ?^MED-I deletion, the polyadenylation signal (poly A) mutations α^T-Saudiα and α^T-Turkishα, and Hb Constant Spring (Hb CS) in association with the common ? α^3.7 deletion. This study has revealed that: 1) at least six genotypes are responsible for Hb H disease in the area: ? α^3.7/? ?^MED-I; ? α^3.7/α^T-Saudiα; α^T-Saudiα/α^T-Saudiα; α^CSα/? ?^MED-I; ? ?^MED-I/α^T-Turkishα; and the atypical forms of Hb H disease ? α^3.7/α^CSα. 2) The molecular background of Hb H disease in the southwest area of Iran is more similar to the Mediterranean type than to the Southeast Asian. 3) Hb Bart's hydrops fetalis syndrome and mild, intermediate or severe postnatal Hb H disease conditions can be expected, but at a relatively low incidence. 4) The diagnostic flowchart for patients with microcytic hypochromic anemia should include iron deficiency, β-thal, α⁺- and α⁰-thal analyses.     

应用多重聚合酶链式反应鉴别布鲁杆菌

目的 探讨应用多重聚合酶链式反应(Muhiple-PCR)进行布鲁杆菌株种型的鉴别.方法 以6株布鲁杆菌标准菌株(牛种、羊种、猪种、犬种、绵羊附睾种、沙林鼠种布鲁杆菌标准菌株)作为阳性对照,以大肠埃希菌O∶157和小肠结肠炎耶尔森菌O∶9作为阴性对照,待测布鲁杆菌株29株.先用布鲁杆菌属特异性聚合酶链式反应(BCSP31-PCR)扩增上述待测布鲁杆菌株,扩增结果为阳性的菌株再用Muhiple-PCR方法进行布鲁杆菌种型鉴别.结果 29株待测布鲁杆菌株BCSP31-PCR方法扩增均为阳性,进一步Multiple-PCR方法扩增均为阳性,其中有20株鉴定为羊种菌,5株鉴定为猪种菌、3株鉴定为牛种菌、1株鉴定为犬种菌.结论 Multiple-PCR方法是一种快速、特异、简便、低风险的布鲁杆菌种型鉴定方法.     

结核杆菌DNA检测在肠结核与克罗恩病鉴别诊断中的价值

目的：探讨聚合酶链反应（PCR）技术对肠结核和克罗恩病（Crohn's disease,CD)的鉴别诊断价值。并对其内镜活检组织的病理改变进行比较,方法：C地38例肠结核和30例CD活检组织进行病理分析、抗酸染色镜检和PCR技术检测结核杆菌DNA。结果：（1）淋巴细胞聚集在CD远较肠结核多见（P＜0．05）;而肉芽肿,尤其是干酪样肉芽肿和肉芽肿的融合在肠结核却更多见（P＜0．05）。两者其他病理特点的差异在活检组织中不明显或难以发现;（2）PCR技术检测结核杆菌DNA总阳性率为63．2％（24／38例）;在与CD肉芽肿形态相同的肠结核组中阳性率为71．4％（10／14例）;在无肉芽肿瘤变的肠结核组中阳性率为64．7（11／17例）。而该技术在CD组无1例阳性。抗酸染色镜检总阳性率为21．1％（8／38例）。结论：内镜活检病理对肠结核与CD的鉴别诊断有一定局限。PCR技术是鉴别肠结核和CD极有价值的一种新方法。     

RT-PCR检测白血病患者mdr1表达的临床研究

目的：应用逆转录－聚合酶链反应（ＲＴ－ＰＣＲ）方法检测多药耐药基因（ｍｄｒ１）ｍＲＮＡ的表达,以探讨白血病细胞的抗药性及化疗效果的关系。方法：采用ＲＴ－ＰＣＲ法检测３３例不同类型白血病患者ｍｄｒ１、ｍＲＮＡ的表达,同时检测２０例正常骨髓做对照。结论：对照均为阴性表达,而初始组ｍｄｒ１的阳性表达率为３５．００％,复发、难治组ｍｄｒ１阳性表达率为８４．６２％（Ｐ〈０．０１）,ｍｄｒ１阳性与ｍｄｒ１阴性     

Rapid detection of chimeric bcr/abl mRNAs in acute lymphoblastic and chronic myeloid leukemia by the polymerase chain reaction

Summary We have developed a rapid method for the detection of bcr/abl mRNAs, the products of the BCR/ABL fusion genes. The method is based on the polymerasechain-reaction (PCR). Through the use of additional internal primers it is possible to detect directly a single Ph¹-positive cell among 10⁵ unaffected cells thus omitting time-consuming blotting procedures. The whole analytical procedure starting from RNA isolation to agarose gel electrophoresis including two rounds of PCR can be performed in less than six hours.Supported by the Wilhelm Sander-Stiftung, Neustadt/Donau and the Deutsche Krebsgesellschaft, Landesverband Berlin     

实时定量多聚酶链反应技术在诊断曲霉菌感染中的应用

近年曲霉菌感染的发病率急剧上升,曲霉病的病死率亦高居不下。早期诊断曲霉菌感染是改善预后的关键。曲霉病临床表现无特异性,早期诊断困难。最新的实时定量聚合酶链反应技术集快速、灵敏、特异、定量等优点于一体,在曲霉菌感染的诊断中有着广阔的应用前景。     

二步法聚合酶链反应检测血液恶性肿瘤患者曲霉菌感染的研究

目的探讨二步法PCR检测曲霉菌的敏感性和特异性及其在血液肿瘤患者侵袭性曲霉菌感染(IFI)中的诊断价值。方法二步法PCR检测有真菌感染高危因素的41例患者外周血中曲霉菌18SrRNA基因,结合CT检查、曲霉菌培养、患者骨髓抑制状况和抗真菌治疗效果,分析二步法PCR的诊断价值。结果多种曲霉菌菌株均能扩增出特异性条带,PCR产物测序结果与曲霉菌18SrRNA基因序列高度同源。41例中16例PCR阳性,25例阴性。阳性组中8例有曲霉菌感染的影像学表现,各有1例从胸腔积液、鼻分泌物培养出曲霉菌,2例血培养阳性。而12例对照者PCR均阴性。PCR阳性组和阴性组患者化疗后WBC数最低值分别为(0·30±0·14)×109/L、(0·50±0·26)×109/L,差异有统计学意义(P=0·0326)。两组患者WBC低于1·0×109/L的持续时间分别为(19·00±8·31)d、(12·69±6·95)d,差异有统计学意义(P=0·0472)。对PCR阳性者拟诊IFI,予以抗真菌治疗后,化疗相关早期死亡仅1例。结论二步法PCR检测对曲霉菌诊断的敏感性和特异性较高,假阳性和假阴性率均较低,结合临床资料,对IFI的早期诊断以及指导临床选择抗真菌治疗有较好的价值。