PCR／SSCP技术在检测HLA基因点突变中的应用

聚合酶链反应－单链构象多态性（ＰＣＲ／ＳＳＣＰ）是根据核苷酸序列不同的单链ＤＮＡ，可形成不同构象，从而在不含变性剂的聚丙烯酰胺凝胶中泳动速度不同的原理，分离鉴定等长ＤＮＡ单链的方法，本文应用ＰＣＲ／ＳＳＣＰ方法检测了６０例无亲属关系个体白细胞抗原Ⅱ类基因（ＨＬＡ－ＤＱＡ１）５－上游调控区的等位基因多态性，并以ＰＣＲ直接测序证明每种ＳＳＣＰ带型都与不同位置单个碱基取代相关联。     

采用通用引物PCR配合SSCP及RFLP技术快速检测常见病原菌

目的 建立一种快速检测病原菌的方法以利于临床诊断。方法 选取10种具有代表性的病原菌,采用通用引物PCR（UPPCR）对其16SrRNA基因进行扩增,并对PCR产物进行限制性酶切片段长度多态性分析（RFLP）和单链构象多态性分析（SSCP）。结果 RFLP电泳图谱呈现多态性,但半数菌的图谱两两相同或相似;SSCP电泳图谱各异,可以相互区分。结论 UPPCR-SSCP技术能快速简便地检测病原菌。     

PCR—SSCP在乙肝垂直传递研究中的应用

为了探讨乙型肝炎（ＨＢ）父子间的垂直传递,我们应用ＰＣＲ结合ＰＣＲＳＳＣＰ技术,研究了２９ 例ＨＢｓＡｇ 阳性的男性乙肝患者（ＨＢＰ） 及其发病前后出生子女血清游离型及外周血单核细胞（ＰＢＭＣ）基因组内整合乙肝病毒（ＨＢＶ） 存在的情况,表明：ＨＢＰ与其发病后出生子女可具有完全一致的单链泳动变位结果。本研究表明：ＨＢＶ 感染可以在父子间垂直传递,为乙肝的遗传传递学说提供了分子遗传学上的证据。     

应用PCR—SSCP法检测LDL受体基因多态性

利用聚合酶链反应（ＰＣＲ）从人白细胞ＤＮＡ中扩增到一条长为１７２碱基的ＬＤＬ受体基因外显子２ＤＮＡ片段，对３０个ＰＣＲ扩增产物进行了单链构象多态性分析（ＳＳＣＰ），其中２个图谱出现异常，序列证实是由ＬＤＬ受体基因外显子２第１４位碱基因Ｔ置换由Ｃ，形成多态性，建立的这种方法可以快速，简便地分析了ＬＤＬ受体因外显子２的多态性。     

应用PCR—SSCP技术进行PKU基因突变检测

本文应用ＰＣＲ－ＳＳＣＰ（聚合酶链反应合并单链构象多态性方法）对２０个苯丙酮尿症家系的先证者和其父母的含密码子ＣＧＡ（Ａｒｇ２４３）的外显子７扩增片断进行检测，共检测到８个家系中１８人含有ＣＧＡＣＡＡ突变，检出率为３０％，为这些家系的第二胎产前诊断提供了依据。     

应用PCR－SSCP方法区分HLADNA纯合基因型与杂合空白基因型

在ＰＣＲ／ＳＳＯ分型的基础上，用一对ＰＣＲ引物扩增ＤＰＢ１基因后作ＳＳＣＰ分析，发现该方法能够确定那些用ＰＣＲ／ＳＳＯ方法只检出一个等位基因的样本为纯合基因型或是含一个空白基因的杂合子，９个用ＰＣＲ／ＳＳＯ方法只检出一个ＤＰＢ１等位基因的湖南汉族人样本中，用ＰＣＲ－ＳＳＣＰ方法发现１个样本表现为杂合子。用银染的方法使得ＳＳＣＰ更快速、可靠和没有同位素污染，并为探测新的ＨＬＡ等位基因提供了可能性。也为精确统计等位基因频率或单倍型频率提供了保证     

JWA基因启动子-76G→C多态性和膀胱癌易感性关系的配对研究

目的检测JWA启动子单核苷酸多态性（single nucleotide polymorphisms，SNPs），探讨其对基因转录活性的影响和与膀胱癌遗传易感性的相关性。方法应用聚合酶链反应-单链构象多态性和DNA测序的方法，检测了155例膀胱癌病例和155例非肿瘤对照人群的JWA基因启动子多态性位点；构建启动子多态性片段氯霉素乙酰转移酶报告基因重组质粒，瞬时转染NIH．3T3细胞，检测启动子多态性片段对基因转录活性的影响。结果检测到一个新SNP-76G→C；C等位基因和GC基因型频率在膀胱癌病例组为10．00％、20．00％，比正常对照组（5．16％、10．32％）高，差异有统计学意义（P〈0．05）；与GG基因型相比，GC基因型启动子的转录活性显著降低（P〈0．01）。Logistic多元回归分析结果（P〈0．05，OR=2．05）显示这一多态性可能是形成膀胱癌新的独立危险因素。结论JWA基因启动子-76G→C多态性可能影响JWA基因转录和表达，从而增加膀胱癌易感性。     

用非同位素PCR—SSCP方法检测石蜡包埋乳腺癌P53基因点突变

应用－非同位素ＰＣＲ－ＳＳＣＰ方法检测常规石蜡包埋乳腺癌组织中Ｐ５３基因点突变，结果显示６０例乳腺癌中，１４例有异常电泳带，表明这些病例相应ＤＮＡ片段中发生了点突变，其中４例位于第５－６外显子，３例位于第７外显子，７例位于第８外显子，免疫组化法检测突变型Ｐ５３蛋白的表达，１３例为阳性，其中１１例ＳＳＣＰ检测到异常电泳带，另２例突变可能发生在所检测的基因片段以外，而５例ＳＳＣＰ分析发现基因突变者，免     

双重实时荧光定量PCR法鉴定克柔念珠菌和光滑念珠菌

目的 建立一种快速、灵敏、特异的鉴定克柔念珠菌和光滑念珠菌的双重实时荧光定量PCR方法.方法 以核糖体基因内转录间隔区Ⅱ(ITSⅡ)为靶目标,设计并合成分别针对克柔念珠菌、光滑念珠菌的种特异引物和探针.建立双重实时荧光定量PCR反应体系,并用该体系对呼吸道相关致病菌进行检测.鉴定结果与临床常规鉴定方法对照,评价其敏感度、特异度及重复性.结果 通过对100例样品的检测,结果显示该双重实时荧光定量PCR法检测标本的鉴定结果与常规鉴定方法的结果对照,特异度为100%,敏感度为100%;最小能检测到10个拷贝数的重组质粒;批内重复实验和批间重复实验结果均与常规鉴定方法结果相符.结论 双重荧光定量PCR法鉴定克柔念珠菌和光滑念珠菌,特异度和敏感度高,重复性好,且快速、简便,该方法将有助于念珠菌病的早期诊断和针对性治疗.     

Simplified sunflower (Helianthus annuus) seed agar for differentiation of Candida dubliniensis from Candida albicans

This study evaluated sunflower (Helianthus annuus) seed agar (SSA) for differentiation of Candida dubliniensis from Candida albicans on the basis of colony characteristics and chlamydospore production. Simplified SSA without creatinine and KH(2)PO(4) was also used. On both media, C. dubliniensis isolates (n = 25) developed rough colonies and formed abundant chlamydospores after incubation for 24-48 h at 28 degrees C, while C. albicans isolates (n = 53) showed smooth colonies with no evidence of chlamydospore formation. Cryptococcus neoformans isolates (n = 10) formed brown colonies on both media. Simplified SSA offers a simple and inexpensive tool for presumptive differentiation of C. dubliniensis from C. albicans in clinical microbiology laboratories.     

Candida sake: a relevant species in the context of HIV-associated oropharyngeal candidosis?

  Candida sake is routinely identified in the oral cavity of patients infected with the human immunodeficiency virus (HIV) using the commercial identification system ATB 32 C. To establish the prevalence of C. sake and to evaluate this designation repeatedly found using the ATB 32 C system, 94 HIV-infected patients were investigated for the presence of oral candidosis based on clinical and microbiological grounds. A total of 186 Candida isolates from 62 patients were obtained. Using the assimilation assay, C. sake was suspected in 49 isolates, but only seven strains were positively identified according to ATB 32 C. With respect to antifungal susceptibility and clinical parameters the 49 strains did not differ markedly from the other strains. Only antifungal susceptibility to amphotericin B, ketoconazole, and flucytosine was increased in C. sake strains when the positively and equivocally identified strains by ATB 32 C were taken together. In addition, amplifying genomic DNA with primers T3B and AP3, C. sake could not be identified in four strains and in one strain, respectively. Therefore biochemical identification of C. sake seems to be misleading and clinical relevance may be lacking. Received: 13 February 1997 / Accepted: 16 September 1997     

Genomic heterogeneity in the yeast Candida parapsilosis

Candida parapsilosis shows a wide intraspecies variation in chromosome/homolog size distribution. As a prerequisite for delineating modes of transmission, we have undertaken an analysis of genetic variation at different levels. In the present study we have observed that a majority of isolates display similar electrophoretic karyotype patterns consistent for the species, with variations in the smaller group of chromosomes. In two strains we observed phenotypic switching; one of these also exhibited a mixed karyotypic subpopulation. In contrast, a few isolates displayed a greater degree of chromosome/ homolog size variation. We also observed, through randomly amplified polymorphic DNA (RAPD) analysis, results consistent with those of pulsed-field electrophoresis. Isolates displaying a high degree of chromosome/homolog variation also displayed a high degree of variation in genomic fingerprints. Polymorphisms, although present, were much reduced in the majority of isolates. These parallel observations suggest a common underlying mechanism. Our results are consistent with the hypothesis that chromosome-sized variations in C. parapsilosis are due to random genetic events. A similar mechanism has been hypothesized for the taxonomically related yeast Candida albicans.     

Construction of stable episomes in Cryptococcus neoformans

We report the generation of stable plasmids constructed by inserting specific DNA sequences into previously known unstable vectors. These sequences were obtained from a DNA library recovered from a previously reported stable minichromosome created by electroporative transformation in Cryptococcus neoformans (Varma and Kwon-Chung 1994). A 6-kb insert from this minichromosome significantly enhanced both the frequencies at which URA5 transformants were obtained as well as the stability of their uracil prototrophy on non-selective media. A 1.5-kb sequence of this insert contained telomeric sequence repeats which when introduced into plasmids resulted in significant increases in transformation frequency. A 1081-bp sequence (STAB), present in the remainder of the insert, had an ARS-like function enhancing the episomal maintenance of plasmids in the transformants regardless of the gene (ADE2/URA5) used as a selection marker. Received: 4 November 1997 / 11 May 1998     

A high yield DNA extraction method for medically important Candida species: A comparison of manual versus QIAcube-based automated system

The prognosis of infected individuals with candidemia depends on rapid and precise diagnosis which enables optimising treatment. Three fungal DNA extraction protocols have been compared in this study for medically important Candida species. The quality and quantity of the DNA extracted by physical, chemical and automated protocols was compared using NanoDrop ND-2000 spectrophotometer. It was found that the yield and purity (260/230) ratio of extracted DNA was significantly high in the physical treatment-based protocol as compared to chemical based or automated protocol. Extracted DNA-based real time-polymerase chain reaction showed an analytical sensitivity of 10³ cfu/mL. The result of this study suggests physical treatment is the most successful extraction technique compared to other two protocols.     

新型隐球菌与肺泡上皮细胞的体外相互作用

目的研究新型隐球菌与肺泡上皮细胞的体外相互作用,探讨隐球菌肺部感染的发病机制。方法体外培养Ⅱ型肺泡上皮细胞A549(ATCC CCL-185),检测新型隐球菌2种变种对细胞的时间/浓度黏附率、通过率;检测新型隐球菌对细胞的损伤作用;透射电镜观察相互作用的超微结构。结果2种变种的新型隐球菌可以对A549细胞产生黏附与侵袭,黏附率与侵袭率呈现时间依赖性;同时还可以使A549细胞凋亡率升高,对其造成损伤,这与菌体的活力相关。超微结构可见隐球菌与肺泡上皮细胞的黏附与侵袭过程。2种变种之间在黏附率、通过率及对细胞的损伤作用方面差异无统计学意义。结论活的隐球菌黏附与侵袭肺泡上皮细胞是隐球菌感染肺部的重要条件,不同变种对肺部的易感性可能不存在差异。进一步明确二者的作用机制对隐球菌的发病机制研究具有重要意义。     

新型隐球菌野生株和荚膜缺陷株抗吞噬细胞吞噬作用的比较

目的　研究荚膜对新型隐球菌在巨噬细胞内外活力的影响 ,探讨荚膜在抗吞噬中的作用。方法　通过新型隐球菌标准野生株B35 0 1和荚膜缺陷株Cap6 0与小鼠巨噬细胞系J774细胞共孵育后 ,检测其出芽率、凋亡率 ,通过电镜观察隐球菌在J774细胞内的超微结构。结果　J774细胞对B35 0 1菌株的吞噬指数在 (5 .6 7± 1.2 9) %～ (8.76± 3.0 9) % ,对Cap6 0株的吞噬指数为 (31.6 2±12 .70 ) %～ (4 1.86± 9.74 ) % ;B35 0 1菌在J774细胞内的出芽率较高 ,可达 (4 6 .85± 6 .6 3) % ,而Cap6 0的胞内出芽率较低 ,仅达 (10 .73± 0 .39) % ;B35 0 1从共孵育时间达 4h后 ,出芽率开始下降 ,而Cap6 0从共孵育时间达 2h后 ,出芽率开始下降。流式细胞仪检测示J774细胞内、外的B35 0 1和Cap6 0菌株的凋亡率分别是 6 .4 %、4 .7%和 15 .4 %、9.6 %。结论　新型隐球菌可在J774巨噬细胞内存活并繁殖 ,荚膜不仅有抗吞噬的作用 ,而且有抗杀菌的作用     

Comparison of adherence of Candida albicans and Candida parapsilosis to silicone catheters in vitro and in vivo

Objective  Although Candida parapsilosis has been associated with device-related infections in the clinical settings, factors that contribute to this association have not been previously examined. The objectives of this study were to compare in vitro and in vivo the adherence to silicone catheters of: (1) Candida albicans vs. C. parapsilosis , and (2) invasive vs. colonizing isolates of C. albicans and C. parapsilosis .
Methods  The records of 840 patients who had had Candida species isolated at three teaching hospitals during a three-month period were reviewed. A total of 20 clinical isolates of each of C. parapsilosis and C. albicans were examined for their adherence to silicone catheters in vitro and in a rabbit model of percutaneously placed catheters. For each Candida species, ten invasive isolates that had caused clinical device-related infection and 10 colonizing isolates that had caused only device colonization were studied.
Results  Candida parapsilosis accounted for <5% of yeast isolates from all sites, while three-quarters were C. albicans . Candida parapsilosis was isolated proportionately more often from blood and/or devices than C. albicans (34.3% vs. 8.5%, respectively, P  < 0.0001). There were no significant differences in the degrees of adherence in vitro and in vivo between C. albicans and C. parapsilosis or between invasive and colonizing Candida .
Conclusion  Although C. parapsilosis was isolated proportionately more often from blood and/or devices than C. albicans in our studied population, there was no significant difference in the adherence of the two Candida species to silicone, nor between invasive and colonizing Candida in our in vitro and in vivo models. Factors other than microbial adherence may help explain the observed association of C. parapsilosis with device-related infections.