蜡样芽胞杆菌DNA分子分型研究

目的 建立蜡样芽胞杆菌分子分型方法，用于蜡样芽胞杆菌食物中毒的快速溯源。方法15株蜡样芽胞杆菌进行生化分型，同时进行vrrA基因PCR扩增和聚丙烯酰胺凝胶电泳-银染检测PCR扩增产物，所有PCR扩增产物进行序列分析，并用ClustalW(ebi．ac．uk)分析软件对DNA序列进行同源性比较。结果传统生化分型：15株蜡样芽胞杆菌中12株可分为3个型，3株不能分型；主要生化型为1型、2型和4型。分子分型：15株蜡样芽胞杆菌都可分为7个型。结论、vrrA基因可作为蜡样芽胞杆菌分子分型的一个多态性遗传标记。蜡样芽胞杆菌分子分型方法与传统生化分型方法相比，将传统生化分型所需的48h甚至更长时间缩短到5h，具有简便快速准确的优点，可做到快速溯源。     

蜡样芽胞杆菌溶血性肠毒素的提取及诊断方法的建立

蜡样芽胞杆菌是引起食物中毒的细菌之一〔1〕。蜡样芽胞杆菌的溶血素具有肠毒素的性质〔2〕,提取其溶血素可作为实验室诊断蜡样芽胞杆菌食物中毒和分析食品中残留毒素的诊断试剂。材料和方法蜡样芽胞杆菌溶血素的制备 :试验用的菌株接种肠毒素产毒培养液 ,经 37℃、16ｈ静置培养后 ,振荡培养 10ｈ。培养液基本按Ｂｅｅｃｈｅｒ等〔3〕的方法提取成粗制溶血素。进一步提纯则采用制备性ＳＤＳ ＰＡＧＥ的方法 ,切下含有 3种溶血素成分的凝胶 ,利用蛋白质沉淀或冷冻干燥的方法浓缩后得到分析测定用的溶血素。溶血素的测定1 用紫外吸收法测定蛋…     

一起蜡样芽孢杆菌10型引起食物中毒的快速诊断与分子分型研究

目的对引起一起食物中毒的蜡样芽孢杆菌进行快速诊断和分子分型。方法对中毒人员的呕吐物、肛拭子、可疑食品等样品用国标方法进行实验室检测,同时采用自主建立的荧光PCR方法进行快速检测。所分离的菌株PCR扩增vrrA基因多态性位点,产物进行基因双向测序和比较分析。结果在10份呕吐物、肛拭子、可疑食品中用传统方法和荧光PCR方法同时检出8份蜡样芽孢杆菌阳性,生物分型为10型。PCR测序结果显示它们有99%的同源性。结论这是一起食用了由蜡样芽孢杆菌10型污染的食品引起的呕吐型食物中毒,用蜡样芽孢杆菌呕吐毒素基因cesB为检测靶标的荧光PCR方法可以快速灵敏地对此类食物中毒进行初步诊断。以vrrA基因为多态性位点的PCR基因测序方法可用于蜡样芽孢杆菌的分子分型和食品污染源的准确追踪。     

地衣芽胞杆菌联合幽门螺旋杆菌补救疗法的临床观察

探讨地衣芽胞杆菌联合幽门螺旋杆菌补救治疗的临床疗效及不良反应。方法 将胃镜及呼气试验确认的Hp感染者予标准三联疗法治疗清除2周,4周后复查Hp,采用随机数字表法将202例首次清除Hp失败者分为地衣芽胞杆菌联合补救疗法的观察组（n=102）和非联合疗法的对照组（n=100）,对比分析根治率及不良反应率;结果 观察组根治率优于对照组（89.13% vs 80.65%）,但差异无统计学意义（P＞0.05）,不良反应发生率低于对照组（5.43% vs 19.35%）,差异有统计学意义（P＜0.05）;结论 地衣芽孢杆菌联合补救疗法清除HP不能加强根治效果,但可以减少药物引起的不良反应。     

实时定量PCR检测白血病中BCL11B基因的表达水平

目的建立BCL11B基因的定量检测方法,并分析其在白血病中的表达水平。方法利用实时定量RTPCR分析TALL(12例)、BALL(8例)、BCLL(6例)、AML(7例)和正常对照(10例)外周血单个核细胞(PBMC)中BCL11B基因的表达水平,以β2微球蛋白(β2M)基因表达水平作为内对照。结果TALL患者PBMC中BCL11B表达水平(553.84±564.01拷贝105β2M拷贝)明显高于正常对照和其他白血病患者(P=0.006,P=0.013,P=0.031,P=0.020);而AML组BCL11B表达水平(0.02±0.04拷贝105β2M拷贝)则明显低于正常对照组(P=0.000)、BALL组(P=0.006)和TALL组(P=0.020);BALL(1.99±1.59拷贝105β2M拷贝)和BCLL(2.26±3.57拷贝105β2M拷贝)中BCL11B表达水平与正常对照(2.20±1.01拷贝105β2M拷贝)无显著差别。结论建立了实时定量RTPCR检测BCL11B方法,TALL中BCL11B高表达可能与其发病有一定关系。     

基于双基因分析的结肠癌标志基因选择

针对结肠癌标志基因选择问题,引入一种最高得分对(TSP)方法,处理一组包含40个肿瘤和22个正常样本的结肠癌微阵列数据,得到标志基因对(VIP,DARS)并构建双基因分类器.结果显示,该分类器对62例样本的分类准确率可达93.55%.进一步,利用荧光实时定量PCR在10个独立样本上检验所选基因对的分类效果,正确率为100%,表明该方法能有效地选择结肠癌标志基因,对临床诊断有积极的意义.     

实时定量RT—PCR技术检测白血病融合基因的临床应用价值

目的评估实时定量RT—PCR技术检测白血病融合基因在辅助白血病临床诊断和药物疗效判定方面的价值。方法56例慢性粒细胞白血病（CML）患者，其中男性31例，女性25例，中位年龄54（18～80）岁；9例急性早幼粒细胞白血病（APL）患者，其中男性4例，女性5例，中位年龄47（2～64）岁。另有22例非CML或APL患者（男性12例,女性10例，年龄6～55岁）。应用实时定量RT—PCR技术检测临床患者骨髓或外周血单个核细胞中白血病融合基因M—bcr／abl或PMIdRARα的表达，结合临床资料进行分析。结果①56例CML患者的M-bcr／abl融合基因检测阳性率为96．4％（54／56）；9例APL患者的PMIdRARa融合基因检测阳性率为77．8％（7／9）。②5例CML患者应用甲磺酸-伊马替尼治疗后定量检测M—bcr／abl融合基因的拷贝数较治疗前明显降低或转阴（4／5），伊马替尼治疗CML效果优于应用羟基脲和干扰素治疗的患者。结论实时定量RT—PCR技术操作简便，检测白血病融合基因的准确性高。在辅助临床诊断和白血病患者药物疗效判定方面都有较大的应用价值。     

实时荧光定量PCR检测鼻咽癌组织中PTCH-1和SMO基因的表达

目的:检测Hedgehog信号通路基因PTCH-1和SMO在鼻咽癌(NPC)组织中的表达及其生物学意义。方法:利用实时荧光定量PCR分别检测32例鼻咽癌组织及32例鼻咽炎组织中PTCH-1和SMO基因的表达情况。根据Livak(2-△△Ct)法进行相对定量表达分析,以鼻咽炎组织为对照,计算鼻咽癌组织中PTCH-1和SMO表达水平。结果:在所检测的全部鼻咽癌组织和鼻咽炎组织中PTCH-1和SMO都有表达。与鼻咽炎组织相比,有75%(24/32)鼻咽癌组织的PTCH-1基因表达水平下调,有68.8%(22/32)鼻咽癌组织的SMO表达水平上调,差异均具有统计学意义(P0.05)。此外,有18.7%(6/32)鼻咽癌组织PTCH-1基因表达水平上调,有15.6%(5/32)鼻咽癌组织SMO基因表达水平下调,差异均无统计学意义(P0.05)。结论:在鼻咽癌中SMO基因普遍具有较高的表达水平,PTCH-1基因的表达水平则相对较低,而SMO基因表达水平的上调及PTCH-1基因表达水平的下调,可引起Hedgehog信号通路在鼻咽癌中的异常激活,Hedgehog信号通路的异常激活可能与鼻咽癌的发生发展过程相关。     

建立实时定量PCR检测急性早幼粒细胞白血病PML/RARa融合基因转录本

目的 建立实时定量PCR(real-time quantitative PCR,RQ-PCR)法榆测急性早幼粒细胞白血病(acute promyelocytic leukemia,APL)PML/RARa融合基因转录本,评价其在APL患者诊断和微小残留病(minimal residual disease,MRD)监测中的意义.方法 设计TaqMan探针和引物,建立RQ-PCR法对长型(L-form)、短型(S-form)、变异型(V-form)不同类型PML/RARa阳性模板进行扩增,并检测6例APL患者PML/RARa融合基因转录本含量.结果 RQ-PCR法最低可检测到10个拷贝/μL的阳性模板,但重复性较差,而108～102拷贝/μL阳模的重复性良好,止常对照无扩增信号.6例初诊APL患者不同类型PML/RARa融合基因转录本绝对含量为4.27×103～3.36×105拷贝/50 ng(中位4.33×104拷贝/50 ng),ABL纠正后的相对含量为29.38%～600.53%(中位48.12%).1例患者诱导缓解后PML/RAHa融合基因转录本下降,复发时义明显升高.结论 RQ-PCR方法敏感、可靠,可用于APL患者的诊断和MRD监测.     

应用实时定量PCR进行肿瘤表达研究中内参基因的选择

实时定量PCR(real-time quantitative PCR,RT-qPCR)是检测肿瘤基因表达水平的一种有效方法,在其中应用较多的相对定量方法需要一种能够在不同的组织、甚至不同的实验条件下表达恒定的理想的内参基因.但是很多研究都已经表明内参基因的表达是可变的.所以为了获得适用于肿瘤表达研究的内参基因,越来越多的研究在近期涌现出来.该文将会对近期关于肿瘤表达研究中内参基因选择的文章进行综述.     

Development of a real-time PCR targeting the yidC gene for the detection of Mycoplasma hominis and comparison with quantitative culture

Mycoplasma hominis is an opportunistic human mycoplasma species that can be either commensal or pathogenic. Its detection by culture is considered to comprise the reference technique. Previously reported PCR techniques target the 16S rRNA or the gap gene, although sequence variations among clinical isolates may lead to variations in clinical sensitivity. The present study aimed to develop a specific TaqMan quantitative real-time PCR assay, targeting a gene conserved in all M. hominis isolates, and to compare it with quantitative culture. With the knowledge of the M. hominis PG21 genome sequence, the yidC gene, encoding a membrane protein translocase, was chosen as target. Its intraspecies heterogeneity was checked at the nucleotide level using 31 reference or clinical strains. The limit of detection, the analytical specificity and the reproducibility of the assay were assessed. Moreover, PCR and culture results were compared using 153 urogenital specimens. The limit of detection was seven copies/μL. The analytical specificity was 100%, with good inter- and intra-assay reproducibility. Among the 153 urogenital specimens, the yidC PCR and culture allowed detection of 55 and 45 M. hominis-positive samples, respectively. Comparison of the bacterial load among the 45 specimens found to be M. hominis-positive by both techniques revealed a statistically significant association between the quantitative results obtained. In conclusion, we developed a specific, sensitive and reproducible real-time PCR to detect all M. hominis clinical isolates. This PCR was shown to have higher sensitivity than culture, although both methods were correlated for quantification of M. hominis loads in urogenital specimens.     

应用实时荧光定量PCR快速分子诊断唐氏综合征

目的探讨一种快速、准确诊断唐氏综合征的方法。方法采用实时荧光定量PCR技术，对25例唐氏患者、50名正常人外周血标本，扩增21号及1号、19号染色体上的多态位点，定量分析比较正常组及唐氏患者组的4对△Ct值。结果唐氏患者组△Ct值明显低于正常组，两组比较差异有统计学意义（P〈0．001）。初步建立了临床应用的参考值范围，可以有效区分出唐氏样本和正常样本。结论应用实时荧光定量PCR技术可快速、准确诊断唐氏综合征，为唐氏综合征的快速产前诊断开辟了新的途径。     

Triplex PCR‐based detection of enterotoxigenic Bacillus cereus ATCC 14579 in nonfat dry milk

Although many strains of Bacillaceae are considered nonpathogenic, Bacillus cereus is recognized worldwide as a bacterial pathogen in a variety of foods. The ability of B. cereus to cause gastroenteritis following ingestion of contaminated food is due to the production of enterotoxins. The ubiquity of this genus makes it a persistent problem for quality assurance in food processing environments. The primary objective of this study was to develop and apply a multiplex real‐time PCR‐based assay for rapid and sensitive detection of enterotoxigenic B. cereus. Template DNA was separately extracted from tryptic soy broth (TSB)‐grown and 2.5% Nonfat Dry Milk (NFDM)‐grown B. cereus using a commercial system. Three enterotoxin gene fragments (hblC, nheA, and hblA) were simultaneously amplified in real‐time followed by melting curve analysis to confirm amplicon identity. Resolution of melting curves (characteristic T_m) was achieved for each amplicon (hblC = 74.5 °C; nheA = 78 °C; and hblA = 85.5 °C in TSB and 84 °C in NFDM) with an assay sensitivities of 10¹ CFU/ml for both TSB and NFDM‐grown B. cereus compared to 10⁴ CFU/ml in either matrix using gel electrophoresis. The results demonstrate the potential sensitivity of real‐time bacterial detection methods in a heterogenous food matrix using real‐time PCR. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

实时荧光定量PCR在周围髓鞘蛋白22基因重复或缺失检测中的应用

目的 应用实时荧光定量PCR在腓骨肌萎缩症和遗传性压力易感性神经病患者中检测周围髓鞘蛋白22基因（peripheral myelin protein 22，PMP22）重复或缺失。方法 采用实时荧光定量PCR检测113个腓骨肌萎缩症家系先证者、4个遗传性压力易感性神经病家系先证者和50名正常人PMP22基因重复或缺失突变。结果 113个腓骨肌萎缩症家系中发现有36个存在PMP22基因重复，4个遗传性压力易感性神经病先证者均存在PMP22基因缺失，50名正常人未发现异常。结论 我国PMP22基因重复突变的致病频率为31．9％（36／113），PMP22基因缺失突变是遗传性压力易感性神经病常见的致病原因。     

用实时荧光定量PCR方法检测母血中的胎儿SRY基因

目的　从孕妇外周血浆中分离出胎儿DNA ,并加以鉴定 ,预防X连锁遗传病患儿的出生。方法　从孕早期、中期共 3 0 0名孕妇外周血浆中分离胎儿DNA ,用实时荧光定量聚合酶链反应 (fluorescencequantitativepolymerasechainreaction ,FQ PCR)的方法检测其中的Y性别决定区 (sex determiningregionY ,SRY)基因。结果　孕早期怀男胎的孕妇 82名 ,70名SRY基因阳性 ,其平均浓度为 ( 5 8.82± 2 0 .90 )拷贝 /ml,中位数为 5 8.5 0拷贝 /ml。孕中期怀男胎的孕妇 90名 ,SRY基因均为阳性 ,平均为 ( 15 2 .0 8± 62 61)拷贝 /ml,中位数为 14 9.3 5拷贝 /ml。怀女胎的孕妇均为阴性。结论　用实时荧光定量PCR的方法最早在孕42天的孕妇外周血浆中就可以检测到胎儿SRY基因 ,随孕周的增加 ,母血中胎儿DNA的量也在逐渐增加。实时荧光定量PCR技术在进行无创伤性产前性别诊断中有重要的价值。     

Effectiveness of real-time quantitative PCR compare to repeat PCR for the diagnosis of Charcot-Marie-Tooth Type 1A and hereditary neuropathy with liability to pressure palsies

The majority of cases of Charcot-Marie-Tooth type 1A (CMT1A) and of hereditary neuropathy with a liability to pressure palsies (HNPP) are the result of heterozygosity for the duplication or deletion of peripheral myelin protein 22 gene (PMP22) on 17p11.2. Southern blots, pulsed-field gel electrophoresis (PFGE), fluorescence in situ hybridization (FISH) and polymorphic marker analysis are currently used diagnostic methods. But they are time-consuming, labor-intensive and have some significant limitations. We describe a rapid real- time quantitative PCR method for determining gene copy number for the identification of DNA duplication or deletion occurring in CMT1A or HNPP and compare the results obtained with REP-PCR. Six patients with CMT1A and 14 patients with HNPP [confirmed by Repeat (REP)-PCR], and 16 patients with suspicious CMT1A and 13 patients with suspicious HNPP [negative REP-PCR], and 15 normal controls were studied. We performed REP-PCR, which amplified a 3.6 Kb region (including a 1.7Kb recombination hotspot), using specific CMT1A-REP and real-time quantitative PCR on the LightCycler system. Using a comparative threshold cycle (Ct) method and beta -globin as a reference gene, the gene copy number of the PMP22 gene was quantified. The PMP22 duplication ratio ranged from 1.35 to 1.74, and the PMP22 deletion ratio from 0.41 to 0.53. The PMP22 ratio in normal controls ranged from 0.81 to 1.12. All 6 patients with CMT1A and 14 patients with HNPP confirmed by REP-PCR were positive by real-time quantitative PCR. Among the 16 suspicious CMT1A and 13 suspicious HNPP with negative REP-PCR, 2 and 4 samples, respectively, were positive by real-time quantitative PCR. Real-time quantitative PCR is a more sensitive and more accurate method than REP-PCR for the detection of PMP22 duplications or deletions, and it is also faster and easier than currently available methods. Therefore, we believe that the real-time quantitative method is useful for diagnosing CMT1A and HNPP.