Leishmania identification by PCR of Giemsa-stained lesion imprint slides stored for up to 36 years

This study examined the ability of PCR to amplify Leishmania DNA, stored on Giemsa-stained slides, from American cutaneous leishmaniasis (ACL) patients. In total, 475 slides stored for up to 36 years were obtained from an outpatient clinic in a Brazilian ACL-endemic region, and Leishmania DNA was amplified from 395 (83.2%) of the DNA samples using primers specific for the minicircle kinetoplast DNA. Restriction fragment length polymorphism analysis of these amplicons demonstrated that Leishmania (Viannia) braziliensis was the only species present in these samples. The results demonstrated that archived Giemsa-stained slides can provide a Leishmania DNA source for performing clinical and epidemiological studies of leishmaniasis.     

Development and evaluation of a new PCR assay for detection of Pseudomonas aeruginosa D genotype

This report describes a new PCR-based assay for the detection of Pseudomonas aeruginosa genotype D in occupational saturation diving systems in the North Sea. This genotype has persisted in these systems for 11 years (1993-2003) and represents 18% of isolates from infections analysed during this period. The new PCR assay was based on sequences obtained after randomly amplified polymorphic DNA (RAPD)-PCR analysis of a group of isolates related to diving that had been identified previously by pulsed-field gel electrophoresis (PFGE). The primer set for the D genotype targets a gene that codes for a hypothetical class 4 protein in the P. aeruginosa PAO1 genome. A primer set able to detect P. aeruginosa at the species level was also designed, based on the 23S-5S rDNA spacer region. The two assays produced 382-bp and 192-bp amplicons, respectively. The PCR assay was evaluated by analysing 100 P. aeruginosa isolates related to diving, representing 28 PFGE genotypes, and 38 clinical and community P. aeruginosa isolates and strains from other species. The assay identified all of the genotype D isolates tested. Two additional diving-relevant genotypes (TP2 and TP27) were also identified, as well as three isolates of non-diving origin. It was concluded that the new PCR assay is a useful tool for early detection and prevention of infections with the D genotype.     

石家庄地区汉族人群DIS80(pMCT118)位点VNTR基因频率分布调查

本文采用ＰＣＲ技术对石家庄地区２１６名无关个体ＤＩＳ８０（ｐＭＣＴ１１８）位点扩增片段长度多态性的等位基因频率进行调查。ＰＣＲ扩增产物采用聚丙烯酰胺凝胶电泳分型及溴化乙锭染色，共检出７０种基因型，２５个等位基因，其中以１８、２４、３０三个等位基因频率最高。将本文结果与日本人群在ＤＩＳ８０（ｐＭＣＴ１１８）位点多态性结果进行比较，石家庄地区汉族人群ＤＩＳ８０（ｐＭＣＴ１１８）位点基因频率分布符合ＨａｒｄｙＷｅｉｎｂｅｒｇ平衡定律，该位点非父排除率为０．７１，个体识别率为０．９，杂合度为０．８５。     

PCR和ELISA法在检测HBV-DNA、HCV-RNA和结核分枝杆菌中的作用

目的:探讨PCR技术和ELISA法在检测HBV-DNA、HCV-RNA和结核分枝杆菌中的作用,以及与直接涂片法的对比结果.方法:分别用两法对临床确诊的感染病例和非感染者同时进行检测,并进行对比分析.结果:HBeAg阳性者HBV-DNA阳性,检出率为96.8%,HCV-RNA阳性检出率为19.4%.不同标本TB-DNA平均阳性检出率为39.98%.结论:PCR法灵敏度和特异度分别高于ELISA法和涂片查菌法.     

套式PCR检测细菌16SrRNA基因

目的　应用套式PCR建立快速检查方法来检测血液中是否含有细菌。方法　采用细菌 1 6SrRNA基因通用引物 ,通过套式PCR方法对细菌进行扩增 ,并对其灵敏度、特异性作一评价。结果　临床常见细菌扩增反应为阳性 ,其他病原微生物及人类基因组DNA为阴性。结论　本法具有敏感、快速、特异、准确的优点 ,是一种可靠的实验手段     

人肿瘤特异抗原MAGE-3基因疫苗的构建和表达

目的：克隆肿瘤特异性共享抗原mage-3基因，制备基于α－病毒复制酶的高效黑色素瘤特异性基因疫苗。方法：用RT-PCR方法从黑色素瘤细胞系LB373中制备mage-3基因，以含α－病毒复制酶的哺乳细胞高效表达质粒pSMART2a为载体，构建重组DNA疫苗。重组子用载体中的T7和T3启动子序列为测序引物进行自动测序。再将鉴定过的重组质粒用脂质体法转化293细胞，用免疫印迹法和免疫组化法鉴定转化细胞中mage-3蛋白的表达。结果：正确构建了mage-3/pSMART2a重组质粒，并且在转化此质粒的293细胞中检测出了mage-3蛋白的表达。结论：此重组mage-3/pSMART2a质粒可以作为肿瘤特异的DNA疫苗，可进行下一步的肿瘤动物模型的疫苗接种及相关免疫学效应研究。     

SA脂质体介导GFP／bFGF基因在豚鼠耳蜗中的表达

目的 :观察天然碱性脂 (Stearylamine,SA)脂质体介导绿色荧光蛋白 /碱性成纤维细胞生长因子(GFP/bFGF)基因于不同时间段豚鼠耳蜗中的表达 ,为进一步研究耳聋的基因治疗提供实验基础。方法 :取豚鼠 1 6只 ,分成 4组 ,每组 4只。其中 3只右耳圆窗内注入SA -GFP/bFGF复合物 ,1只同法注入生理盐水作为对照。分别于术后第 2、7、1 4、2 1天取材。在荧光显微镜下观察GFP的表达 ,用免疫组化法检测bFGF的转导情况。结果 :荧光显微镜下见双侧耳蜗于术后第 2天开始部分细胞发出绿色荧光 ,第 7天达到高峰 ,支持细胞及内外毛细胞均显荧光 ,细胞轮廓清晰 ;第 1 4天开始减弱 ,第 2 1天消失。免疫组化染色显示 ,除血管纹外 ,耳蜗各回Corti器、螺旋韧带、螺旋缘及螺旋神经节细胞均有高浓度的表达产物 ,对照动物呈阴性表达。结论 :SA脂质体介导的GFP/bFGF基因单耳给药双侧耳蜗均有高效表达 ,为进一步研究基因治疗耳聋提供了可能。     

Presence of beta human papillomaviruses in nonmelanoma skin cancer from organ transplant recipients and immunocompetent patients in the West of Scotland

Background  Nonmelanoma skin cancer (NMSC) has been linked to cutaneous human papillomaviruses of the genus beta (betaPV).
Objectives  We sought to assess the presence of betaPV in NMSC biopsies from a group of Scottish skin cancer patients, both immunocompetent (IC) patients and immunosuppressed (IS) organ transplant recipients.
Methods  One hundred and twenty-one paraffin-embedded skin tumours (27 actinic keratosis, 41 intraepidermal carcinoma, 53 squamous cell carcinoma) and 11 normal skin samples were analysed for the presence of betaPV by a polymerase chain reaction–reverse hybridization assay designed to detect the presence of the 25 known betaPV genotypes.
Results  In IC patients, betaPV was detected in 30 of 59 (51%) tumours and two of 11 (18%) normal skin samples ( P  =   0·046). In IS patients, betaPV was found in 27 of 62 (44%) tumours; no normal skin samples were available for comparison. The most frequently found genotypes were HPV-24, HPV-15 and HPV-38. Of those tumours infected with betaPV, 28 of 57 (49%) were infected with more than one genotype (range 2–8). Tumours from IS patients were from a younger age group (mean age 57·4 years) than IC patients (mean age 73·8 years). Multiple infections were more common in tumours from IC patients (21 of 30; 70%) compared with those from IS patients (seven of 27; 26%) ( P  <   0·001). In the IC group, age did not appear to influence the distribution of single and multiple infections whereas in IS patients the proportion of multiple infections to single infections increased with age. There were no multiple infections in normal skin.
Conclusions  A wide spectrum of betaPV types was detected in our samples. Further characterization of betaPV in vivo is needed in order to determine the mechanisms by which the virus contributes to cutaneous carcinogenesis.     

Differential Regulation of RGS-2 by Constant and Oscillating PTH Concentrations

PTH has diverse effects on bone metabolism: anabolic when given intermittently, catabolic when given continuously. The cellular mechanisms underlying the varying target cell response are not clear yet. PTH induces RGS-2, a member of the Regulator of G-protein Signaling protein family, via cAMP/PKA, and inactivates PKC-mediated signaling. To investigate intracellular signaling pathways with different PTH concentration-time patterns, we treated UMR 106-01 osteoblast-like cells in a perfusion system. PTH was administered intermittently (4 min/h, 10⁻⁷ M) or continuously at an equivalent cumulative dose (6.6 × 10⁻⁹ M). cAMP was measured using radioimmunoassay, mRNA levels using real-time rtPCR and ribonuclease protection assay, and protein levels using Western immunoblotting. A single PTH pulse transiently increased cAMP levels by 2000% ± 1200%. In contrast to continuous PTH exposure, cAMP induction remained unchanged with intermittent PTH, ruling out desensitization of the PTH receptor. In continuously perfused cells, RGS-2 abundance was three to five times higher than in cells intermittently exposed to PTH for up to 12 h. MKP-1 and -3 were significantly less induced with pulsatile PTH; exposure-mode-dependent differences in MMP-13 and IGFBP-5 were small. Pulsatile but not continuous PTH administration prevents PTHrP receptor desensitization and accumulation of RGS-2 in osteoblasts, which should preserve PKC-dependent signaling.