广西1例人田鼠巴贝虫感染巢式PCR鉴定及其同事感染调查

目的确诊1例国内感染田鼠巴贝虫的病例,对患者周边同事进行调查,初步了解田鼠巴贝虫在广西人群中的分布情况。方法提取患者及相关人士血液DNA,共计121份,进行巢式PCR鉴定。以扩增患者18S rRNA基因为分子标记,与GenBank中各巴贝虫序列进行同源性分析,采用最大似然率法(maximum likelihood)构建系统进化树,分析亲缘关系。结果患者感染田鼠巴贝虫病,患者周边同事共40人感染,阳性率为33.06%(40/121);田鼠巴贝虫的系统分类属于感染人的巴贝虫类,与感染牛、犬的巴贝虫亲缘关系较远。结论田鼠巴贝虫在患者周边同事中感染率较高,对接触人员具潜在的感染风险,应加强预防和控制。     

两种PCR方法检测狂犬病毒的敏感性和特异性比较

目的比较Nested-PCR和SYBR GreenⅠReal-Time PCR两种方法检测狂犬病毒的敏感性、特异性和时效性。方法对10倍连续稀释的狂犬病毒（疫苗株）核酸样品,采用Nested-PCR和SYBR GreenⅠReal-Time PCR方法进行平行检测,比较两种方法的敏感性;同时以登革热2型病毒、诺如病毒、星状病毒、EV71和乙型脑炎病毒核酸对两种方法的特异性进行评价。结果 SYBR GreenⅠReal-Time PCR方法可检测出2.3×106copies/μl核酸分子,与Nested-PCR方法相比,敏感度提高10倍。两种检测方法的特异性均好,与登革热2型病毒、诺如病毒、星状病毒、EV71和乙型脑炎病毒核酸均无交叉反应。SYBR GreenⅠReal-Time PCR方法检测花费3h,检测时间比Nested-PCR方法节省2h。结论与Nested-PCR相比,SYBR GreenⅠReal-Time PCR是相对高效的检测狂犬病毒的方法。     

用巢式聚合酶链式反应从全血扩增麻风菌特异片段提高麻风病确诊率

目的用巢式聚合酶链式反应(Nested-PCR)检测全血中麻风菌特异片段,以提高麻风病诊断水平。方法收集多菌型(multibacillary leprosy,MB)、少菌型(paucibacillary leprosy,PB)麻风病患者、其家庭内接触者以及麻风病流行区、非流行区的正常人全血和血清样本,建立巢式PCR,扩增麻风菌重复序列的特异片段,PCR阳性产物直接测序鉴定扩增片段的特异性。结果在35例MB患者中巢式PCR有34例阳性;26例PB患者中19例阳性;23例家庭内接触者中2例阳性。流行区和非流行区正常人中未见阳性。此结果与血清学结果进行比较,发现血清学检测MB患者阳性率稍高于巢式PCR;值得注意的是,对PB患者中细菌密度指数(BI)为阴性者,巢式PCR阳性检测率高达78.57%。结论巢式PCR对于麻风病MB患者的诊断敏感性不亚于血清学抗体诊断,但可提高麻风病特别是对PB患者诊断的敏感性;对高危人群尤其是家庭内接触者的监测有较大的实用价值。     

骨髓移植患者病房空气中呼吸道腺病毒的检测研究

目的建立医院环境空气中呼吸道腺病毒的采集检测工作模型. 方法利用Teflo滤膜过滤式采集病房空气中腺病毒,采用套式PCR方法,从滤膜分离标本中进行腺病毒hexon基因中特异性片段的扩增;同时,对可能采集到的腺病毒进行细胞培养,观察病毒细胞毒效应. 结果准确检测到骨髓移植后呼吸道腺病毒感染患者房间中的腺病毒存在;对照患者发生了医院获得性呼吸道腺病毒感染,其病房空气中腺病毒分布为阳性,但造成此医院感染的腺病毒的传播途径尚不清楚. 结论 Teflo滤膜采集及套式PCR方法,用于医院病房空气中腺病毒颗粒监测切实可行,具有较高的灵敏度;这将为研究分析腺病毒气溶胶造成医院感染的传播途径等奠定良好的技术基础.     

脆性X综合征的筛查及基因诊断

目的：建立一种简便快速初步筛查脆性X综合征智力缺陷基因FMR－1突变的方法。方法：采用套式PCR技术对新生儿及婴幼儿的足跟血X染色体上基因FMR－1CGG重复序列进行扩增,通过以其拷贝数的鉴定筛查其突变型。结果：共筛查5200全新生儿和婴幼儿,查出1例男婴患者,其母亲是携带者。结论：套式PCR能简便快速地初筛出人群中携带者和可疑患者,对脆性X综合征的早期诊断和产前诊断有应用价值。     

湖南省HIV-1分子流行病学研究

目的 了解HIV毒株在湖南省的亚型别特点和流行规律。方法 收集湖南省不同地区40名既往已确认为HIV-1感染者的抗凝全血样品，提取前病毒DNA，用套式聚合酶链反应方法（nested-PCR）扩增病毒膜蛋白env基因的C2-V3区，并进行序列测定。序列的排列、比较和同源性分析使用威斯康星GCG公司软件完成，确定亚型。结果 通过PCR扩增得到33份样品的结果，依据env区基因序列，与HIV-1各个亚型国际参考株比较，通过系统进化分析，最后确定湖南省样品分属6个亚型，分别为HIV-1B亚型中的泰国B（B’）亚型10份，欧美B亚型3份，流行重组型CRF-BC5份，流行重组型CRF01-AE13份，A亚型1份和D亚型1份。结论 湖南省已存在多种HIV-1亚型，应加强对HIV-1毒株亚型变异的监测，及时调整防治策略。     

中缅边境(西段)传疟媒介的初步调查

目的了解中缅边境(西段)传疟媒介的分布与构成。方法 2008年8～9月,在中缅边境的中国云南省盈江县及其相邻的缅甸昔懂县6个自然村,用诱蚊灯在人房和牛棚共进行20次通宵诱捕。将捕获的蚊虫以传统方法进行形态学鉴定,然后用复合PCR法鉴别微小按蚊、乌头按蚊和杰普按蚊。同时,抽提部分蚊虫标本总基因组DNA,以巢式PCR方法检测蚊体内的疟原虫感染情况。结果共捕获各类蚊虫4 571只,隶属9属50种,其中按蚊属是优势蚊种,占总量的54.32%(2 483/4 571)。人房和牛棚的按蚊蚊种构成差异有统计学意义,其中人房以腹簇按蚊、微小按蚊和中华按蚊为主,而牛棚以腹簇按蚊(223只)、环纹按蚊(184只)、迷走按蚊(131只)和杰普按蚊(129只)为主。对比有牛村和无牛村中人房的蚊种构成发现,有牛村的人房以微小按蚊(260只)和腹簇按蚊(49只)为主,而无牛村人房则以腹簇按蚊(481只)和中华按蚊(124只)为主。巢式PCR检测1 075只按蚊,其中9只检出疟原虫阳性,分别为微小按蚊(7/408)、乌头按蚊(1/125)和伪威氏按蚊(1/101)。经测序鉴定均为恶性疟原虫感染,目的条带长204 bp。结论中缅边境(西段)...     

Emerging Intestinal Microsporidia Infection in HIV+/AIDS Patients in Iran: Microscopic and Molecular Detection

Background

Species of Microsporidia have been known as opportunistic obligate intracellular parasites particularly in immunocompromised patients. Enterocytozoon bieneusi is one of most prevalent intestinal microsporida parasites in HIV⁺/AIDS patients. In this study, intestinal microsporidia infection was determined in HIV⁺/AIDS patients using microscopic and molecular methods.

Methods

Stool samples were collected from HIV⁺/AIDS patients during 12 months. All of the stool specimens washed with PBS (pH: 7.5). Slim slides were prepared from each sample and were examined using light microscope with 1000X magnification. DNA extraction carried out in microscopic positive samples. DNA amplification and genus/species identification also performed by Nested-PCR and sequencing techniques.

Results

From 81 stool samples, 25 were infected with microsporidia species and E. bieneusi were identified in all of positive samples. No Encephalitozoon spp. was identified in 81 collected samples using specific primers.

Conclusion

E. bieneusi is the most prevalent intestinal microsporidia in immunocompromised patients of Iran. On the other hand, Nested-PCR using specific primers for ssu rRNA gene is an appropriate molecular method for identification of E. bieneusi.     

Comparison of loop-mediated isothermal amplification (LAMP) and nested-PCR assay targeting the RE and B1 gene for detection of Toxoplasma gondii in blood samples of children with leukaemia

Toxoplasmosis diagnosis constitutes an important measure for disease prevention and control. In this paper, a newly described DNA amplification technique, loop-mediated isothermal amplification (LAMP), and nested-PCR targeting the repeated element (RE) and B1 gene, were compared to each other for the detection of Toxoplasma gondii DNA in blood samples of children with leukaemia. One hundred ten blood samples from these patients were analyzed by LAMP and nested-PCR. Out of 50 seropositive samples (IgM+, IgG+), positive results were obtained with 92% and 86% on RE, B1-LAMP and 82% and 68% on RE, B1-nested PCR analyses, respectively. Of the 50 seronegative samples, three, two and one samples were detected positive by RE-LAMP, B1-LAMP and RE-nested PCR assays, respectively, while none were detected positive by B1-nested PCR. None of the 10 IgM−, IgG+ samples was detected positive after testing LAMP and nested-PCR assays in duplicate. This is the first report of a study in which the LAMP method was applied with high sensitivity and efficacy for the diagnosis of T. gonii in blood samples of children with leukaemia.     

围产期解脲脲原体感染的分析

目的：探讨围产期解脲脲原体(UU)感染与不良妊娠结局、母婴间的垂直传播及对受感染儿的远期影响。方法：用套式PCR技术对280位临产妇、28例新生儿(母亲UU阳性)进行UU检测。结果：产妇中UU的感染率为10％，母婴间垂直传播率为35．71％(10例)。产妇感染UU与胎儿早产、出生低体重有相关性；UU在早产儿、低出生体重儿、新生儿窒息者中母婴垂直传播率高于正常足月儿；母婴垂直传播所致受感染新生儿今后呼吸道系统疾病的感染率高于未受感染儿。结论：产妇UU感染易出现早产及低出生体重儿；早产儿、低出生体重儿及窒息新生儿易被UU传染；受UU母婴垂直传播新生儿易发生呼吸道系统疾病。