用PCR检测临床标本中人类微小病毒B19 DNA

以自行设计及合成的两对引物,利用聚合酶链反应（ＰＣＲ）检测临床标本中人类微小病毒Ｂ１９ＤＮＡ。结果：两对引物的ＰＣＲ检测结果无显著差异性。自然流产组Ｂ１９病毒ＤＮＡ检出率为３４．６％（９／２６）,明显高于人工流产组５％（１／２０）,进一步证明Ｂ１９病毒感染与自然流产的发生密切相关。本方法亦为Ｂ１９病毒感染的临床诊断提供了新的手段。     

犬细小病毒中国内蒙株VP2基因克隆及序列分析

从我国内蒙古地区流行的犬细小病毒病病犬的肠溶物中分离提纯犬细小病毒（ＣＰＶ）。提取病毒基因组ＤＮＡ,并以此ＤＮＡ为模板,采用人工合成的引物进行ＰＣＲ扩增,ＰＣＲ产物经ＢａｍＨＩ、ＳａｃＩ双酶切后,克隆于ｐＵＣ１９质粒的ＢａｍＨＩ／ＳａｃＩ位点。重组质粒ｐＵＣＶＰ２经ＰＣＲ鉴定、限制酶切分析和序列分析,结果表明：获得了犬细小病毒内蒙株（ＣＰＶ－ＩＭ）ＶＰ２基因的全长克隆,ＶＰ２基因全长１７５５ｎｔ,     

多聚酶链反应技术在研究人微小病毒B19中的应用

本文概述了人微小病毒Ｂ１９的基本情况,详细介绍了多聚酶链反应（ＰＣＲ）技术在Ｂ１９病毒引起的各类感染、血制品等的检测,寻找整合状态的Ｂ１９病毒和其他微小病毒,以及研究Ｂ１９病毒在组织培养中的复制过程等方面的应用,并对其作了初步的评价。     

人细小病毒B19基因变异的研究进展

人细小病毒Ｂ１９简称Ｂ１９病毒,是细小病毒中惟一能感染人类的病毒,也是动物病毒中对人类具有致病性的最小单链线状ＤＮＡ病毒,其与儿童及成人的多种疾病密切相关。Ｂ１９病毒的基因变异对其致病、传播等具有重要影响,且与Ｂ１９病毒诊断试剂,疫苗的制备等关系密切。本文就Ｂ１９病毒基因组的变异状况,基因变异研究方法及研究Ｂ１９病毒基因变异的意义作一概述。     

鼻咽癌Epstein—Barr病毒受体（EBVR／CR2）基因的病毒结合区的序列测定

Ｅｐｓｔｅｉｎ－Ｂａｒｒ病毒（ＥＢＶ）进入鼻咽上皮细胞的途径,是人们研究ＥＢＶ与鼻咽癌（ＮＰＣ）的病因关系时所必须回答的一个问题。用抗ＥＢＣ受体（ＥＢＶＲ／ＣＲ２）单抗检测上皮细胞中该受体的表达已有报道,但对上皮细胞中ＥＢＶＲ／ＣＲ２的基因结构仍需研究。我们采用ＰＣＲ扩增和不对称ＰＣＲ直接测序法,首次检测了１０例ＮＰＣ及３例正常人胚鼻咽上皮（Ｈｕｍａｎ ｅｍｂｒｙｏｎｉｃ ｎａｓｏｐｈａｒｙｎｇｅ     

人细小病毒B19感染及其防治

人细小病毒Ｂ１９（简称Ｂ１９病毒）感染遍布世界各地,但大多数表现为无症状感染或轻微症状感染,而孕妇感染该病毒后可导致早产、流产、非免疫性胎儿水肿和死胎等。本文对Ｂ１９病毒的流行病学、致病机制、临床表现及防治措施等研究作一介绍。     

应用PCR技术检测细小病毒H－1DNA在人肝癌与裸鼠正常组织中复制的差异

应用ＰＣＲ技术检测细小病毒Ｈ－１ＤＮＡ在人肝癌与裸鼠正常组织中复制的差异黄青山,马承武,郭兰萍,陈献华,罗祖玉（上海复旦大学生理与生物物理学系,上海２００４３３）关键词：自主性细小病毒Ｈ－１及ＭＶＭ,聚合酶链式反应（ＰＣＲ）,人肝癌模型,抑瘤作用,肿...     

两个地区食管鳞癌中Epstein—Barr病毒的PCR检测

应用聚合酶链反应技术（ＰＣＲ）检测两个不同地区４８例食管鳞状细胞癌中ＥＢ病毒的感染。结果广州食管鳞癌中ＥＢ病毒ＤＮＡ检出率为９５．８％（２３／２４）；郑州食管鳞癌中ＥＢ病毒ＤＮＡ检出率为１９．６％（４／２４）。两者差异有显著性（Ｐ〈０．０５）。提示食管鳞癌的病因在不同的地区可能有所差异。郑州食管癌的发生可能是由其它致癌物质引起而与ＥＢ病毒感染无关；广州食管癌的发生可能与ＥＢ病毒的感染有一定的关系。     

鼻咽癌Epstein－Barr病毒受体（EBVR／CR＿2）基因的病毒结合区的序列测定

Ｅｐｓｔｅｉｎ－Ｂａｒｒ病毒（ＥＢＶ）进入鼻咽上皮细胞的途径,是人们研究ＥＢＶ与鼻咽癌（ＮＰＣ）的病因关系时所必须回答的一个问题。用抗ＥＢＶ受体（ＥＢＶＲ／ＣＲ＿２）单抗检测上皮细胞中该受体的表达已有报道,但对上皮细胞中ＥＢＶＲ／ＣＲ＿２的基因结构仍需研究。我们采用ＰＣＲ扩增和不对称ＰＣＲ直接测序法,首次检测了１０例ＮＰＣ及３例正常人胚鼻咽上皮（Ｈｕｍａｎｅｍｂｒｙｏｎｉｃｎａｓｏｐａｒｙｎｇｅａｌｅｐｉｔｈｅｌｉｕｍ,ＨＥＮＥ）组织样本中ＥＢＶＲ／ＣＲ＿２的ＥＢＶ结合区的编码序列。这一片段的ＤＮＡ序列测定结果显示,人ＮＰＣ细胞和正常ＨＥＮＥ细胞的ＥＢＶＲ／ＣＲ＿２的ＥＢＶ结合区的编码序列,与正常人Ｂ淋巴细胞的ＥＢＶＲ／ＣＲ＿２的相应序列完全相同,提示ＥＢＶ感染鼻咽上皮细胞可能与ＥＢＶＲ／ＣＲ＿２基因的ＥＢＶ结合区结构改变无直接关系。     

HBV－DNA的PCR二步法扩增及其快速检测

ＨＢＶ－ＤＮＡ的ＰＣＲ二步法扩增及其快速检测方勤,吴云涛,蔡宜权（中国科学院武汉病毒研究所,武汉４３００７１）关键词ＨＢＶ－ＤＮＡ,二步温控ＰＣＲ,Ｓｏｕｔｈｅｒｎ杂交,生物素寡聚核苷酸杂交乙型肝炎是危害人类健康的主要疾病之一,其病原常用的检测方法多...     

Application of Polymerase Chain Reaction to Detect HIV-1 DNA in Pools of Dried Blood Spots

Nucleic acid tests that detect HIV infection at an early phase are available and have been applied on individual dried blood spot (DBS). The present study was undertaken with an aim to evaluate the feasibility of performing PCR for HIV-1 DNA on pools of DBS as an alternative to individual testing. Standardization of PCR by a modified Amplicor HIV-1 DNA assay version 1.5 (Roche molecular diagnostics, USA), on pooled DBS was performed using five confirmed HIV reactive samples with known low viral load of HIV-1 and HIV non-reactive samples in pools of 5, 10 and 20 DBS. After successful standardization of pooling procedure, a total of 183 pools (of 10 DBS each) were prepared from 1,823 DBS samples, collected from a population-based study that tested negative for HIV antibodies and p24 antigen. All these pools were screened for HIV-1 DNA by the Amplicor assay. Standardization of pooling procedure indicated that pooling of 10 DBS gave an optimum result. Out of 183 pools tested, one pool of 10 samples was positive and of these ten DBS that were tested individually to identify the positive DBS, one sample was detected to be positive for HIV-1 DNA. Our study demonstrates that PCR for HIV-1 DNA can be successfully performed on pools of DBS. However, this may be needed only on specialized studies of HIV and not for routine epidemiology studies as only a very small fraction of cases would be missed if only antibody/antigen testing were done.     

Problems of isolating Borrelia burgdorferi from ticks collected in United Kingdom foci of Lyme disease

Abstract. Many isolates of Borrelia burgdorferi have been obtained from ticks and vertebrate tissues collected in North America and continental Europe but only one established culture of United Kingdom Borrelia burgdorferi has been recorded. In this paper we report the isolation of B.burgdorferi from one of 108 tick pools representing 733 ticks and eighty-four tissue samples from twenty-six rodents collected in the U.K., and the subsequent failure to establish the isolate (from ticks collected in Fordingbridge) in culture. In contrast, using identical techniques and culture medium, B.burgdorferi was isolated from one of seven tick pools collected in Switzerland, and from a single pool of ticks collected in Slovakia, and both isolates were successfully passaged. Analysis of questing I.ricinus collected from Fordingbridge by direct immunofluorescence showed 6/32 (19%) of adults and 8/108 (7%) of nymphs were positive for B. burgdorferi , although only one nymph contained ≥ 1000 spirochaetes. To examine further the problem of isolating U.K. B.burgdorferi , twelve Ixodes ricinus tick samples from Fordingbridge, a recognized focus of Lyme disease, were subjected to isolation and culturing techniques, and the procedures monitored by use of the polymerase chain reaction (PCR). Whereas 11/12 samples were PCR positive after 2 weeks in culture, only one was PCR positive after 4 weeks. Motile spirochaetes were not visible by dark-field microscopy in any of the cultures. The results indicate that the standard BSK II medium routinely used to isolate and culture B. burgdorferi does not readily support the replication of the Borrelia species endemic to the U.K.     

Cat scratch disease. Survey on the presence of Bartonella henselae among cats of Tuscany

To verify the presence of Bartonella henselae-infection in cats living in Tuscany (central Italy) serological and bacteriological surveys were carried out. The blood serum samples of 427 cats, 254 living in private houses and gardens and 173 in public or private catteries, were tested for anti-B. henselae antibodies by indirect immunofluorescence assay (IFA). Among these samples, 35 were examined by IFA to detect antibodies against Bartonella quintana. Bacteriological examinations were performed on the blood samples, collected in EDTA (ethylene diaminetetraacetic acid), of 18 cats (10 seropositive to B. henselae and 8 negative). From each of the same 18 specimens DNA was extracted and used as template in polymerase chain reaction (PCR). The primers p24E and p12B were employed in the PCR assay to amplify a 296 bp fragment of the Bartonella 16S rRNA gene. IFA detected 98 (22.95%) B. henselae-positive serum samples (40-40.82% from cats living in houses and gardens and 58-59.18% from cats of catteries) at different antibody titers (70 at 1:64 titer, 4 at 1:128, 22 at 1:256, 2 at 1:512). Among the 35 sera tested to detect antibodies against B. quintana, 9 (25.71%) resulted positive at 1:64 titer; all these samples showed higher antibody titers to B. henselae. Out of the 26 negative sera, 20 were negative to B. henselae too and 6 had antibodies against B. henselae at 1:64. Hemocultures gave negative results. PCR scored positive with DNA of 4 B. henselae-seropositive cats, two of which belonged to two children with cat scratch disease (CSD).     

The potential of TaqMan Array Cards for detection of multiple biological agents by real-time PCR

The TaqMan Array Card architecture, normally used for gene expression studies, was evaluated for its potential to detect multiple bacterial agents by real-time PCR. Ten PCR assays targeting five biological agents (Bacillus anthracis, Burkholderia mallei, Burkholderia pseudomallei, Francisella tularensis, and Yersinia pestis) were incorporated onto Array Cards. A comparison of PCR performance of each PCR in Array Card and singleplex format was conducted using DNA extracted from pure bacterial cultures. When 100 fg of agent DNA was added to Array Card channels the following levels of agent detection (where at least one agent PCR replicate returned a positive result) were observed: Y. pestis 100%, B. mallei & F. tularensis 93%; B. anthracis 71%; B. pseudomallei 43%. For B. mallei & pseudomallei detection the BPM2 PCR, which detects both species, outperformed PCR assays specific to each organism indicating identification of the respective species would not be reproducible at the 100 fg level. Near 100% levels of detection were observed when 100 fg of DNA was added to each PCR in singleplex format with singleplex PCRs also returning sporadic positives at the 10 fg per PCR level. Before evaluating the use of Array Cards for the testing of environmental and clinical sample types, with potential levels of background DNA and PCR inhibitors, users would therefore have to accept a 10-fold reduction in sensitivity of PCR assays on the Array Card format, in order to benefit for the capacity to test multiple samples for multiple agents. A two PCR per agent strategy would allow the testing of 7 samples for the presence of 11 biological agents or 3 samples for 23 biological agents per card (with negative control channels).     

中国美利奴细毛羊BAC文库的三维PCR筛选

本研究利用中国美利奴细毛羊全基因组BAC文库,构建了可供快速筛选的两级水平的混合池,一级混合池和二级混合池(Primary pools and secondary pools).一级混合池基于每一384-well盘而构建,由盘、行,列三维混合池组成,二级混合池基于整个BAC文库而构建.设计了一种基于PCR技术的快速筛选方法,先筛选二级混合池m再根据结果筛选相应的一级混合池.利用此方法只需一步共66个PCR反应即可从BAC丈库中7.4万个克隆中筛选出1个阳性克隆,或三步100个以内的PCR反应筛选出多个阳性克隆.以绵羊基因组多态性分子标记BF94-1为引物,用一步共66个PCR反应成功筛选到1个阳性克隆373D13.     

Polymerase chain reaction for detection of Clostridium botulinum types A, B and E in food, soil and infant faeces

The application of the polymerase chain reaction (PCR) for detection of Clostridium botulinum types A, B and E in foods, environmental and clinical samples was evaluated and compared to the mouse bioassay. Samples inoculated with 10, 100 and 1000 spores of Cl. botulinum types A and B included pasteurized milk, UHT milk, infant formula, infant faeces, meat juice, canned tuna, mushrooms, blood sausage and soil. Clostridium botulinum type E spores were inoculated into fish eggs, canned tuna, picked herring, raw fish and soil at similar levels. Spores were added to 2.5 g of each sample with the exception of soil which was inoculated in 10 g samples. The presence of Cl. botulinum in sample enrichments was determined by both PCR and the bioassay. An overall correlation of 95.6% was observed between PCR results and the mouse bioassay. Of the total of 114 samples tested there was disparity between the mouse bioassay and the PCR in three samples of soil inoculated with 100 type A or E spores and 10 type B spores per 10 g, respectively, and two samples of infant faeces inoculated with 10 type A or B spores per 2.5 g. All of these samples gave negative animal results and positive PCR results.     

Prevalence of hepatitis B virus in chronic hepatitis B patients

The aim of the current study was to detect HBV by Real time - PCR in chronic hepatitis B patients. Fifty-eight sera of chronic hepatitis B patients were subjected during the period March 2009 to April 2010 in Ilam cities in West of Iran. Sera assayed by real-time PCR and ELISA methods. Twenty serum samples from healthy volunteers and non-hepatitis B patients and negative for hepatitis B seromarkers served as negative controls for the study. Among fifty-eight sera, ELISA showed fifty-five (94.8%) of the samples were positive for HBsAg and three (5.2%) negative results obtained while real-time PCR specified fifty-eight (100%) positive results in chronic hepatitis B patients. HBsAg status did not necessarily reflect HBV DNA level in the serum, as 5.2% of chronic Hepatitis B patients were positive for HBV DNA but negative for HBsAg. HBV DNA was not found to be positive amongst any of the negative controls. Real time - PCR is a sensitive and reproducible assay for HBV DNA quantization.     

Identification of Naegleria fowleri in domestic water sources by nested PCR

The free-living amoeboflagellate Naegleria fowleri is the causative agent of primary amoebic meningoencephalitis (PAM), a rapidly fatal disease of the central nervous system. In the United States, the disease is generally acquired while swimming and diving in freshwater lakes and ponds. In addition to swimming, exposure to N. fowleri and the associated disease can occur by total submersion in bathwater or small backyard wading pools. In the present study, swipe samples and residual pipe water from homes in Arizona were examined for N. fowleri by nested PCR due to the death of two previously healthy children from PAM. Since neither child had a history of swimming in a freshwater lake or pond prior to the onset of disease symptoms, the domestic water supply was the suspected source of infection. Of 19 samples collected from bathroom and kitchen pipes and sink traps, 17 samples were positive for N. fowleri by PCR. A sample from a Micro-Wynd II filter was obtained by passing water from bathtubs through the filter. Organisms attached to the filter also tested positive by PCR. The two samples that tested negative for N. fowleri were one that was obtained from a kitchen sink trap and a swipe sample from the garbage disposal of one home.     

Comparison of different PCR methods for detection of Brucella spp. in human blood samples

For detection of Brucella species by PCR four DNA extraction methods and four targets were compared using pure culture of Brucella melitensis and the best conditions were applied in clinical samples. It was found that the MagNA Pure LC method was the most efficient and sensitive method showing a positive PCR reaction with DNA extracted from as low as 25 and 100 CFU suspended in one ml blood and one ml water, respectively. Detection of Brucella spp. by conventional PCR was investigated using four different targets. The results indicated that The B4-B5 amplification method was the most sensitive one as it could amplify DNA extracted from as a low as 25 and 100 CFU/ml suspended in one ml water and blood, respectively. Furthermore real-time PCR was able to detect Brucella using DNA extracted from as low as 50 CFU/ml blood and 15 CFU/ml water, respectively. The best and optimum detection conditions were applied to the clinical samples. Evaluation of conventional PCR assays on blood specimens confirmed 72% of the results obtained by conventional blood culture methods with a specificity of 95%, while serum samples had a sensitivity of 54% and specificity of 100%. Real-time PCR was generally found to be more sensitive and specific for detecting Brucella spp. in blood and serum samples compared to conventional PCR. The real-time PCR done on blood specimens confirmed 77.5% of the results obtained by conventional blood culture methods with specificity of 100%, while 60% of serum samples were found to be positive with specificity of 100%. These results suggest that serum and blood analysis by conventional and real time PCR is a convenient and safe method for rapid and accurate diagnosis of brucellosis.     

A significant age shift of the human parvovirus B19 antibody prevalence among young adults in Japan observed in a decade.

A seroepidemiological study on human parvovirus (B19) in Japan was undertaken with serum samples randomly collected from healthy Japanese populations (416 in 1973, 675 in 1984 and 508 in 1987/88). All samples were tested for anti-B19 IgG antibody by the indirect antigen-capture ELISA. The antibody prevalence for ages 0-9 years old in 1984 was significantly higher (16%) than that in 1973 (2%), whereas those for ages 20-29 years and 30-39 years were significantly lower in 1984 (20% and 56%) than in 1973 (67% and 80%) (p < 0.005). After the erythema infectiosum (EI) outbreak in 1986/87, the antibody prevalences for ages 5-9, 10-14 and 15-19 years were 40-85% in Fukuoka, 0-10% in Gunma, and 21-41% in Chiba reflecting each EI incidence in these three prefectures, whereas those for ages 20-29 years remained low (< 20%). These data indicate that B19 virus was transmitted mainly among children and no significant incidence of B19 virus infection in adults has occurred since 1973, resulting in a notable shifting of B19 susceptibility toward older ages including child-bearing females.