BK病毒DNA:一种人肿瘤病毒的完整核苷酸序列

本文介绍了人乳头多瘤空泡病毒BK病毒(BKV)的完整DNA序列。由于BKV(MM株)有4963个碱基对,故可认为SKV至少有五种蛋白:肽链氨基末端相同的T抗原和t抗原;VP2和VP3[有232个氨基酸与VP2相同]蛋白以及VP1蛋白。 VP1编码序列与VP2、VP3重叠113个核甙酸,但读码不同。BKV基因位点和排列与SV40极为类似,BKV蛋白质的氨基酸排列顺序有73％与SV40同源,而且两者的DNA序列70％相同,提示它们之间的进化关系密切。但两种病毒非编码区的DNA重复序列不同。     

恶性间皮瘤可能与致瘤性猿猴病毒SV40无关

目的探讨致瘤性猿猴病毒SV40（simian virus 40）是否与中国人恶性间皮瘤的发生相关。方法从蜡块中提取17例恶性间皮瘤组织中的DNA后,用三组引物对SV40大T抗原（TAg）的基因片段分别进行聚合酶链反应（PCR）扩增,另外,用两种SV40相关抗体（Pab101和Ab-2）分别进行免疫组织化学染色,检测肿瘤组织中是否存在SV40 TAg。结果（1）一组引物的PCR反应仅有3例扩增出了SV40TAg的基因片段,其余两组引物的PCR反应均为阴性。（2）两种抗体的免疫组织化学染色均未检测出SV40TAg。结论中国人恶性间皮瘤与SV40感染的关系可能不密切。     

小儿病毒性脑炎基因分型在诊断中应用的探讨

目的 探讨一种新的肠道病毒分型诊断方法的临床应用价值.方法 选取我院2006.06～2009.08期间诊断为病毒性脑炎的住院患儿共50例,均于入院的当天抽取脑脊液.试验先经种属特异性引物扩增,筛选出肠道病毒样本,然后分别用测序VP1及VP2序列的方法进行分型.分型结果最后经回顾性中和试验进行确认.试验同时设立阳性对照及阴性对照.结果 50例临床样本中,共有45例临床样本检测出EV RNA.其中,经VP1序列分型方法可成功扩增分型40例,反应产物为357 bp的片段.经VP2序列分型方法可成功扩增分型38例,扩增产物为584 bp的片段.微量中和试验及BLAST比对结果显示:两者在分型结果上有较高的一致性及互补性.结论 ①经VP1序列分型方法可以成功分型临床常见肠道病毒中的大多数血清型.②联合VP2序列分型方法较单用VP1序列分型方法,分型率及敏感性更高,值得临床上进一步推广.     

小儿病毒性脑炎基因分型在诊断中应用的探讨

目的 探讨一种新的肠道病毒分型诊断方法的临床应用价值.方法 选取我院2006.06～2009.08期间诊断为病毒性脑炎的住院患儿共50例,均于入院的当天抽取脑脊液.试验先经种属特异性引物扩增,筛选出肠道病毒样本,然后分别用测序VP1及VP2序列的方法进行分型.分型结果最后经回顾性中和试验进行确认.试验同时设立阳性对照及阴性对照.结果 50例临床样本中,共有45例临床样本检测出EV RNA.其中,经VP1序列分型方法可成功扩增分型40例,反应产物为357 bp的片段.经VP2序列分型方法可成功扩增分型38例,扩增产物为584 bp的片段.微量中和试验及BLAST比对结果显示:两者在分型结果上有较高的一致性及互补性.结论 ①经VP1序列分型方法可以成功分型临床常见肠道病毒中的大多数血清型.②联合VP2序列分型方法较单用VP1序列分型方法,分型率及敏感性更高,值得临床上进一步推广.     

真菌通用引物结合高分辨熔解曲线分析检测鉴定常见曲霉菌

目的探索一种能够快速检测鉴定临床常见侵袭性曲霉菌病原的新型分子生物学方法。方法以真菌核糖体RNA基因的内转录间隔区2为靶基因,在5.8S、28S rRNA基因上设计泛真菌通用引物,扩增临床常见曲霉菌。同时用新生隐球菌基因组DNA、人基因组DNA及临床常见细菌基因组DNA进行引物特异性检测。将通用真菌引物扩增产物进行高分辨熔解曲线分析。以新鲜提取的烟曲霉基因组DNA为模板按照1∶10的比例梯度稀释,进行敏感性检测。检测7个浓度梯度的烟曲霉基因组DNA。结果设计的真菌通用引物可以扩增临床常见的4种曲霉菌及新生隐球菌,但与人基因组DNA和临床常见细菌基因组DNA无扩增反应,检测限可低至1.5 pg/μl,且4种曲霉菌及新生隐球菌扩增产物的熔解曲线不同。该方法敏感性和特异性均较好。结论利用真菌通用引物的扩增产物结合高分辨熔解曲线分析可以达到检测鉴定临床常见4种曲霉菌的目的,此方法对侵袭性曲霉菌的快速检测鉴定具有重要意义。     

多特异性内参照模板的构建及应用

目的：构建多特异性内参照模板，用于Fas、FasL、GB、P、TIA-1和β-actin的竞争性PCR定量。方法：体外合成DNA单链，PCR扩增后，得到2个片段。片段1长。145bp，含有Fas、FasL、GB、P、TIA-1和pactin的5’引物序列；片段2长147bp，含有相同基因的3’引物序列。将片段1、2分别克隆在载体PKF3上。结果：克隆载体经酶切鉴定及DNA序列测定，证实为本实验所设计的多特异性内参照模板，以此为模板，用不同的引物进行PCR扩增，即可得到相应的竞争内参照。应用多特异性内参照模板对移植肾急性排斥患者外周血淋巴细胞中TIA-1进行定量，结果显示，底物量在一定范围内，靶序列和竞争性内参照模板的扩增效率基本一致，尤其是在指数期。这一结果同样适用于Fas、FasL、GB、P和β-actin的定量。结论：本实验构建的多特异性内参照模板可用于6种基因的竞争性PCR检测，为进一步研究打下了基础。     

PCR结合寡核苷酸探针杂交检测临床常见真菌的实验研究

目的 建立PCR结合生物标记的寡核甘酸探针斑点杂交技术,鉴定临床常见的真菌。方法 首先用真菌通用引物扩增白念球菌、热带念球菌、假热带念球菌、近平滑念球菌、光滑念球菌、解脂念球菌、克鲁斯念球菌、季也蒙念球菌、黄曲 霉、烟曲霉的核糖体大亚单位基因的保守区序列,然后用生物素标记的种特异性寡核苷酸探针与扩增产物杂交,并将此方法用于临床标本和临床分离菌株的检测。结果 通用引物可以扩增上述11种临床常见真菌的DNA,扩增片段长度在260bp左右。9种特异性探针分别与11种真菌标准菌株的PCR扩增产物杂交,结果表明每种探针都具有高度特异性。斑点杂交法和Southerm杂交法检测敏感性相同,为100fg;琼脂糖凝胶电泳法检测敏感性为1pg。通过69例临床标本和31例临床分析菌株的检测,PCR－杂交法的结果和真菌培养法的结果基本一致。结论 PCR结合生物素标记的寡核苷酸探针杂交技术可将9种临床常见真菌鉴定到种,方法快速、敏感、特异。     

信号放大系统的原理及在病毒核酸检测中的应用

在病毒性疾病诊断中 ,核酸检测是最直接、最确切的手段。对难于分离培养 ,或不易用免疫学方法检测特异性抗原、抗体的病毒 ,更为重要。但单纯核酸探针的敏感性不高 ,为提高检测敏感性 ,有两种主要途径 :1模板扩增技术 ,它通过扩增靶序列来提高敏感性 ;2信号放大系统 ,它通过放大杂交后信号强度来提高敏感性。它反应迅速 ,简便易行 ,没有模板扩增的干扰因素 ,近年来在核酸检测越来越受到重视。常用的信号放大系统有分支 DNA探针 ,3DNA树状体 ,DNA标记脂质体等 ,本文拟就常用信号放大系统的原理及其在病毒核酸检测中的应用作一综述     

脑膜炎奈瑟菌DNA片段的引物设计及其临床应用

用聚合酶链反应(PCR)检测脑膜炎奈瑟菌,在染色体DNA中的保守区域选择一段核苷酸作为靶DNA,用PCR技术进行扩增.引物的核苷酸序列为20个碱基对,其GC比值在引物1、2分别是55%和50%,符合设计要求.将此片段94C变性,55C退火,72℃延伸35个循环可将极微量细菌扩增到能通过电泳凝胶检测出特异性产物.本方法快速、敏感、特异地检测脑膜炎奈瑟菌,具有重要的应用价值.     

应用聚合酶链式反应鉴定实验细胞的来源种属

目的 应用聚合酶链式反应(PCR)鉴定常见细胞的种属来源,以判断培养细胞是否存在种间的交叉污染.方法 根据文献报道和NCBI数据库我们得到了32对种属特异性引物,并分别针对10种常见的细胞种属,对这些引物进行特异性和敏感性的筛选;以待检测细胞的基因组DNA为模板进行PCR及后续的琼脂糖凝胶电泳;以不同种属来源细胞的DNA混合物作为阳性对照模板,以水作为阴性对照模板.结果 针对上述10种常见细胞来源的种属分别得到了1对特异性和敏感性均较好的引物,经PCR扩增和琼脂糖凝胶电泳后可以准确鉴定待检测细胞的种属来源,并判断该细胞是否存在种间污染.结论 应用聚合酶链式反应可以简便、快速地鉴定实验细胞的种属,并判断该细胞是否存在种间的交叉污染.     

Nested polymerase chain reaction and in situ hybridization for detection of nucleopolyhedrosis

A nested polymerase chain reaction (PCR) and in situ hybridization were developed for detection of baculoviruses in insects or other arthropods with nucleopolyhedrosis. The nested PCR was based on the sequences of polyhedrin genes from baculoviruses. Two sets of primers were designed, primers set, 35/36, was for the first step of amplification and yielded a product of around 680 bp, the second primer, 35-1/36-1, was designed to yield a product of around 335bp from the fragment amplified by the first primer set. The sensitivity of this two-step amplification was 100 to 1000 times higher than that of the one-step amplification by primer set (35/36). Samples which contained baculovirus DNA yielded an amplification product showing the expected DNA fragment mobility, whereas nucleic acid extracted from tissue samples of clinically healthy insects or uninfected cells showed no such DNA fragment, thereby confirming the specificity of the primers. Using the 35/36 amplicon as a probe, the PenuNPV-infected cells show positive reaction by in situ hybridization. Two-step DNA amplification and in situ hybridization with the DNA probe developed in the present paper provide effective detection and diagnostic tools for screening insects or other arthropods, especially crustacean species, crabs and shrimps, for baculovirus infections, and may be important in preventing (and/or controlling/enhancing) the infection of baculoviruses.     

Absence of simian virus 40 (SV40) DNA in lymphoma samples from Tasmania, Australia

AIMS: It has been postulated that the recent world-wide increase in the incidence of non-Hodgkin's lymphoma (NHL) may have been caused by human infection with simian virus 40 (SV40) (a lymphotropic monkey virus that was introduced to man from contaminated poliovirus vaccines between 1955 and 1963); therefore, we set out to determine the incidence of SV40 DNA positivity in lymphoma samples from patients in Tasmania, Australia. METHODS: One hundred lymph node samples, 50 from patients with lymphomas and 50 from controls, were tested using PCR amplification of three SV40-specific primer pairs followed by dot-blot hybridisation. RESULTS: All of the samples tested contained amplifiable DNA, but none contained amplifiable SV40 sequences with any of the primer sets used. CONCLUSIONS: Our results demonstrate absence of SV40 in the lymphoid tissues of our study population in Tasmania, Australia. SV40 does not explain the increasing incidence of NHL in our population.     

Investigation of simian virus 40 large T antigen in 18 autopsied malignant mesothelioma patients in Japan

It has been reported that Simian virus 40 (SV40) is linked to human beings by inoculation of contaminated poliovaccines and may have a role in the etiology of malignant mesothelioma. However, there have been no reports describing the relationship between SV40 and malignant mesothelioma in Japan. A study was undertaken to investigate whether SV40 was related to patients of malignant mesothelioma in Japan by the polymerase chain reaction (PCR) assay, DNA sequence analysis, and immunohistochemical methods. Paraffin-embedded samples of the 18 autopsied patients with pleural malignant mesothelioma were collected from five hospitals in Japan. After isolation of DNA from paraffin blocks, PCR analyses followed by sequencing were performed using three different sets of primers for detection of SV40 large T antigen (TAg) gene. All 18 malignant mesothelioma samples were also immunohistochemically evaluated for expression of SV40 TAg protein with two different anti-SV40 TAg antibodies. SV40 TAg genome was detected in eight malignant mesothelioma cases. Only one of three primer pairs successfully amplified SV40 genome in the samples, whereas all pairs yielded a PCR product in the controls, suggesting a low content of virus DNA. No immunopositive staining for SV40 TAg was found in any of the samples. This study shows that SV40 genome was present in a subset of Japanese malignant mesothelioma patients who were unlikely to have received a contaminated polio vaccine based on their age.     

Nested polymerase chain reaction for detection of human immunodeficiency virus type 1 DNA in clinical specimens.

A highly sensitive two-step polymerase chain reaction (PCR) method was evaluated for detection of human immunodeficiency virus type 1 (HIV-1) DNA in clinical specimens. The product resulting from the first amplification reaction is used as the template for the second PCR with an internal (nested) primer pair. Even when starting from a single copy of HIV-1 DNA, the double PCR product was readily detected by direct visualization in ethidium bromide-stained agarose gels. Amplification of minute amounts of HIV-1 DNA was successful in a considerable excess of HIV-1 negative DNA than reported previously. All of 85 HIV-1-infected individuals were PCR-positive with at least two of the three sets of primers used, 252 of 255 amplifications allowing unambiguous visualization of a unique DNA fragment of the expected size. The two-step amplification protocol is simple and rapid and fulfills the requirements of sensitivity and specificity for use in a clinical laboratory.     

Highly Sensitive Method for Amplification of Human Immunodeficiency Virus Type 2 DNA

We evaluated a new human immunodeficiency virus type 2 (HIV-2) DNA amplification strategy based on peripheral blood mononuclear cell long PCR (XL PCR) followed by nested PCR amplification. The primers used were located in the highly conserved long terminal repeat and in the pol regions of the genome. Five primer pairs corresponding to different regions of the HIV-2 env gene were used in the nested step. Samples from 42 patients were tested, which yielded positive amplification with at least two primer pairs in 40 (95%) samples. A primer pair (EB2/EB5) located on the V3 region succeeded in amplifying proviral DNA in 40 samples.     

Comparison of PCR assays for detection of the agent of human granulocytic ehrlichiosis,Anaplasma phagocytophilum

Human granulocytic ehrlichiosis is an emerging infectious disease in the United States and Europe, and PCR methods have been shown to be effective for the diagnosis of acute infections. Numerous PCR assays and primer sets have been reported in the literature. The analytical sensitivities (limits of detection) of 13 published PCR primer sets were compared using DNA extracted from serial dilutions of Anaplasma phagocytophilum-infected HL-60 cells. The specificity of the assays that were able to detect 相似文献   

Detection of Haemophilus influenzae in cerebrospinal fluids by polymerase chain reaction DNA amplification

Two primer sets were chosen for the detection of Haemophilus influenzae in cerebrospinal fluid by polymerase chain reaction (PCR) DNA amplification. One primer set was selected from sequences encoding a capsulation-associated protein and reacted with target DNA from all 15 capsulate H. influenzae strains (all serotypes) examined. The other primer set was selected from the DNA sequence of a gene encoding for outer-membrane protein P6 and reacted with the 15 capsulate and 10 non-capsulate strains of H. influenzae tested. This primer set also reacted with the closely related species H. haemolyticus and H. aegyptius, and with two of nine H. parainfluenzae strains. In reconstruction experiments, PCR DNA amplification was able to detect as few as five H. influenzae cells when 40 cycles of amplification were used. Two hundred cerebrospinal fluid (CSF) samples collected consecutively from patients suffering from meningitis were investigated by PCR; 40 were culture-positive for H. influenzae and 39 of these were also clearly positive in the PCR test with both primer sets. Contamination occurred to some extent with 40 cycles of amplification but was completely eliminated when the number of cycles was reduced to 35. We conclude that the two primer sets are appropriate for the detection of H. influenzae by PCR, each having its own specificity. When these two primer sets are used, PCR is a technique of equivalent sensitivity to culture for the detection of H. influenzae in CSF.     

An improved technique for the in situ detection of DNA after polymerase chain reaction amplification.

In situ detection of polymerase chain reaction (PCR)-amplified DNA in cell and tissue preparations previously required 5 to 7 primer pairs designed to generate a long (greater than 1,000 base pair) product. The authors describe a nonisotopic PCR in situ technique, employing a single primer pair and target sequences as short as 115 base pairs, that can detect one target molecule per cell. The essential procedural change is to withhold the DNA polymerase or primers until the reaction temperature approaches 80 degrees C. The Hot-Start method greatly increased amplification specificity which, more than product size, appears to determine success of in situ PCR. The marked improvement in specificity may permit target detection by direct incorporation of labeled nucleotides.