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用~(32)P和生物素标记的克隆化的人巨细胞病毒(HCMV)AD169株DNA片段作探针,采用DNA-DNA斑点杂交法,对照检测了27例婴儿肝炎综合症患者(血清学检测为非甲非乙型肝炎者)临床血、尿标本的HCMV-DNA。其中16份血标本呈阳性,占59%;9份尿标本呈阳性,占33%。初步结果表明,血标本中HCMV DNA检出率比尿标本检出率高26%。标记的~(32)P探针可检测10pg同源DNA,生物素探针可检测50Pg同源DNA,均不与其它疱疹病毒及未感染的人胚肺细胞DNA杂交。将其中26份血标本的HCMV DNA杂交结果与抗HCMV IgM ELISA检测结果相比较,符合率为65%。 相似文献
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本文用光敏生物素标记的R3 cDNA探针杂交检测了HFRS患者外周血淋巴细胞、血清和尿沉淀细胞中HFRS病毒核酸。结果42份淋巴细胞标本中30份为阳性,48份血清标本中有24份出现阳性杂交信号,81份尿沉淀细胞标本中53份为阳性,表明将此光敏生物素标记的cDNA探针用于HFRS实验诊断和致病机理的研究是可行的。 相似文献
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本文将柯萨奇B组3型病毒(CVB3)cDNA的重组质粒DNA(pGP51B)转化到E.coli HB101菌株中.筛选转化阳性菌株,经培养扩增后,提取重组质粒DNA,用缺口求移法制备生物素标记探针,通过原位杂交技术检测CVB1.CVB3感染的Hela细胞及正常Hela细胞对照.结果该探针只与CVB杂交,而不与细胞对照杂交,且可检测出病毒感染5h尚未出现病变细胞中的病毒核酸.表明该探针具有良好的特异性和敏感性. 相似文献
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PCR-微孔板杂交-ELISA法检测巨细胞病毒感染 总被引:3,自引:0,他引:3
建立PCR-微孔板杂交-ELISA法检测人血清标本中巨细胞病毒DNA.将5'端标记生物素的PCR扩增产物与5'端标记地高辛的探针呈液相混合,90℃ 2min,55℃,1min杂交.杂交后产物被链霉亲和素酶标板固定,经酶标记抗地高辛标记抗体结合显色.本试剂检测灵敏度为2.5×104 copies/mL,比传统PCR和ELISA敏度性高,特异性强,与单纯疱疹病毒Ⅰ型和Ⅱ型,风疹病毒、EB病毒、腺病毒核酸无交叉反应,批内CV为8.9%,批间CV为10.5%.该法可用于定性和定量检测HCMV DNA. 相似文献
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建立PCR-微孔板杂交-ELISA法检测人血清标本中巨细胞病毒DNA。将5’端标记生物素的PCR扩增产物与5’端标记地高辛的探针呈液相混合,90℃ 2min,55℃,1min杂交。杂交后产物被链霉亲和素酶标板固定,经酶标记抗地高辛标记抗体结合显色。本试剂检测灵敏度为2.5×104 copies/mL,比传统PCR和ELISA敏度性高,特异性强,与单纯疱疹病毒Ⅰ型和Ⅱ型,风疹病毒、EB病毒、腺病毒核酸无交叉反应,批内CV为8.9%,批间CV为10.5%。该法可用于定性和定量检测HCMV DNA。 相似文献
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本文用生物素标记柯萨奇B3病毒(Cox B3)cDNA(530bp)制备探针,检测不同病毒(Cox A9,Cox B1—B6,Polio1,ECHO1,AD3,HSV-1,HSV-2,VV)感染的Hela细胞,结果表明该探针只与肠道病毒核酸杂交而不与非肠道病毒核酸杂交,且可检测出Cox B3感染Hela细胞后3小时细胞病变阴性(CPE~-)细胞内的肠道病毒核酸,表明该探针具有良好的特异性和敏感性.同时用Cox B1感染BalB/c小鼠,建立小鼠心肌炎模型,取心肌组织与该探针进行原位杂交,成功地检测出心肌组织中的肠道病毒核酸.这对于用该探针检测人类病毒性心肌炎组织中的肠道病毒核酸,对病毒性心肌炎进行特异的早期诊断积累了经验. 相似文献
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应用聚合酶链式反应扩增和光敏生物素标记的cDNA探针检测马铃薯纺锤块茎类病毒 总被引:2,自引:0,他引:2
以含马铃薯纺锤块茎类病毒(potato spindle tuber viroid,PSTV)RNA的总核酸为模板,加入人工合成的互补DNA引物,用反转录酶合成PSTV cDNA;在聚合酶链式反应系统中,用两个PSTV特异性引物进行cDNA扩增,用以制备光敏生物素标记的PSTV cDNA探针。用此探针进行斑点杂交检测含PSTV的马铃薯核酸提取液和汁液均出现阳性杂交信号,而健康马铃薯的核酸提取液和汁液的结果均为阴性。光敏生物素标记探针检测纯化PSTV的灵敏度可达5pg;检测感染PSTV的马铃薯块茎汁液的可测出最高稀释度为1:400。 相似文献
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Holger Hackstein Gerhard Jahn Holger Kirchner Gregor Bein 《Histochemistry and cell biology》1996,106(2):229-234
Radioactive in situ hybridization techniques or enzymatic detection procedures of hapten-modified human cytomegalovirus (HCMV)
probes have been widely used for studying the infection of peripheral blood leukocytes with HCMV. This report describes significant
improvements in terms of signal resolution which can be obtained by applying a highly sensitive fluorescence in situ hybridization
(FISH) technique in conjunction with a large subgenomic HCMV DNA probe. Three cosmid clones spanning 119.1 kb of the HCMV
genome (230 kb) were used to construct the digoxigenin-11-dUTP-labeled probe which was found to be superior to a total HCMV
probe representing the entire genome. Crucial hybridization parameters were analyzed systematically in order to ensure optimal
resolution power and sensitivity. The protocol was successfully applied to HCMV-infected fibroblasts and peripheral blood
leukocytes of 12 transplant patients and unambiguously facilitated the precise intracellular localization of HCMV genomes
in infected cells. Because of its excellent resolution properties, accompanied by the virtual absence by any background staining,
we recommend the use of this protocol as a sensitive approach for further virological analyses of the interactions between
HCMV and peripheral blood leukocytes at the single-cell level.
Accepted: 16 February 1996 相似文献
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Cytokine response and polymerase chain reaction study of peripheral blood mononuclear cells in infants with human cytomegalovirus infection 总被引:2,自引:0,他引:2
Hideomi Asanuma Kei Numazaki Nobuo Nagata Shunzo Chiba 《FEMS immunology and medical microbiology》1995,12(2):153-158
Abstract We tried to detect human cytomegalovirus (HCMV) DNA in CD4 + and CD8 + T lymphocytes from fourteen infants with HCMV hepatitis using polymerase chain reaction (PCR) assay. HCMV was isolated from their urine and anti-HCMV IgM antibody was detected in their sera. One set of primers were designed from a region — a major immediate early (IE) gene. We detected HCMV IE DNA in the specimens obtained from six infants. HCMV IE DNA was detected from CD4 + cells in two cases and from CD8 + cells in one. In three cases, HCMV IE DNA was detected from both CD4 + and CD8 + cells. We also studied the relationship between HCMV infection and serum levels of cytokines. We determined serum levels of interleukin-4 (IL-4), tumor necrosis factor alpha (TNF-α) and soluble interleukin 2 receptor (sIL-2R) which were associated with the activation of T lymphocytes by enzyme immunoassay. In the acute phase of HCMV infection, titers of sIL-2R were correlated with serum levels of liver enzymes in some cases. IL-4 and TNF-α activities were not detected in sera. It is likely that expression of viral genome on T lymphocytes as well as activities of some cytokines are associated with active HCMV infection. 相似文献
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A biotin-labelled DNA probe was used in a dot-blot hybridization test to demonstrate the presence of Escherichia coli in a variety of artificially contaminated foodstuffs. Positive hybridization was detected by using a streptavidine/polyalkaline phosphatase conjugate to generate an insoluble coloured precipitate in the presence of an appropriate dye. The colour intensity was measured with a computer-controlled image analysis system which assessed objectively the hybridization signal produced by each sample. The method was capable of distinguishing positive hybridization at cell concentrations exceeding 10(4) cells/dot-blot, equivalent to 2 x 10(7) cells/g food, and had none of the drawbacks normally associated with the use of radioactively labelled DNA in hybridization techniques. The procedure is highly specific and takes less than 30 h. Many samples can be screened simultaneously and the procedure can be used to detect any species for which a suitable DNA probe is available. 相似文献
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Wen-jun L qu-lian G Hong-ying C Yan Z Mei-Xian H 《Cell biochemistry and biophysics》2012,63(2):133-141
We investigated the role of homeobox B4 (HOXB4) mRNA/protein expression induced by human cytomegalovirus (HCMV) and/or all-trans retinoic acid (ATRA) in proliferation and committed differentiation of human cord blood hematopoietic stem cells (HSCs) into colony-forming-units of T-lymphocyte (CFU-TL) and erythroid (CFU-E) progenitors in vitro. Twelve cord blood samples were collected from the fetal placenta umbilical vein and cultured in vitro. The proliferation and differentiation of cord blood HSCs into CFU-TL and CFU-E were continuously disrupted with HCMV-AD169 and/or 6 × 10(-8) mol/l of ATRA. HOXB4 mRNA/protein expression in CFU-TL and CFU-E was detected in control, ATRA, HCMV and ATRA + HCMV groups on days 3, 7, and 12 of culture by fluorescent qRT-PCR/western blot. We found that HOXB4 mRNA/protein expression was detectable on day 3, increased on day 7 and was highest on day 12. HOXB4 mRNA/protein expression in HCMV group was downregulated compared with control group (P < 0.05). However, the levels were significantly upregulated in HCMV + ATRA group compared with HCMV group (P < 0.05). We concluded that the abnormal HOXB4 mRNA/protein expression induced by HCMV could play a role in hematopoietic damage. ATRA, at the concentration used, significantly up-regulated HOXB4 mRNA/protein expression in normal lymphocyte and erythrocyte progenitor cells as well as in HCMV-infected cells. 相似文献
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用显微切割技术和催化信号扩增法检测何杰金病瘤细胞(H/RS)中的人巨细胞病毒 总被引:2,自引:0,他引:2
为明确何杰金病 (Hodgkin’sdisease ,HD)瘤细胞中是否存在人巨细胞病毒 (humancytomegalovirus,HCMV)感染 ,采用显微切割技术结合PCR方法检测HD组织及其瘤细胞Hodgkin/Reed Sternberg(H/RS)中的HCMV核酸 ;采用免疫组织化学催化信号扩增 (catalysedsignalamplification ,CSA)法检测HD中HCMV的立即早期抗原、早期抗原和基质蛋白。结果在 5 4例HD中选取 13例HD的H/RS细胞 ,经显微切割分离后有 4例 (30 8% )经PCR扩增出HCMV的核酸 ;5 4例HD组织中 ,经PCR检测有 10例 (18 5 % )扩增出HCMV的核酸 ;CSA染色显示有 6例(11 1% )HCMV立即早期抗原和早期抗原 (DDG9/CCH2 )阳性 ,5例 (9 3 % )基质蛋白 (AAC10 )阳性。对照的 17例反应性增生淋巴结中HCMV核酸的PCR检测 ,以及三种HCMV抗原的CSA检测 ,均为阴性。表明HD组织的H/RS细胞中存在HCMV核酸和抗原 ,而反应性增生淋巴结中不存在 ,提示HCMV可能参与了何杰金病的发病过程。 相似文献
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Detection of Leptospira interrogans in clinical specimens by in situ hybridization using biotin-labelled DNA probes 总被引:4,自引:0,他引:4
W J Terpstra G J Schoone G S Ligthart J ter Schegget 《Journal of general microbiology》1987,133(4):911-914
In situ DNA hybridization using biotin-labelled leptospiral DNA was performed on clinical specimens to investigate its usefulness as a technique for the identification of Leptospira interrogans. The applicability of this test in blood, urine and liver smears was demonstrated. In situ DNA hybridization can be completed in only 4 h and it combines the advantage of visualization of the leptospiral morphology with the specificity of the hybridization reaction. No cross-hybridization was observed with other bacteria. This study shows that hybridization in situ can be simple to perform and may contribute to a rapid diagnosis. 相似文献
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A biotin-labelled DNA probe was used in a dot-blot hybridization test to demonstrate the presence of Escherichia coli in a variety of artificially contaminated foodstuffs. Positive hybridization was detected by using a streptavidine/polyalkaline phosphatase conjugate to generate an insoluble coloured precipitate in the presence of an appropriate dye. The colour intensity was measured with a computer-controlled image analysis system which assessed objectively the hybridization signal produced by each sample. The method was capable of distinguishing positive hybridization at cell concentrations exceeding 104 cells/dot-blot, equivalent to 2×107 cells/g food, and had none of the drawbacks normally associated with the use of radioactively labelled DNA in hybridization techniques. The procedure is highly specific and takes less than 30 h. Many samples can be screened simultaneously and the procedure can be used to detect any species for which a suitable DNA probe is available 相似文献