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A Simple Method for Detection of Multiple Chemical-Specific IgGs in Serum Based on Dot Blotting
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作者 Mayumi Tsuji Hsu-Sheng Yu +4 位作者 Yasuhiro Ishihara Toyohi Isse Nami Ikeda-Ishihara Takuto Tuchiya Toshihiro Kawamoto 《Health》 CAS 2016年第15期1645-1653,共10页
Plastic resins are known to cause occupational allergies. Therefore, serum-specific antibodies against plastic resins have been widely investigated as diagnostic markers for occupational allergies. In this study, we a... Plastic resins are known to cause occupational allergies. Therefore, serum-specific antibodies against plastic resins have been widely investigated as diagnostic markers for occupational allergies. In this study, we aimed to establish a convenient method for detection of multiple chemical-specific IgG antibodies in human serum based on dot blot analysis. Toluene diisocyanate (TDI), phthalic anhydride (PA), and formaldehyde (FA), which are frequently used to synthesize various resins, reacted well with lysine residues of human serum albumin (HSA) under alkaline conditions. Native polyacrylamide gel electrophoresis (PAGE) showed that the structures of chemical adducts of HSA were different from those of native HSA. Therefore, we performed dot blot assays using these adducts as artificial antigens. Serum samples from workers at plants utilizing plastic resins strongly reacted with TDI, PA, and FA adducts in HSA, while reduced signals were detecting using the serum from unexposed workers. These results suggested that dot blot assays using chemical-HSA adducts as antigens could be beneficial for simultaneously measuring multiple chemical-specific IgGs. 展开更多
关键词 IGG dot Blot Assay Plastic Resin
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番茄斑萎病毒外壳蛋白原核表达及Dot-blotELISA检测方法的建立 被引量:9
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作者 于翠 邓凤林 +1 位作者 杨翠云 吴建祥 《浙江大学学报(农业与生命科学版)》 CAS CSCD 北大核心 2008年第6期597-601,共5页
将番茄斑萎病毒(Tomato spotted wilt virus,TSWV)全长外壳蛋白基因亚克隆到原核表达载体pET-32a中,经限制性酶切分析及测序结果表明插入方向正确且阅读框架无移码突变.将重组表达质粒转化大肠杆菌BL21(DE3),SDS-PAGE分析结果证实IPTG... 将番茄斑萎病毒(Tomato spotted wilt virus,TSWV)全长外壳蛋白基因亚克隆到原核表达载体pET-32a中,经限制性酶切分析及测序结果表明插入方向正确且阅读框架无移码突变.将重组表达质粒转化大肠杆菌BL21(DE3),SDS-PAGE分析结果证实IPTG可诱导1个分子量约为48 kDa的融合蛋白表达.利用6×His标签单抗和TSWV FoPaTs1多抗证实所表达的蛋白为TSWV外壳蛋白.以纯化的重组外壳蛋白免疫小鼠,制备杂交瘤细胞培养上清单抗,并用杂交瘤细胞培养液上清建立了可靠、有效检测TSWV的Dot-blot ELISA方法. 展开更多
关键词 番茄斑萎病毒 原核表达 dot—blot EL/SA方法
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Comparison of the Tellgenplex HPV DNA test with the PCR-reverse dot blot assay for human papillomavirus genotyping 被引量:2
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作者 Ya-Chao Yao Nan Li +2 位作者 Liang-Shan Hu Ya-Hong Li Zhi Zhang 《Asian Pacific Journal of Tropical Medicine》 SCIE CAS 2018年第2期141-146,共6页
Objective: To access the performance of the Tellgenplex human papillomavirus(HPV) DNA test compared to the polymerase chain reaction-reverse dot blot(PCR-RDB) assay for the HPV genotyping.Methods: Sixty cervical swab ... Objective: To access the performance of the Tellgenplex human papillomavirus(HPV) DNA test compared to the polymerase chain reaction-reverse dot blot(PCR-RDB) assay for the HPV genotyping.Methods: Sixty cervical swab samples were genotyped by the Tellgenplex HPV DNA test and the PCR-RDB assay.The Tellgenplex HPV DNA test and the PCR-RDB assay can detect 26 and 23 HPV genotypes, respectively.Each sample showed discrepancy was genotyped using sequencing.Results: The percent agreement between the two tests ranged from 83.3% to 100.0% according to different genotype.This showed perfect agreement(>0.81) for high-risk HPV genotypes(35, 39, 45, 53, 56, 59, 66, 68, and 82), substantial agreement(>0.65) for high-risk HPV genotypes(16, 18, 33, 52, and 58) and low-risk HPV genotype 43 between the two assays by the kappa analysis.The positive rates of the two assays for frequent HPV genotypes(16, 35, 39, 45, 52, 53, 58, 59, 66, and 82) were not statistically different, but the PCR-RDB assay showed higher positive rates than the Tellgenplex HPV DNA test for HPV genotypes 81(P<0.05).As for more than 10 positive results by the Tellgenplex HPV DNA test and/or the PCR-RDB assay, the PCR-RDB assay showed higher relative sensitivity and specificity than the Tellgenplex HPV DNA test for the three HPV genotypes(16, 52, and 81).All HPV genotypes that can be detected by only the Tellgenplex HPV DNA test(HPV genotypes 44 and 55) were confirmed by sequencing.Conclusions: In conclusion, our results demonstrated that the PCR-RDB assay which can detect more multiple HPV genotypes in each specimen shows higher relative sensitivity and specificity than the Tellgenplex HPV DNA test, which makes it a better option for routine clinical use. 展开更多
关键词 Human papillomavirus Genotying Polymerase chain reaction-reverse dot blot Flowcytometry fluorescence hybridization
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Rapid Detection of Rifampin-resistant Clinical Isolates of Mycobacterium tuberculosis by Reverse Dot Blot Hybridization 被引量:1
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作者 GUO Qian YU Yan +7 位作者 ZHU Yan Ling ZHAO Xiu Qin LIU Zhi Guang ZHANG Yuan Yuan LI Gui Lian WEI Jian Hao WU Yi Mou WAN Kang Lin 《Biomedical and Environmental Sciences》 SCIE CAS CSCD 2015年第1期25-35,共11页
Objective A PCR-reverse dot blot hybridization (RDBH) assay was developed for rapid detection of rpoB gene mutations in 'hot mutation region' of Mycobacterium tuberculosis (M. tuberculosis). Methods 12 oligonucl... Objective A PCR-reverse dot blot hybridization (RDBH) assay was developed for rapid detection of rpoB gene mutations in 'hot mutation region' of Mycobacterium tuberculosis (M. tuberculosis). Methods 12 oligonucleotide probes based on the wild-type and mutant genotype rpoB sequences of M. tuberculosis were designed to screen the most frequent wild-type and mutant genotypes for diagnosing RIF resistance. 300 M. tuberculosis clinical isolates were detected by RDBH, conventional drug-susceptibility testing (DST) and DNA sequencing to evaluate the RDBH assay. Results The sensitivity and specificity of the RDBH assay were 91.2% (165/181) and 98.3% (117/119), respectively, as compared to DST. When compared with DNA sequencing, the accuracy, positive predictive value (PPV) and negative predictive value (NPV) of the RDBH assay were 97.7% (293/300), 98.2% (164/167), and 97.0% (129/133), respectively. Furthermore, the results indicated that the most common mutations were in codons 531 (48.6%), 526 (25.4%), 516 (8.8%), and 511 (6.6%), and the combinative mutation rate was 15 (8.3%). One and two strains of insertion and deletion were found among all strains, respectively. Conclusion Our findings demonstrate that the RDBH assay is a rapid, simple and sensitive method for diagnosing RIF-resistant tuberculosis. 展开更多
关键词 Mycobacterium tuberculosis Rifampin-resistance Reverse dot blot hybridization
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用抑制消减杂交法分离巴西橡胶树胶乳特异表达基因 被引量:8
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作者 邓柳红 罗明武 +2 位作者 曾会才 杨卫帆 张春发 《林业科学》 EI CAS CSCD 北大核心 2006年第6期32-36,共5页
采用巴西橡胶树常割树胶乳的Poly(A+)RNA为Tester,叶片Poly(A+)RNA为Driver,通过抑制消减杂交法(suppressivesubtractivehybridization,SSH)构建了一个胶乳特异表达基因差减文库,通过菌落PCR及反式NorthernDot-Blot筛选鉴定阳性差异片段... 采用巴西橡胶树常割树胶乳的Poly(A+)RNA为Tester,叶片Poly(A+)RNA为Driver,通过抑制消减杂交法(suppressivesubtractivehybridization,SSH)构建了一个胶乳特异表达基因差减文库,通过菌落PCR及反式NorthernDot-Blot筛选鉴定阳性差异片段,获得79个胶乳特异表达阳性克隆,部分阳性克隆还通过NorthernBlot杂交及RT-PCR进一步验证。随机挑选部分阳性克隆序列比较分析,结果表明有部分克隆与巴西橡胶树中已知的基因相同,而多数克隆在巴西橡胶树中是新发现,它们与橡胶生物合成、物质代谢运输、信号传导和形态建成等相关。 展开更多
关键词 巴西橡胶树 抑制消减杂交法 胶乳 反式Northern dot—Blot
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柚(Citrus grandis Osbeck)单染色体LA-PCR-AFLP分子标记体系的建立 被引量:1
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作者 王发明 王平 +2 位作者 唐琦 柳燕贞 陈伟 《热带作物学报》 CSCD 2010年第10期1778-1784,共7页
通过微分离和微克隆技术,对柚单染色体LA-PCR-AFLP技术体系中反应程序、模板浓度等关键参数进行了优化和引物筛选。结果表明:退火温度较高的程序TD3[退火梯度:72℃→65℃30 s(-0.5℃/cyc)]扩增效果较好;稀释100倍(10 ng/μL)为最适模板... 通过微分离和微克隆技术,对柚单染色体LA-PCR-AFLP技术体系中反应程序、模板浓度等关键参数进行了优化和引物筛选。结果表明:退火温度较高的程序TD3[退火梯度:72℃→65℃30 s(-0.5℃/cyc)]扩增效果较好;稀释100倍(10 ng/μL)为最适模板量;10个引物对组合扩增多态性较好。对22条单染色体的AFLP多态性分析和dot-blot杂交验证,表明较好地构建了柚单染色体LA-PCR-AFLP体系。这为柚单染色体的识别和文库构建提供了试验基础。 展开更多
关键词 单染色体 AFLP体系建立 dot—blot
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Mutations in pre-core and basic core promoter regions of hepatitis B virus in chronic hepatitis B patients 被引量:12
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作者 Xiao-Ling Wang Jian-Ping Ren +3 位作者 Xue-Qing Wang Xiao-Hong Wang Shao-Fang Yang Yi Xiong 《World Journal of Gastroenterology》 SCIE CAS 2016年第11期3268-3274,共7页
AIM: To investigate the frequency of mutations in pre-core (pre-C) and basic core promoter (BCP) regions of hepatitis B virus (HBV) from Shanxi Province, and the association between mutations and disease related index... AIM: To investigate the frequency of mutations in pre-core (pre-C) and basic core promoter (BCP) regions of hepatitis B virus (HBV) from Shanxi Province, and the association between mutations and disease related indexes.METHODS: One hundred chronic hepatitis B patients treated at Shanxi Province Hospital of Traditional Chinese Medicine were included in this study. PCR-reverse dot blot hybridization and mismatch amplification mutation assay (MAMA)-PCR were used to detect the mutations in the HBV pre-C and BCP regions. HBV DNA content and liver function were compared between patients with mutant HBV pre-C and BCP loci and those with wild-type loci. The consistency between PCR-reverse dot blot hybridization and MAMA-PCR for detecting mutations in the HBV pre-C and BCP regions was assessed.RESULTS: Of the 100 serum samples detected, 9.38% had single mutations in the pre-C region, 29.17% had single mutations in the BCP region, 41.67% had mutations in both BCP and pre-C regions, and 19.79% had wild-type loci. The rates of BCP and pre-C mutations were 65.7% and 34.3%, respectively, in hepatitis B e antigen (HBeAg) positive patients, and 84.6% and 96.2%, respectively, in HBeAg negative patients. The rate of pre-C mutations was significantly higher in HBeAg negative patients than in HBeAg positive patients (&#x003c7;<sup>2</sup> = 26.62, P = 0.00), but there was no significant difference in the distribution of mutations in the BCP region between HBeAg positive and negative patients (&#x003c7;<sup>2</sup> = 2.43, P = 0.12). The presence of mutations in the pre-C (Wilcoxon W = 1802.5, P = 0.00) and BCP regions (Wilcoxon W = 2906.5, P = 0.00) was more common in patients with low HBV DNA content. Both AST and GGT were significantly higher in patients with mutant pre-C and BCP loci than in those with wild-type loci (P &#x0003c; 0.05). PCR-reverse dot blot hybridization and MAMA-PCR for detection of mutations in the BCP and pre-C regions had good consistency, and the Kappa values obtained were 0.91 and 0.58, respectively.CONCLUSION: HBeAg negative patients tend to have HBV pre-C mutations. However, these mutations do not cause increased DNA copies, but associate with damage of liver function. 展开更多
关键词 Basic core promoter region Pre-core region Liver injury Reverse dot blot hybridization Mismatch amplification mutation assay
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Profile, spectrum and significance of hepatitisB virus genotypes in chronic HBV-infected patients in Yunnan, China 被引量:9
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作者 Hutcha Sriplung Virasakdi Chongsuvivatwong Alan Geater 《Hepatobiliary & Pancreatic Diseases International》 SCIE CAS 2008年第3期271-279,共9页
BACKGROUND: There are significant variations in the geographical distribution of hepatitis B virus (HBV) genotypes throughout the world, and some genotypes are associated with different clinical outcomes. Eight genoty... BACKGROUND: There are significant variations in the geographical distribution of hepatitis B virus (HBV) genotypes throughout the world, and some genotypes are associated with different clinical outcomes. Eight genotypes of human HBV (designated A-H) have been reported. The present study was designed to examine the distribution of HBV genotypes among patients at various stages of chronic type B liver disease in Yunnan Province, China, and to explore its significance and the relationship of HBV genotype with gender and age, clinical spectrum of chronic HBV infection, and viral replicative activity. METHODS: Serum samples from 126 patients with chronic HBV infection from Yunnan Province, including 26 chronic asymptomatic HBV carriers (ASC), 61 patients with chronic hepatitis B (CHB) (21 mild, 30 moderate and 10 severe), 20 patients with chronic fulminant hepatic failure (CFHF), 12 patients with HBV-related liver cirrhosis (LC) and 7 patients with HBV-related hepatocellular carcinoma (HCC) were analyzed using reverse dot blot (RDB) methodology, which is based on the reverse hybridization principle for HBV genotyping. The relations of HBV genotype with gender and age, clinical patterns, and serological data of the patients were analyzed. RESULTS: In this series, genotypes A, B, C, and D were found. 38.1% patients (48/126) belonged to B, 54.8% (69/126) to C, 0.8% (1/126) to D, 1.6% (2/126) to a mixture of B and C, and 1.6% (2/126) to a mixture of A and C. 3.2% patients (4/126) had unknown genotypes. No other genotypes (E, F, G, and H) were found. Genotypes B and C were predominant. There was a statistically significant difference in the distributions of genotypes C and B (chi(2)=7.04, P=0.008), and C was the dominant genotype in all patient categories. The rate of genotype B in the mild CHB group was significantly higher than that in the moderate and severe groups (chi(2)=12.16, P=0.0001; chi(2)=11.98, P=0.001, respectively), the ASC group (chi(2)=5.46, P=0.02), the CFHF group (chi(2)=5.53, P=0.019), and the LC/HCC group (chi(2)=12.13, P=0.001). The rate of genotype C in the LC/HCC group and the severe CHB group were significantly higher than that in the mild group (chi(2)=9.95, P=0.002; chi(2)=8.78, P=0.003, respectively). HBV DNA positivity and HBeAg positivity were higher in genotype C than in genotype B (chi(2)=9.81, P=0.002; chi(2)=3.85, P=0.05, respectively). The prevalence of genotype C showed an increasing trend in lowest-, middle- and highest-level groups of HBV replication (25.0%, 70.0%, and 55.6%, respectively); in contrast, the prevalence of genotype B showed an opposite trend in the same order (62.5%, 30.0%, and 37.0%, respectively). The rate of genotype C in the highest-level group of HBV replication was higher than genotype B (chi(2)=7.45, P=0.006). The rate of genotype C in the over-30 age group was higher than that in the below-30 age group (chi(2)=3.7, P=0.05). There was no difference between the sexes (P>0.05). More severe liver damage was found in genotype C than in genotype B (P<0.05). CONCLUSIONS: The predominant HBV genotypes in chronic HBV-infected patients are B and C, and C is the most prevalent genotype in Yunnan Province, China. HBV genotype C is associated with the development of more severe liver disease and a higher level of HBV viral replication, and genotype B has a relatively good progress. 展开更多
关键词 hepatitis B virus GENOTYPE reverse dot blot distribution clinical interrelation
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Cloning and Sequence Analysis of Genome from the Inner Mongolia Strain of the Endogenous Betaretroviruses (enJSRV) 被引量:4
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作者 Yu WANG Shu-ying LIU +2 位作者 Jian-yun LI Min HAN Zhen-ling WANG 《Virologica Sinica》 SCIE CAS CSCD 2008年第1期15-24,共10页
In order to amplify the complete genome of enJSRV from the strain of Inner Mongolia (enJSRV-NM), we used enJSRV-specific and JSRV-specific DNA probes in dot blot hybridization. Seven pairs of primers were designed bas... In order to amplify the complete genome of enJSRV from the strain of Inner Mongolia (enJSRV-NM), we used enJSRV-specific and JSRV-specific DNA probes in dot blot hybridization. Seven pairs of primers were designed based on Genbank sequences. Seven fragments were obtained by PCR and were cloned into the PMD19-T vectors. The recombinant plasmids were sequenced and analyzed. The results showed that the genome was 7 942 bp in length and contained four overlapping open reading frames corresponding to the gag, pro, pol and env genes as well as an additional open reading frame (orf-x) that overlaps the 3' end of the pol gene. The nucleotide acid sequences of the enJSRV-NM loci were compared with the sequences of South Africa enJS56A1 strain (Accession No. AF153615) and USA JSRV21 strain (Accession No. AF105220). The nucleotide acid identities were 99.2% and 92.3% respectively. Two zinc fingers were found in the NC region in the predicted amino acid sequence. However, the YXXM motif, which is a reliable molecular marker for the infectious exogenous virus, was not found in the TM region. It was found that the enJSRV-NM region was 90%-98% identical at the amino acid level to its exogenous infectious counterparts in most of the retroviral genome. This is the first nucleotide sequence of enJSRV reported in P.R China. The resource work has provided a wide range of information useful not only for expression genomics and annotation of genomic DNA sequence, but also for further research on the clinical diagnosis of OPA. 展开更多
关键词 Jaagsiekte sheep retrovirus (JSRV) Endogenous betaretroviruses (enJSRV) Ovine pulmonaryadenocarcinoma (OPA) dot blot hybridization
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Differential gene expression between asymptomatic HBV carriers and normal adults 被引量:2
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作者 Tang, Cui-Lan Chen, Zhi 《Hepatobiliary & Pancreatic Diseases International》 SCIE CAS 2009年第4期383-388,共6页
BACKGROUND: There are more than 100 million wa symptomatic HBV carriers (ASCs) in China and they are at a high risk of developing liver disease, which creates a serious health problem. But more than 90% of normal adul... BACKGROUND: There are more than 100 million wa symptomatic HBV carriers (ASCs) in China and they are at a high risk of developing liver disease, which creates a serious health problem. But more than 90% of normal adults clear virus from primary HBV infection, so the difference of gene expression between ASCs and normal adults determines the differential outcomes. To identify differentially expressed genes in ASCs compared to normal adults, we used suppression subtractive hybridization to compare gene expression. METHODS: cDNA subtracted libraries were constructed by suppression subtracted hybridization from peripheral blood monocytes of ASCs and normal adults, the subtracted clones were prescreened by dot blot hybridization, and the levels of genes of interest were confirmed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: One hundred and two and 96 positive clones were acquired from the A-N and N-A libraries, respectively, and 14 and 11 clones were identified by dot blot hybridization in the A-N and N-A libraries. Two genes were confirmed as differentially expressed between a set of ASC and normal adult samples by real-time RT-PCR. CONCLUSIONS: Differentially expressed genes in peripheral blood mononuclear cells between ASCs and normal adults were isolated by suppression subtractive hybridization, and included some new genes. Of the upregulated genes in ASCs, checkpoint suppressor I is associated with DNA damage-induced cell cycle arrest. Perforin I is associated with inflammation. The information about such alterations in gene expression could be useful for elucidating the mechanisms of ASC pathogenesis. 展开更多
关键词 hepatitis B virus asymptomatic HBV carriers suppression subtractive hybridization dot blot hybridization reverse transcriptase-polymerase chain reaction
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Effect of Jianpiyiqi Prescription on the Expression of Heat Shock Proteins in Acetic Acid-induced Chronic Gastric Ulcer Rats
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作者 陶秀良 李国成 罗树星 《Journal of Huazhong University of Science and Technology(Medical Sciences)》 SCIE CAS 2001年第2期105-107,共3页
To investigate the effect of Jianpiyiqi prescription on the expression of heat shock proteins and the changes of HSPs in gastric ulcerated rats, the rat model of chronic gastric ulcer was induced by acetic acid. The... To investigate the effect of Jianpiyiqi prescription on the expression of heat shock proteins and the changes of HSPs in gastric ulcerated rats, the rat model of chronic gastric ulcer was induced by acetic acid. The SABC immunohistochemical method was used to observe HSP70 of mucosa around the gastric ulcer. Imaging analysis was performed. Western dot blot was used to detect HSPs contents in the plasma and gastric mucosal homogenate in each group. The results showed that HSP70 contents of the mucosa around the gastric ulcer in the model group and ranitidine treated group were increased as compared with control group . Jianpiyiqi could increase the expression of HSP70 of the mucosa around the gastric ulcer further as compared with that in the model group and ranitidine treated group . The HSP70 contents in the serum and mucosa in the model group and ranitidine treated group were increased as compared with control group ( P <0.01, P <0 05 respectively). HSP70 of serum and mucosa in the Jianpiyiqi treated group was higher than in the model group and ranitidine treated group ( P <0.05, P <0.01 respectively).It was concluded that HSP might play a role in the process of pathophysiology of gastric ulcer. Jianpiyiqi could enhance gastric ulcer healing through the protective mechanism of HSPs. 展开更多
关键词 peptic ulcer heat shock proteins acetic acid IMMUNOHISTOCHEMISTRY western dot blot Jianpiyiqi
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Isolation, Cloning, and Identification of Expressed Sequence Tags from the Backfat Tissue in Duroc and Tongcheng Pigs by mRNA Differential Display
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作者 REN Zhu-qing XIONG Yuan-zhu DENG Chang-yan LEI Ming-gang ZUO Bo LI Feng-e ZHENG Rong XU De-quan 《Agricultural Sciences in China》 CAS CSCD 2006年第2期141-145,共5页
mRNA differential display technique was performed to discuss the differential expression of genes in fat tissue between introduced European and Chinese indigenous pigs. Four anchor primers in combination with five arb... mRNA differential display technique was performed to discuss the differential expression of genes in fat tissue between introduced European and Chinese indigenous pigs. Four anchor primers in combination with five arbitrary primers (20 sets in total) were used and nearly 300 bands were observed in polyacrylamide gel, among which 29 differential display bands were obtained. Twelve of 29 cDNA fragments were identified using reverse Northern dot blot, and subsequently cloned and sequenced. Eight of 12 cDNAs had no matches in GenBank and were submitted to GenBank, and the other 4 showed similarity to identified genes from GenBank. Three among 8 novel ESTs were selected to be further identified by semiquantitative RT-PCR. In our experiment, silver staining DDRT-PCR and DIG primer DNA labeling reverse Northern dot blot were used to avoid radioactive pollution. The result showed that the expressions of 5 among 8 novel ESTs were stronger in the backfat of Tongcheng pigs and the others were weaker than that in Duroc pigs. These novel ESTs were prepared for selecting genes related to adipose cells. 展开更多
关键词 ESTS fat tissue reverse Northern dot blot semi-quantitative RT-PCR mRNA differential display
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Carrier Screening and Prenatal Gene Diagnosis of β-thalassemia by PCR-RDB Technique
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作者 张宏秀 单可人 +6 位作者 惠春林 何燕 袁筑华 窦友莲 曾金琳 谢渊 修瑾 《Journal of Huazhong University of Science and Technology(Medical Sciences)》 SCIE CAS 2003年第1期55-56,共2页
In order to identify the distribution of gene types of β-thalassemia and reduce the birthrates of β-thalassemia major in Guiyang area, 1054 pregnant women and their spouses from Affiliated Hospital, Guiyang Medical ... In order to identify the distribution of gene types of β-thalassemia and reduce the birthrates of β-thalassemia major in Guiyang area, 1054 pregnant women and their spouses from Affiliated Hospital, Guiyang Medical College were screened. The positive samples were analyzed with polymerase chain reaction and reverse dot blot method (PCR-RDB). When both partners were heterozygous identified as carriers for β- thalassemia, the risk of having a fetus who was homozygous or compound heterozygous was 2.66 %; the ratio of male to female was 1/1.15. Seven types of mutation were identified. CD17 and CD41-42 were dominant among them. Among the 4 cases subject to prenatal gene diagnosis, one fetus was completely normal and 3 fetuses were diagnosed as having β-thalassemia major (1 homozygous and 2 compound heterozygous). The fetuses diagnosed as β-thalassemia major were selectively terminated within two weeks. It was concluded that the birthrate of β-thalassemia major in Guiyang area was reduced and the target of improving birth outcome and child development has been achieved. 展开更多
关键词 THALASSEMIA polymerase chain reaction reverse dot blot prenatal gene diagnosis
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Construction of white spot syndrome virus (WSSV) whole genome phage display library
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作者 ZHU Yanbing YANG Feng 《Acta Oceanologica Sinica》 SCIE CAS CSCD 2007年第2期75-83,共9页
A rebuilt vector pCANTAB 5 EE was obtained by inserting a 34 bp double-stranded oligonucleotide which contained a EcoRV recognition sequence into pCANTAB 5 E. White spot syndrome virus (WSSV) genome DNA was fragment... A rebuilt vector pCANTAB 5 EE was obtained by inserting a 34 bp double-stranded oligonucleotide which contained a EcoRV recognition sequence into pCANTAB 5 E. White spot syndrome virus (WSSV) genome DNA was fragmented by sonication to isolate fragments mainly in the range of 0.8 ~2.0 kb, then the fragments were blunt-ended with T4 DNA polymerase and cloned into the EcoRV site of pCANTAB 5 EE. The primary recombinant clone of the library was 3.0 × 10^5.Colony PCR of random selected recombinants showed that the size of the inserts was 0.12 ~ 1.77 kb. After the whole library recombinant phages infected Escherichia coli HB2151 cells, the extracellular and periplasmic extracts were dropped on PVDF membranes to perform dot blot, using polyclonal mouse anti-VP24 serum,anti-WSV026 serum,anti-WSV063 serum,anti-WSV069 serum,anti-WSV112 serum, anti WSV238 serum,anti-WSV303 serum and anti-VP26 serum as the primary antibody, respectively. The results showed that the display library could express the viral proteins. 展开更多
关键词 white spot syndrome virus genome phage display library dot blot
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Expression and Cellular Localization of Interleukin-6 mRNA in Ovariectomized Rats
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作者 涂意辉 杜靖远 杨安礼 《Journal of Huazhong University of Science and Technology(Medical Sciences)》 SCIE CAS 2000年第2期145-147,共3页
In order to observe the expression and cellular localization of interleukin-6 (IL-6) mRNA in bone tissue, ovaria of the rats were excised to develop osteoporosis model. The expression of IL-6 mRNA in bone tissues was ... In order to observe the expression and cellular localization of interleukin-6 (IL-6) mRNA in bone tissue, ovaria of the rats were excised to develop osteoporosis model. The expression of IL-6 mRNA in bone tissues was detected by using dot blot hybridization assay and the cells producing IL6 identified and localized by using in situ hybridization respectively. The results showed that the expression of IL-6 mRNA was significantly increased in the ovariectomized rats as compared with that in normal control rats and strong IL-6 mRNA hybridization signals were detected in lining cells, osteoblasts and osteocytes. It was suggested that loss of ovarian function induced in vivo osteoblast lineage increased IL-6 mRNA expression. IL-6 might play important roles in the development of bone loss following ovariectomy. 展开更多
关键词 INTERLEUKIN-6 OVARIECTOMY dot blot hybridization in situ hybridization
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Detectingβ-thalassaemia mutations from a single cell by PEP and RDB
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作者 易萍 李力 +3 位作者 姚宏 周元国 邓兵 陈竹钦 《Journal of Medical Colleges of PLA(China)》 CAS 2006年第3期158-163,共6页
Objective :To evaluate the possibility of the technology involving PEP and RDB for detectingβ-thalassaemia multipoint mutations from a single cell simultaneously. Methods: A set of allele specific oligonucleotide (AS... Objective :To evaluate the possibility of the technology involving PEP and RDB for detectingβ-thalassaemia multipoint mutations from a single cell simultaneously. Methods: A set of allele specific oligonucleotide (ASO) probes used for detecting 8 familiarβ-thalassaemia mutations (CD41-42. IVS-Ⅱ-654, CD17, TATA box nt-28, CD71-72, TATA box nt-29, CD26, IVS-Ⅰ-5) were immobilized on a strip of nylon membrane. The genome of a individual cell was amplified by primer extension preamplification (PEP) with the mixture of 15-base random oligonucleotides. The aliquots from PEP were used to amplify the objective gene fractions ofβ-thalassaemia gene by nested or semi-nested PCR. The membrane was hybridized with the final amplified products and then treated with Streptavidin-HRP and color development. Results:Totally 30 lymphocytes were picked up from blood samples of 1 healthy female and 4 patients with knownβ-thalassaemia mutations respectively. Each single lymphocyte was lysed in the proteinase K buffer. The amplification efficacy was 94. 0% and alle drop-out (ADO) rate was 8. 0%. Revert dot blot (RDB) was applied to the final amplified products from the 5 participants. The results of diagnosis were the same to the expected, and their genotypes were N/N, CD17(A→T)/N, IVS-Ⅱ-654(C→T)/CD17(A→T), CD41-42(-CTTT)/N and TATA box nt-28(A→G)/N, respectively. Conclusion: The technology involving PEP and RDB could detect multipleβ-thalassaemia mutations from a single cell simultaneously, and the research provides experimental evidences for the feasibility of applying PEP and DNA array technology to screening multiple genetic mutations from a single cell, and will be applied to preimplantation genetic diagnosis and non-invasive prenatal diagnosis forβ-thalassaemia. 展开更多
关键词 β-thalassaemia preamplification reverse dot blot
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Temporal analysis of mRNA expression of endogenous TGFβ And its typeⅠ,typeⅡreceptor on burn wounds
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作者 韦多 葛绳德 +2 位作者 陈玉林 戴方平 苏波 《Journal of Medical Colleges of PLA(China)》 CAS 1996年第3期205-209,共5页
Recent studies have shown that application of exogenous transforming growth factor?(TGF?)to a wound has a beneficial effect. However,little is known about the roles of endogenous TGF? and its receptors in the course o... Recent studies have shown that application of exogenous transforming growth factor?(TGF?)to a wound has a beneficial effect. However,little is known about the roles of endogenous TGF? and its receptors in the course of wound healing. In the present study, mRNA expression of endogenous TGF? and its type Ⅰ,type Ⅱreceptors on wounds of burned rats was observed by using dot blot hybridization.Thermal injury could induce expression of TGF ?, and its type Ⅰ,type Ⅱ receptor genes, and their expression appeared to be regulated. The expression was the strongest at 5 and 7 d postscalding when the repair of wounds was most active.The study suggests that TGF? plays an important role in epidermis regeneration, and that expression of TGF ? receptors is one of factors regulating wound healing. The expression discrepancy between TGF ? and its type Ⅰ,type Ⅱ receptor genes at the later stages of wound healing implies the presence of a well-controlled mechanism to limit excessive effect of endogenous TGF ? on repair cells. 展开更多
关键词 BURNS wound healing transforming growth factor β ENDOGENOUS RNA messenger dot blot hyBridization
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Screening of Species-specific DNA Probes for Identification of Fallopia multiflora
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作者 Chuanjin ZHENG Nian CHEN 《Agricultural Biotechnology》 CAS 2014年第1期22-25,30,共5页
To screen species-specific DNA probes for identification of Fallopia muhiflora, the genomic DNA (gDNA) suppression subtraction hybridization (SSH) between F. muhiflora and F. muhiflora var. ciliinervis was firstly... To screen species-specific DNA probes for identification of Fallopia muhiflora, the genomic DNA (gDNA) suppression subtraction hybridization (SSH) between F. muhiflora and F. muhiflora var. ciliinervis was firstly performed. The obtained differential gDNA fragments by SSH were then hybridized with gDNA ar- rays consisting of multiple whole genomes of several species (adulterants and/or closely related species of F. muhiflora) and four differential fragments were screened uniquely representing F. muhiflora, which could be used as F. muhiflora species-specific probes. The screened DNA probes were tested by reverse dot blot hybridization and the results demonstrated that these probes could be used reliably to identify F, muhiflora. The species-specific DNA probes obtained in this study exhibited broad application prospects in the preparation of gene chips for identifying Chinese traditional medicines and the authentication of germplasm re- sources and crude drugs of F. muhiflora. 展开更多
关键词 Fallopia muhiflora DNA probe Species identification Reverse dot blot hybridization
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Detection of Human Papillomavirus Type 16 DNA in Cervical Carcinomas
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作者 高基民 徐钤 陈碧魂 《Journal of Medical Colleges of PLA(China)》 CAS 1989年第3期187-190,共4页
Accurate typing of the different human papillomavirus types is csscntial in view of the differ-ent pathological potential of the common virus types of human papillomavirus (HPV) present in thecervix. We have develop... Accurate typing of the different human papillomavirus types is csscntial in view of the differ-ent pathological potential of the common virus types of human papillomavirus (HPV) present in thecervix. We have developed hybridization, washing and autoradiography conditions that minimize thecross-hybridization among different specific types of HPV so as to allow clear - cut type assignmentthrough practical dot blot hybridization technique using nylon membrane and <sup>35</sup>S - labeled HPV - 16DNA probe. Under these conditions seventeen of thirty (56.7%) of squamous cell carcinomas of thecervix uteri obtained from Tianjin women were detected in the presence of HPV - 16 DNA. 展开更多
关键词 human papillomavirus cervical carcinoma dot blot bybridization ^(35)S-labeled HPV DNA probe
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Accuracy of a reverse dot blot hybridization assay for simultaneous detection of the resistance of four anti-tuberculosis drugs in Mycobacterium tuberculosis isolated from China 被引量:2
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作者 Li Wan Qian Guo +9 位作者 Jian-Hao Wei Hai-Can Liu Ma-Chao Li Yi Jiang Li-Li Zhao Xiu-Qin Zhao Zhi-Guang Liu Kang-Lin Wan Gui-Lian Li Cha-Xiang Guan 《Infectious Diseases of Poverty》 SCIE 2020年第2期100-101,共2页
Background:Drug resistant tuberculosis poses a great challenge for tuberculosis control worldwide.Timely determination of drug resistance and effective individual treatment are essential for blocking the transmission ... Background:Drug resistant tuberculosis poses a great challenge for tuberculosis control worldwide.Timely determination of drug resistance and effective individual treatment are essential for blocking the transmission of drug resistant Mycobacterium tuberculosis.We aimed to establish and evaluate the accuracy of a reverse dot blot hybridization(RDBH)assay to simultaneously detect the resistance of four anti-tuberculosis drugs in M tuberculosis isolated in China.Methods:In this study,we applied a RDBH assay to simultaneously detect the resistance of rifampicin(RIF),isoniazid(INH),streptomycin(SM)and ethambutol(EMB)in 320 clinical M.tuberculosis isolates and compared the results to that from phenotypic drug susceptibility testing(DST) and sequencing.The RDBH assay was designed to test up to 42 samples at a time.Pearson's chi-square test was used to compute the statistical measures of the RDBH assay using the phenotypic DST or sequencing as the gold standard method,and Kappa identity test was used to determine the consistency between the RDBH assay and the phenotypic DST or sequencing.Results:The results showed that the concordances between phenotypic DST and RDBH assay were 95%for RIF,92.8%for INH,84.7%for SM,77.2%for EMB and the concordances between sequencing and RDBH assay were 97.8%for RIF,98.8%for INH,99.1%for SM,93.4%for EMB.Compared to the phenotypic DST results,the sensitivity and specificity of the RDBH assay for resistance detection were 92.4 and 98.5%for RIF,90.3 and 97.3%for INH,77.4 and 91.5%for SM,61.4 and 85.7%for EMB,respeaively;compared to sequencing,the sensitivity and specificity of the RDBH assay were 97.7 and 97.9%for RIF,97.9 and 100.0% for INH,97.8 and 1OO.O% for SM,82.6 and 99.1%for EMB,respectively.The turnaround time of the RDBH assay was 7 h for testing 42 samples.Conclusions:Our data suggested that the RDBH assay could serve as a rapid and efficient method for testing the resistance of M. tuberculosis against RIF,INH,SM and EMB,enabling early administration of appropriate treatment regimens to the affected drug resistant tuberculosis patients. 展开更多
关键词 Mycobacterium tuberculosis Drug resistance Reverse dot blot hybridization ISONIAZID RIFAMPICIN STREPTOMYCIN ETHAMBUTOL
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