戒毒者乙肝病毒标志物检测的分析

我们在１９９８年３月～１０月,对４５０例戒毒者做了血清乙肝病毒标志物的检测,年龄１９～３５岁,现将检测结果报告如下：１ 材料与方法１．１ 标本来源：我院４５０例住院戒毒者的空腹静脉血。１．２ 试剂：由珠海亚利生物工程有限公司提供的快速２０ＥＬＩＳＡ试剂盒（ＨＢｓＡｇ、抗－ＨＢｓ、ＨＢｅＡｇ、抗－ＨＢｅ、抗ＨＢｃ）;卫生部临检中心的临界值质控血清做质控物与标本一起进行检测。１．３ 检测仪器：ＭＲ６００酶标仪。１．４ 方法：各项检测均采用ＥＬＩＳＡ法,操作规程按照试剂盒的说明书方法检测。２ 结果（见附表）…     

聚合酶链反应检测乙肝患者唾液腺组织中的HBV DNA

应用免疫组化技术对１５例乙型肝炎患者的唾液腺组织进行了检测，其中ＨＢｓＡｇ阳性６例，ＨＢｃＡｇ１例，继而采用原位杂交及聚合酶链反应（ＰＣＲ）技术对免疫组化的阳性病例作了进一步检测，其阳性率分别为２／６（３３．３％）及４／６（６６．７％）．结果表明：唾液中的ＨＢＶ源于受染的唾液腺组织，但唾液腺组织中不存在有ＨＢＶ的复制．     

贵州地区不同人群血清庚型肝炎病毒核酸检测和部分核苷酸序列分析

目的了解贵州地区庚型肝炎病毒感染状况,分析庚型肝炎病毒贵州株的基因特点。方法 以庚型肝炎病毒（ＨＧＶ）的ＮＳ３区两对寡核苷酸为引物,用逆转录套式聚合酶链反应（ＲＴ－ｎＰＣＲ）检测不同人血血清中庚型肝炎病毒核酸（ＨＧＶ ＲＮＡ０,并用ＰＣＲ产物直接测序分析３份阳性血清５’－非编码区的部分核苷酸序列。结果６０例正常人,３７例血液病,８６例肝病患者及１０７例静脉药瘾者血清ＨＧＶ ＲＮＡ阳性率分别为１．７     

聚合酶链反应技术的应用及HBV－DNA结果分析

聚合酶链反应技术的应用及ＨＢＶ－ＤＮＡ结果分析金德来（安徽省安庆市传染病医院检验科安庆市２４６０００）聚合酶链反应（ＰＣＲ）是一种将核酸片断在体外无限量的扩增的最新的生物学技术。它采用遗传基因工程技术直接将致病基因或病员体ＤＮＡ（或ＲＮＡ）进行检测，...     

致肾盂肾炎大肠杆菌papA免疫保护作用的初步研究

目的：对致肾盂肾炎大肠杆菌ｐ菌毛的结构基因ｐａｐＡ的免疫保护作用进行研究。方法：用ＰＣＲ方法扩增ｐａｐＡ基因,并将５５４ｂｐ扩增产物克隆入真核表达载体ｐｃＤＮＡ３,构建重组质粒ｐＣＴ３７作为ＤＮＡ疫苗,免疫ＢＡＬＢ／ｃ小鼠。结果：全菌ＥＬＩＳＡ测定显示疫苗接种组小鼠血清效价明显高于对照组,分别为１：５７５０和１：４０００（Ｐ〈０．０１）。尿液和肾脏菌落计数在接种组均明显低于对照组（Ｐ〈０．０５）,     

用裸RNA免疫小鼠产生抗天然HIV-1包膜糖蛋白单克隆抗体

作者介绍了使用西门利克森林病毒（ＳＦＶ）这一新型表达系统产生具有天然构型的１型人类免疫缺陷症病毒（ＨＩＶ－１）包膜（Ｅｎｖ）糖蛋白，以及用裸ＳＦＶ－ｇｐ１６０ＲＮＡ免疫小鼠制备抗Ｅｎｖ单克隆抗体（ＭｃＡｂ）和对１２ＨＺ杂交瘤细胞系进行鉴定的结果。用聚合酶链反应（ＰＣＲ）扩增全长ＨＩＶ－１ＨＸＢ２ｅｎｖ基因并将其插入ｐＳＦＶ－１载体的ＢａｍＨＩ位点，构建表达载体ＰＳＦＶ－ｇｐ１６０．纯比后采用电穿孔技术将ＳＦＶ－ｇｐｌ６０ＲＮＡ转染ＢＨＫ－２１细胞。以蛋白印迹法、ＳＤＳ－ＰＡＧＥ及免疫荧光法检测Ｈ…     

肝炎和巨细胞病毒与再生障碍性贫血相关性研究

应用套叠式聚合酶链反应（ＮＥＳＴ－ＰＣＲ）检测了４６例首发再生障碍性贫血（ＡＡ）和１０５例随机正常人血清中的甲型肝炎病毒ＲＮＡ（ＨＡＶ、ＲＮＡ）、乙型肝炎病毒ＤＮＡ（ＨＢＶ、ＤＮＡ）。丙型肝炎病毒ＲＮＡ（ＨＣＶ、ＲＮＡ）和人类巨细胞病毒ＤＮＡ（ＣＭＶ、ＤＮＡ）。结果显示：ＡＡ组中ＨＡＶ、ＨＢＶ、ＨＣＶ和ＣＭＶ一率分别为８．７％、７８．８９％、４１．３％和６７．３９％;而正常人中ＨＡＶ、ＨＢＶ、睡Ｃ     

FQ-PCR技术检测438例HBV DNA结果分析

荧光定量ＰＣＲ(ＦＱ—ＰＣＲ)技术是近年发展起来的基因检测技术 ,现已在临床检验工作中得到广泛的应用 ,我院开展此项新技术以来 ,共检测ＨＢＶＤＮＡ４３８例 ,现报道如下。１材料及方法１ １材料 :(１)ＰＣＲ扩增仪 (美国ＰＥ９６００)。 (２)ＤＡ６２０微量荧光检测仪 (上海棱光技术有限公司生产)。 (３)ＨＢＶＤＮＡ试剂盒 (深圳市达尔安生物工程有限公司生产 )。 (４)标本来自我院２００１检测ＨＢＶＤＮＡ送检标本共４３８例 ,其中男２８５例 ,女性１５３例。１ ２方法 :(１)用一次性无菌注射器抽静脉血１ｍｌ于Ｅｐ ｐｅｎｄｏｆｆ…     

多巴胺D3受体基因多态性与帕金森病

目的：探讨多巴胺Ｄ３受体（ＤＲ Ｄ３）基因多态性与帕金森病（ｐａｒｋｉｎｓｏｎ ｄｉｓｅａｅ，ＰＤ）遗传易感性的关系。方法：采用聚合酶链反应－限制性片段长度多态性（ＰＣＲ－ＲＦＬＰ）技术分析比较了１３０例ＰＤ病人和１２０名正常对照者的ＤＲＤ３基因第１号外显子ＢａｌＩ多态性和第５号内含子ＭｓｐＩ多态性。结果：ＤＲ Ｄ３基因ＢａｌＩ多态位点和ＭｓｐＩ多态位点各基因型的分布和等位基因频率在ＰＤ组和对照     

PCR法检测慢性乙肝患者HBV－DNA的结果分析

ＰＣＲ法检测慢性乙肝患者ＨＢＶ－ＤＮＡ的结果分析李卓名聚合酶链反应（ＰＣＲ，ＰｏｌｙｍｅｒａｓｅＣｈａｉｎＲｅａｃｔｉｏｎ），于１９８５年首见报道。本文应用此技术检测我院住院的ＨＢＶ－Ｍ阳性的慢性肝病患者血清ＨＢＶ－ＤＮＡ，并与酶联免疫检测ＨＢｓＡｇ...     

Congenital adrenal hyperplasia: epidemiology, management and practical drug treatment

Congenital adrenal hyperplasia (CAH) owing to 21-hydroxylase deficiency is a common disorder, and is characterised by a defect in cortisol biosynthesis with or without a defect in aldosterone synthesis and androgen excess. The classic form, also known as the severe form, occurs in 1:15,000 births worldwide, while the nonclassic or mild form occurs in approximately 1:1,000 births worldwide and is much more common (up to 1:20) in certain ethnic groups. In classic 21-hydroxylase deficiency, glucocorticoids are given in doses sufficient to suppress adrenal androgen secretion, and mineralocorticoids are given to normalise electrolytes and plasma renin activity. The management of CAH may be complicated by iatrogenic Cushing's syndrome, inadequately treated hyperandrogenism, or both. Prenatal treatment may decrease virilisation of the affected female foetus, but the efficacy and safety of treating CAH prenatally remains to be fully defined. Close clinical monitoring of growth and development is essential to optimise treatment outcome. New treatment approaches are currently under investigation in the most severely affected patients, while nonclassic CAH does not always require treatment.     

HLA AND HORMONAL DATA FOR IDENTIFICATION OF HETEROZYGOTES IN 11β- AND 17α-HYDROXYLASE DEFICIENCY SYNDROMES

1. In previous studies, baseline and ACTH-stimulated hormone levels, plus HLA genotyping, have been used to detect heterozygous carriers in congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21-OHDS). 2. In the present study similar parameters were determined in a family of four including two children with CAH due to 11 beta-hydroxylase deficiency (11-OHDS), and a family of twelve including three sibs (two females, one genotypically male) with CAH due to 17 alpha-hydroxylase deficiency (17-OHDS). 3. HLA typing showed affected sibs with 11-OHDS to differ in one of their haplotypes. No significant differences in basal and ACTH-stimulated steroid levels were seen between the parents (obligate heterozygotes) and the general population. 4. In 17-OHDS, affected members differed from one another in one to two haplotypes; one patient had identical HLA profiles with two of the normal siblings, as did the genotypically male patient with two others; each of the other healthy siblings had one haplotype found in two of the affected subjects. The genes responsible for 11-OHDS and 17-OHDS--in contrast with 21-OHDS--do not appear to be HLA-linked. However, the measurement of ACTH-stimulated corticosterone levels may be useful, since the gene responsible for 17-OHDS seems to be expressed hormonally in the heterozygous state.     

Molecular diagnosis of CYP21 mutations in congenital adrenal hyperplasia: implications for genetic counseling

Congenital adrenal hyperplasia (CAH) is an inherited disorder of steroid biosynthesis most often attributable to mutations in CYP21 (also termed CYP21A2) encoding the active steroid 21-hydroxylase enzyme. This review focuses on clinical and genetic aspects of CAH, and updates the reader on current methodology and applications for molecular genetic diagnosis. Genotyping patients with CAH has revealed > 50 mutations within CYP21, yet only 10 mutations account for approximately 95% of affected alleles. Many CYP21 mutations are gene conversions arising via transfer of gene sequences between the non-functional CYP21 pseudogene and CYP21. Phenotype is generally well-correlated with genotype. Historically, CAH has been divided into 3 types of disease: classic salt-wasting, classic simple virilizing (non-salt-wasting), and nonclassic. Recent findings support the notion that rather than discrete phenotypic categories, CAH is better represented as a continuum of phenotypes, from severe to mild. Molecular genetic diagnosis is most effectively employed now in prenatal diagnosis of classic CAH. As newborn screening for CAH becomes more widespread, genotyping may be implemented to resolve diagnostic difficulties encountered with hormonal testing. As automated methods of DNA diagnosis such as microarrays or gene chips are refined, it is likely that genetic screening will become less expensive and more readily available. The clinician should be aware of the potential for both false negatives and false positives with PCR-based gene screening. In short, whereas molecular genetic diagnosis is a valuable tool, it cannot replace clinical acumen and hormonal assays.     

解偶联蛋白2基因多态性与糖尿病并发大血管病变的相关性分析

目的探讨解偶联蛋白2（ucP-2）基因8号外显子3’-末端（3’．UTR）45bp插Ⅳ缺失多态性与糖尿病并发大血管病变的关系。方法182例糖尿病患者中并发大血管病变患者80例（A组）、无大血管病变患者102例（B组）,检测两组基因组DNA,用聚合酶链反应（PCR）方法对ucP-2基因进行扩增,记录各组uCP-2等位基因及基因型频率并进行比较。结果UCP．2基因3’-UTR45bp插入／缺失多态性在A组和B组中基因型分布分别为（II：6．25％、ID：18.75％、DD：75．00％）和（II：9．80％、ID：23．53％、DD：66．67％）,两组差异均无统计学意义（均P〉0．05）,两组等位基因频率分别为（I：15．63％、D：84．37％）和（I：21．57％、D：78．43％）,两组间差异均无统计学意义（均P〉0．05）。结论UCP-2基因8号外显子3’-末端（3’-UTR）45bp插入／缺失多态性与糖尿病并发大血管病变无相关性。     

生长激素受体外显子3缺失多态性在矮小儿童与正常儿童中的分布比较

目的:研究生长激素受体(GHR)基因外显子3缺失多态性在矮小症儿童与正常儿童的人群分布,并比较二者有无差异。方法:选择矮小症儿童143例为试验组,包括生长激素缺乏症(GHD)58例,小于胎龄儿(SGA)40例,特发性矮小症(ISS)45例。采用多重聚合酶链反应(PCR)的方法对GHR外显子3缺失进行多态性分析,与同期健康儿童170例(对照组)对比。结果:试验组GHR外显子3缺失多态性fl/fl、fl/d3、d3/d33种基因型分别为71、58、14例,对照组为110、49、11例,前者fl/d3和d3/d3的比例明显高于后者,2组多态性分布差异有统计学意义(P<0.05)。试验组d3等位基因频率0.301(86/286)高于对照组的0.209(71/340),差异有统计学意义(P<0.05)。不同地区正常人群中3种基因型及等位基因频率分布对比差异有统计学意义(P<0.05),而SGA和GHD患者间分布差异均无统计学意义(均P>0.05)。结论:GHR外显子3多态性在矮小症儿童与正常儿童中的分布存在差异,推测可能与矮小症发病有一定关系。     

先天性肾上腺皮质增生症21羟化酶基因突变的研究

目的：检测32例21－OHD患儿CYP 21 3个点突变。方法：采用PCR－ACRS方法检测了32例21－OHD患儿及其家系3个点突变。结果：3例患儿为CYP 21第2内含子656位点突变，2例患儿为CYP 21第4外显子CD 172点突变，3例患儿为CYP 21第8外显子CD 356点突变。患儿为纯合子，家系成员为杂合子。结论：采用PCR－ACRS检测CYP 21点突变方法简便，结果可靠。     

A 6 bp polymorphism in the thymidylate synthase gene causes message instability and is associated with decreased intratumoral TS mRNA levels

OBJECTIVE: A 6 bp deletion polymorphism in the thymidylate synthase (TS) gene was investigated in order to determine its function. METHODS: A luciferase system was used to investigate the function of the 6 bp/1494 polymorphism in vitro. A group of 43 patients with colorectal carcinoma were evaluated for the 6 bp/1494 polymorphism and for intratumoral TS mRNA levels in vivo. RESULTS: The 3'UTR of TS containing the +6 bp polymorphism resulted in an approximate 35% decrease in luciferase activity and mRNA levels, while the TS-3'UTR bearing the -6 bp deletion resulted in an approximate 70% decrease in luciferase activity and mRNA levels. The TS-3'UTR construct containing the -6 bp/1494 deletion also had a higher rate of message degradation compared to the +6 bp/1494 construct. Individuals homozygous for the insertion (+6 bp/+6 bp) had significantly higher TS mRNA levels compared to individuals that were homozygous for the deletion (-6 bp/-6 bp) (P < 0.007). We determined the frequency of the -6 bp/1494 deletion polymorphism to be 41% in non-Hispanic whites, 26% in Hispanic whites, 52% in African-Americans and 76% in Singapore Chinese. CONCLUSIONS: These results suggest that the -6 bp/1494 deletion polymorphism in the 3'UTR of TS is associated with decreased mRNA stability in vitro and lower intratumoral TS expression in vivo. Further, the 6 bp/1494 polymorphism varies greatly within different ethnic populations and is in linkage disequilibrium with the TS 5' tandem repeat enhancer polymorphism. Taken together, these data suggest that the 6 bp/1494 polymorphism may be a useful screening tool in predicting TS mRNA expression.     

A novel deletion in the flavin-containing monooxygenase gene (FMO3) in a Greek patient with trimethylaminuria

Mutations of the flavin-containing monooxygenase type 3 gene (FMO3) that encode the major functional form present in adult human liver, have been shown to cause trimethylaminuria. We now report a novel homozygous deletion of exons 1 and 2 in an Australian of Greek ancestry with TMAuria, the first report of a deletion causative of trimethylaminuria. The deletion occurs 328 bp upstream from exon 1. The 3'-end of the deletion occurs in intron 2, 10013 base pairs downstream from the end of exon 2. The deletion is 12226 bp long. For the proband homozygous for the human FMO3 gene deletion, it is predicted that in addition to loss of monooxygenase function for human FMO3 substrates, such as TMA and other amines, the proband will exhibit decreased tolerance of biogenic amines, both medicinal and those found in foods.     

CYP2A6 genetic polymorphisms and liver microsomal coumarin and nicotine oxidation activities in Japanese and Caucasians

Genotypes of CYP2A6, namely CYP2A6(*)1 (wild-type), CYP2A6(*)2, and CYP2A6(*)3, were examined in liver DNA of 39 Japanese and 43 Caucasians using two-step polymerase chain reaction (PCR) methods. We first amplified a DNA fragment (1725 bp) located between near middle of exon 1 and end of exon 4 of the CYP2A6 gene and further amplified using a forward primer 't' or 'mut' (middle of exon 3) and a reverse primer 'E3R' (middle of intron 3) for the detection of CYP2A6(*)2-genetic polymorphism. The 1725 bp fragment was also used for the amplification between exon 3 and near middle of intron 3 of the CYP2A6 gene and the fragment thus obtained digested with XcmI or DdeI to detect and confirm the CYP2A6(*)2- and CYP2A6(*)3-types, respectively. Only one DNA sample from a Japanese origin (J18) was not amplified by CYP2A6-specific primers; liver microsomes from this individual had very low activity of coumarin 7-hydroxylation and were devoid of protein(s) immunoreactive to anti-CYP2A6 antibody. Thus, this individual was suggested to be due to the gene deletion in CYP2A6. By analyzing the remaining 38 Japanese and 43 Caucasians, we found that there were no cases of CYP2A6(*)3-type polymorphism in the samples examined in this study, and no cases of CYP2A6(*)2-type polymorphism in the Japanese samples. Of Caucasians studied two individuals were classified into heterozygous CYP2A6(*)1/(*)2-type. Liver microsomal coumarin 7-hydroxylation activities in these two Caucasians were found to be lower than those of the other 41 Caucasians. Kinetic analysis showed that two CYP2A6(*)1/(*)2 individuals had a very low ratio of V(max) to K(m) for nicotine C-oxidation as well as coumarin 7-hydroxylation in liver microsomes, compared with those of homozygous CYP2A6(*)1-type. These results suggest that among 39 Japanese and 43 Caucasians examined one Japanese is classified to be CYP2A6 gene deletion and two Caucasians are heterozygous CYP2A6(*)1/(*)2-genotype. Thus the race-related differences in the occurrence of CYP2A6 genetic polymorphisms were supported.     

Analysis of point mutation in exon 2 of CYP2E1 gene in renal cell/urothelial cancer patients in comparison with control population

OBJECTIVE: Genetic polymorphisms of human cytochrome P450s have been implicated to be of importance for susceptibility to different cancers. Recently, a point mutation was found in the exon 2 of the CYP2E1 gene (CYP2E1*2) [Hu et al. 1997]. In order to evaluate a possible link between the point mutation in exon 2 of the CYP2E1 gene and the susceptibility to renal cell/urothelial cancer, we developed a screening method based on the polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). MATERIAL: DNA of peripheral white blood cells was isolated from 158 renal cell/urothelial cancer patients as well as from 150 controls. METHOD: Primers for PCR were designed by the Primer 3 release 0.1 program. The PCR yield a product of 215 base pairs (bp), which was digested with the restriction enzyme Hha I. The DNA fragments were separated on a 3% agarose gel stained with ethidium bromide. Restriction enzyme digestion of the PCR product obtained from the wild-type DNA resulted in the appearance of a 66 bp, a 43 bp, a 40 bp, a 39 bp and a 28 bp DNA fragment. In contrast to the wild-type, the digestion of the PCR product from DNA carrying the point mutation resulted in the loss of the 39 bp and 40 bp fragments and the appearance of an additional 79 bp fragment. Therefore, the loss of one Hha I restriction site caused by a single nucleotide exchange is suitable for the identification of the point mutation in exon 2 of CYP2E1 gene. RESULTS: However, we could not detect any point mutation in any of the 158 renal cell/urothelial cancer patients or the 150 controls. The distribution of the point mutation in exon 2 of CYP2E1 gene did not show any difference in renal cell/urothelial cancer patients and controls. CONCLUSION: This might indicate a lack of association between this CYP2E polymorphism (CYP2E1*2) and renal cell/urothelial cancer.