幽门螺杆菌鞭毛基因PCR-RFLP分析及其与胃十二指肠疾病关系的研究

目的探讨幽门螺杆菌(Helicobacter pylori)鞭毛基因(flaA和flaB)PCR—RFLP分型及其与胃十二指肠疾病的关系一方法用PCR技术分别扩增46株从消化性溃疡(25株)和非消化性溃疡(21株)患者胃黏膜中分离得到的H.pylori flaA和flaB基因，并用HpaⅡ和HindⅢ消化PCR扩增产物，琼脂糖凝胶电泳分离酶切片段，将酶切片段编码后输入SPSS10．0软件进行聚类分析．并比较不同的基因型别在溃疡组和非溃疡组的分布。结果46株H．pylori菌株flaA基因HpaⅡ和HindⅢ酶切后分别产生4种和3种酶切图谱；flaB基因HpaⅡ和HindⅢ酶切后分别产生1种和2种酶切图谱：综合flaA和flaB基因酶切结果，46株菌株可分为四个基因型别：但这四个基因型别在溃疡组和非溃疡组的分布差异无统计学意义(P=0．513)。结论应用PCR—RFLP方法可以将H.pylori fla基因分为不同的基因型别．但胃十二指肠溃疡和非溃疡组的菌株fla基因型别无差别。     

Species-level identification of clinical staphylococcal isolates based on polymerase chain reaction–restriction fragment length polymorphism analysis of a partial groEL gene sequence

A pair of degenerate primers that amplified, by polymerase chain reaction (PCR), a partial groEL gene sequence (550 bp) was used for the identification of the 12 most common human staphylococcal pathogens. The amplified products were digested by AluI endonuclease, and distinctive PCR restriction fragment length polymorphism (RFLP) patterns for reference strains were obtained. This protocol was validated by the identification of 89 clinical staphylococcal isolates, and the results were compared with those obtained by the reference biochemical identification, showing 100% concordant results. Two species, Staphylococcus aureus and Staphylococcus lugdunensis, showed intraspecies polymorphisms on their PCR-RFLP patterns. All strains were also identified using the API Staph ID test (bioMérieux, Durham, NC) and the MicroScan WalkAway automated system (Dade Behring, West Sacramento, CA). When 17 Staphylococcus isolates were tested in a blind experiment by the PCR-RFLP of the groEL gene method, all strains were also correctly identified. We propose the PCR-RFLP of the groEL gene with AluI as a reliable and reproducible method for identification of Staphylococcus spp.     

A study of point mutation of ras genes in human gastric cancer

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Cystatin C基因5’端-157G/C突变与阿尔茨海默病的相关性研究

目的探讨Cystatin C基因5’端-157G/C突变与中国昆明地区汉族人阿尔茨海默病是否存在关联。方法运用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)分析方法在134例无亲缘关系之中国昆明汉族人(正常对照组73例,阿尔茨海默病患者61例)中对Cystatin C基因变异进行检测。同时检测两组人群血浆Cystatin C之浓度。结果在阿尔茨海默病患者中GG,GC和CC基因型频率分别为16.39%,50.82%和32.79%;正常对照组中分别为26.03%,41.10%和32.87%。G等位基因频率在阿尔茨海默病组中和正常对照组中分别为41.80%和46.58%。基因型频率和等位基因频率在阿尔茨海默病患者与对照组之间无统计学差异。两组人群血浆Cystatin C浓度也无统计学差异。结论在中国昆明汉族人群中存在Cystatin C基因5’端-157G/C变异的多态性,但可能对阿尔茨海默病的发生不起作用。     

Restriction fragment-length polymorphism analysis of 16S rDNA from oral asaccharolytic Eubacterium species amplified by polymerase chain reaction

Restriction fragment-length polymorphism (RFLP) analysis of 16S rDNA amplified by polymerase chain reaction was used to generate restriction profiles of the type strains of oral asaccharolytic Eubacterium species, that is, Eubacterium brachy, Eubacterium exiguum, Eubacterium lentum, Eubacterium minutum, Eubacterium nodatum, Eubacterium saphenum, Eubacterium timidum and 33 asaccharolytic Eubacterium strains isolated from oral sites. The 16S rRNA gene sequences from isolated genomic DNA samples were amplified by polymerase chain reaction (PCR). PCR products were purified and characterized by single digestions with 7 restriction endonucleases. Among the 7 endonucleases, Hpall was found to discriminate the respective reference strains. Twenty-three isolates, out of 33, were assigned to one of the reference species, on the basis of their restriction profiles by digestion with Hpall. The remaining 10 isolates could not be assigned to any of the established species and constituted 4 distinct groups, each of which may be a new species.     

Lewis genotyping by the PCR-RFLP method in a Japanese population and its evaluation in forensic analysis

Lewis phenotyping of red blood cells has many problems such as the influence of many biological conditions, the change during the period from newborn to early childhood and mistyping by non-specific anti-Lewis antibodies. Therefore, it would be useful to determine the Lewis genotype. Recently a method of Le-genotyping by PCR-RFLP was established. We determined the frequencies of Lewis genotypes in a Japanese population and discuss the applicability to paternity tests and other forensic applications. The gene frequencies of Le, le1 and le2 in the Japanese population studied were 0.7032, 0.2358 and 0.0610 respectively. Out of 12 paternity cases where paternity was excluded by other markers, 3 alleged fathers could also be excluded by Lewis genotyping. The genotype from organs of a fetus from a 3-month pregnancy was Le/Le. The determination of Lewis genotypes could play a useful role as a genetic marker in paternity tests and forensic analyses. Received: 9 October 1996 / Received in revised form: 1 April 1997     

Evidence for an association of TP53 codon 72 polymorphism with breast cancer risk

BACKGROUND: A common Arg/Pro polymorphism at codon 72 of the TP53 gene has been investigated as a risk factor for cancer in different populations. So far, the results have been controversial. Our purpose was to investigate the association of this polymorphism with breast carcinoma in women from Southern Brazil, a high-risk area for breast cancer. METHODS: Blood samples collected from 118 women with primary breast carcinoma and from 202 female blood donors were analyzed through polymerase chain reaction-restriction fragment length polymorphism and DNA sequencing. RESULTS: The relative frequency of each allele was 0.75 for Arg and 0.25 for Pro in patients with cancer, and 0.62 for Arg and 0.38 for Pro in normal controls (P < 0.001). The Arg/Arg genotype was significantly associated with an increased risk for breast cancer (OR 2.9; 95% CI 1.43-3.6; P < 0.002). No correlation between the genotype distribution and specific prognostic predictors for the disease outcome was observed. DISCUSSION: TP53 codon 72 polymorphism might be implicated in breast carcinogenesis, with the Arg/Arg genotype being associated with an increased susceptibility for this malignancy.     

新疆水源性隐孢子虫的检测

目的检测新疆15个城市自来水和河水隐孢子虫污染情况。方法采集水样,分别应用改良美国环保局(EPA)1622法及巢式PCR(nested-PCR)方法进行检测。1)改良EPA1622法:水样经微孔滤膜抽滤、淘洗,磁抗体分离法分离纯化后免疫荧光染色鉴定。2)Nested-PCR法:用试剂盒提取纯化的隐孢子虫卵囊基因组DNA,针对隐孢子虫小亚单位核糖体RNA(18SrRNA)部分基因,依据文献设计并合成引物,用巢式PCR扩增,产物纯化后经SspⅠ及VspⅠ单酶切,并进行RFLP分析。结果2种方法检测新疆15个地区的自来水隐孢子虫卵囊均为阴性,改良EPA1622法检测乌鲁木齐市、昌吉市、伊宁市和吐鲁番市的河水隐孢子虫卵囊阳性。巢式PCR检测乌鲁木齐市和伊宁市水样,均扩增出约830 bp的特异片段,RFLP初步鉴定为小鼠隐孢子虫基因;昌吉市和吐鲁番市河水水样PCR检测阴性。结论新疆乌鲁木齐市、伊宁市河水检出隐孢子虫,初步鉴定为小鼠隐孢子虫。而当地的饮用水未受污染。     

Study of hepatitis B virus gene mutations with enzymatic colorimetry-based DNA microarray

OBJECTIVES: To establish a modified microarray method for detecting HBV gene mutations in the clinic. DESIGN AND METHODS: Site-specific oligonucleotide probes were immobilized to microarray slides and hybridized to biotin-labeled HBV gene fragments amplified from two-step PCR. Hybridized targets were transferred to nitrocellulose membranes, followed by intensity measurement using BCIP/NBT colorimetry. RESULTS: HBV genes from 99 Hepatitis B patients and 40 healthy blood donors were analyzed. Mutation frequencies of HBV pre-core/core and basic core promoter (BCP) regions were found to be significantly higher in the patient group (42%, 40% versus 2.5%, 5%, P < 0.01). Compared with a traditional fluorescence method, the colorimetry method exhibited the same level of sensitivity and reproducibility. CONCLUSIONS: An enzymatic colorimetry-based DNA microarray assay was successfully established to monitor HBV mutations. Pre-core/core and BCP mutations of HBV genes could be major causes of HBV infection in HBeAg-negative patients and could also be relevant to chronicity and aggravation of hepatitis B.