一种基于momp基因的衣原体分子生物学甄别技术的初步建立

目的利用PCR方法建立一种基于momp基因的衣原体分子甄别方法。方法根据衣原体momp基因的恒定区和可变区分别设计衣原体科特异性引物和种特异性引物，利用PCR方法对本实验室保存的衣原体进行扩增，以达到甄别的目的。利用限制性片段长度多态性（RFLP）分析科特异性PCR扩增产物，研究momp基因的多态性。结果利用建立的PCR方法对本实验室保存的九株衣原体进行了研究，结果表明八株衣原体都是鹦鹉热嗜衣原体，一株为沙眼衣原体。利用限制性片段长度多态性（RFLP）分析科特异性PCR扩增产物，分析了D34、B11001、CW2和CP12四株衣原体，结果可以产生两种不同的带型。结论利用建立的PCR方法可以达到检测甄别衣原体的目的，并可以通过RFLP分析衣原体的侵袭性。     

结核分支杆菌与非结核分支杆菌快速鉴别的实验研究

目的 探讨提供一种从菌种水平快速检测和鉴别分支杆菌的方法。方法 应用三对分别对应于分支杆菌(分子量65000)表面抗原、结核分支杆菌插入序列IS6110及人类β-珠蛋白基因的部分序列的特异性寡核苷酸引物对受试菌种及临床标本中的DNA进行多重PCR扩增，其扩增产物分别为439bp、268bp及123bp，并对439bp进行BstE Ⅱ和Hae Ⅲ两种限制性内切酶消化，同时，将135例临床标本进行培养、涂片，和多重PCR联合限制性片段长度多态性分析(mPCR-RFLP)检测。结果 mPCR-RFLP方法可将12种受试的标准菌种分别区分到种及亚种水平。本方法在临床实验中亦体现出较高的灵敏性及特异性。结论 mPCR-RFLP分析是对结核分支杆菌与非结核分支杆菌进行菌种分类鉴定的一种快速有效的方法。     

我国与日本山梨不同地域品系日本血吸虫DNA的限制性片段长度多态性分析

目的 :为了进一步了解中国各地日本血吸虫的特性 ,将其与日本山梨的日本血吸虫进行了虫体 DNA限制性片段长度多态性 ( RFLP)的分析和比较。方法 :以 p SM889为探针 ,与我国云南、四川、广西、湖北、湖南、江西、安徽、台湾和日本山梨的日本血吸虫雄虫 DNA进行杂交。结果 :经限制性内切酶 Eco RI酶切的我国 7地日本血吸虫 DNA与探针杂交的图谱显示弱杂交带数目呈现差异 ,而其 4条强杂交带数目则相同。但是,台湾及日本山梨的日本血吸虫 DNA杂交图谱不仅弱杂交带数目呈现差异 ,而且强杂交带数目亦有不同 ;前者的强杂交带数目为 3条 ,后者则为 5条。结论 :我国与日本山梨不同地区的日本血吸虫核基因组存在着遗传变异, 台湾省的日本血吸虫的遗传变异程度远较其他省份的更为显著。     

More Than Half the Sporadic Cases of Hemophilia A in Sweden Are Due to a Recent Mutation

ABSTRACT. The aim of this study was to ascertain how many of the sporadic cases of severe Haemophilia A in Sweden are due to recent mutation, and establish its origin. DNA analysis was performed in 18 randomly selected families with a sporadic case of severe haemophilia A. Restriction fragment length polymorphism (RFLP) patterns were investigated, two intragenic (Bcl I, Xba I/Kpn I) and two extragenic (DX 13, St 14) RFLP being used. In 10/18 families a haemophilia-linked gene was found to have derived from the healthy maternal grandfather; on the basis of clotting and immunological assay results, the odds were high (> 104:1) for maternal carriership in four of these 10 cases, and for maternal non-carriership in two, four being indeterminate. In 4/18 families a haemophilia-linked gene derived from the healthy maternal grandmother; according to clotting and immunological assay results, in two cases the odds were high for maternal non-carriership. In the remaining 4/18 families no conclusions could be drawn from the RFLP pattern as to the origin of mutation. We conclude that at least 55 % of the sporadic cases of severe hemophilia A in Sweden are due to a recent mutation within the last two genrations.     

Detailed deletion mapping of the short arm of chromosome 3 in small cell and non-small cell carcinoma of the lung

We constructed a detailed deletion map of the short arm of chromosome 3 (3p) for 55 lung cancer cases by using 17 restriction fragment length polymorphism (RFLP) probes. Initially, we examined 40 small cell lung cancer (SCLC) cases and found three regions of deletion at 3p25–26, 3p21.3 and 3p14-cen. suggesting the possibility of at least three different tumor-suppressor genes on 3p. In order to obtain more detailed deletion area, and to compare the pattern of 3p deletion, we also examined 15 non-small cell lung cancer (NSCLC) cases. Compared to NSCLC cases, most of SCLC cases have widespread deletion on 3p, suggesting multiple tumor-suppressor genes on 3p may be inactivated in this type of cancer. In 3p21.3 area, minimum overlapping area of deletion lays between two probes which are close to each other. These data will be useful to isolate the putative tumor-suppressor genes located on the chromosome 3p.     

A prospective,multicenter study of laboratory cross-contamination of Mycobacterium tuberculosis cultures

A prospective study of false-positive cultures of Mycobacterium tuberculosis that resulted from laboratory cross-contamination was conducted at three laboratories in California. Laboratory cross-contamination accounted for 2% of the positive cultures. Cross-contamination should be a concern when an isolate matches the genotype of another sample processed during the same period.     

Hypoxanthine-guanine phosphoribosyltransferase gene analysis for Japanese patients with Lesch-Nyhan syndrome

The Lesch-Nyhan syndrome results as a consequence of a severe deficiency of functional activity of purine salvage enzyme, hypoxanthine phosphoribosyltransferase (HPRT). We performed Southern blot analysis for five patients and their families using full length cDNA of the HPRT gene as a probe. Pst I digested Southern blot analysis revealed a large deletion that included exon 2 in patient 3. The size of this deletion was about 4.4 Kb. The mother of this patient had the same mutated allele and a normal one (heterozygote). This type of mutation from a Lesch-Nyhan syndrome patient has not been previously reported. The restriction fragment length polymorphism (RFLP) pattern was analyzed by Bam HI digested Southern blot analysis for one family who had no major gene abnormality. We determined from this analysis that the sister of the patient was a Lesch-Nyhan syndrome carrier and the fetus (brother) was normal for HPRT activity. This study shows RFLP analysis is still useful for carrier detection and prenatal diagnosis of Lesch-Nyhan syndrome.     

不同宿主肝片吸虫DNA限制性片段长度多态性分析

本文首次对分离自黄牛、水牛和牦牛的肝片吸虫的基因组DNA，以BamHⅠ、BglⅡ、EcoRⅠ、HindⅢ和PStⅠ等6种限制性内切酶消化后进行琼脂糖电泳，分析比较它们基因组DNA的限制性片段长度多态性（RestrictionFragmentLengthPolymorphism，RFLP）；同时用地高车标记的牦牛肝片吸虫基因组DNA为探针对不同宿主肝片吸虫DNA进行Southern印迹杂交，限制性内切酸酶切结果显示只有两种限制性内切酶（BamHⅠ和EcoRⅠ）在寄生于牦牛的肝片吸虫中检测到多态性，而水牛肝片吸虫与黄牛肝片吸虫基因组DNA之间没有检测到多态性，根据此结果计算了牦牛肝片吸虫与黄牛肝片吸虫和水牛肝片吸虫基因型间的遗传距离，Southern杂交结果显示牦牛肝片吸虫基因DNA的杂交谱带与黄牛肝片吸虫、水牛肝片吸虫基因组DNA的杂交港带存在一定的差异，本文的实验结果表明牦牛肝片吸虫与黄牛肝片吸虫、水牛肝片吸虫的亲缘关系相对较远，而水牛肝片吸虫与黄牛肝片吸虫的亲缘关系较近。     

A rare genotype of Cryptococcus gattii caused the cryptococcosis outbreak on Vancouver Island (British Columbia, Canada)

Cryptococcus gattii causes life-threatening infection of the pulmonary and central nervous systems in hosts with normal immunity and traditionally has been considered to be restricted geographically to tropical and subtropical climates. The recent outbreak of C. gattii in the temperate climate of Vancouver Island, BC, Canada, led to a collaborative investigation. The objectives of the current study were to ascertain the environmental source of the outbreak infections, survey the molecular types of the outbreak and environmental cryptococcal isolates, and determine the extent of genetic diversity among the isolates. PCR-fingerprinting and amplified fragment length polymorphism (AFLP) were used to examine the genotypes, and mating assays were performed to determine the mating type of the isolates. All outbreak and environmental isolates belonged to C. gattii. Concordant results were obtained by using PCR-fingerprinting and AFLP analysis. The vast majority of clinical and veterinary infections were caused by isolates of the molecular type VGII/AFLP6, but two were caused by molecular type VGI/AFLP4. All environmental isolates belonged to molecular type VGII/AFLP6. Two or three subtypes were observed within VGII/AFLP6 among outbreak and environmental isolates. All mating-competent isolates were of the alpha-mating type. The emergence of this usually tropical pathogen on Vancouver Island highlights the changing distribution of this genotype and emphasizes the importance of an ongoing collaborative effort to monitor the global epidemiology of this yeast.     

Oncogenic beta-catenin and MMP-7 (matrilysin) cosegregate in late-stage clinical colon cancer.

BACKGROUND & AIMS: Recent in vitro studies showed that beta-catenin translocated into the tumor cell nucleus functions as an oncogene by transactivating oncogenes, including MMP-7. We conducted a large-scale analysis of beta-catenin and MMP-7 expression in human colon cancer to determine the potential clinical importance of these molecules. METHODS: In 202 colon cancer patients with known postoperative outcomes, we determined the expression of beta-catenin and MMP-7 in the tumors immunohistochemically and correlated the findings with the patients' clinicopathological characteristics and survival. RESULTS: We found 2 distinct patterns of beta-catenin nuclear accumulation (NA) in the colon cancers: diffuse NA (NAd) in 89 cases (44%) and selective NA at the invasion front (NAinv) in 18 cases (9%). The presence of the NAinv pattern was significantly correlated with advanced Dukes' stage (P = 0.0187) and tumor recurrence (P = 0.0005) as well as with MMP-7 expression in the tumor invasion front (P = 0.0025), resulting in extremely unfavorable clinical outcomes. A multivariate analysis determined that the NAinv expression pattern and Dukes' C stage were independent prognostic factors. CONCLUSIONS: Oncogenic activation of beta-catenin in the tumor invasion front, as represented by its NAinv pattern of expression, may be an independent and reliable indicator of membership in a subset of colon cancer patients who are highly susceptible to tumor recurrence and have a less favorable survival rate.