食品中污染酵母菌的RAPD分类鉴定

目的探索采用RAPD技术对食品污染酵母进行分类鉴定的可行性。方法采用RAPD技术对从食品中分离到的22株污染酵母菌进行分子分型并以UPGMA法构建以上菌株的带型关系系统树状图。结果22株污染酵母菌总共扩增出11种不同的指纹图谱，经聚类分析可分为2个聚类群。结论RAPD技术可以作为一种有效、快速、简便的对污染酵母菌进行分类鉴定的手段。     

RAPD法对肺炎克雷白杆菌的基因分型

为了探讨肺炎克雷白杆菌的基因分型，我们用煮沸法制备细菌基因组ＤＮＡ，进行随机引物－聚合物酶链反应，所有１８个肺炎克雷白杆菌菌株均产生了特异性的，可重复的扩增条带，提示：ＲＡＰＤ方法可用于肺炎克雷白杆菌的分型鉴定和进行分子流行病学研究。     

Assignment of RFLP,RAPD and isoenzyme markers to Aspergillus nidulans chromosomes,using chromosome-substituted segregants of a hybrid of A. nidulans and A. quadrilineatus

Chromosome-substituted haploid segregants were selected from among the benomyl-induced progeny of an interspecific hybrid produced by polyethylene-glycol-induced fusion of protoplasts of an Aspergillus nidulans master strain and an A. quadrilineatus auxotrophic mutant. These segregants were examined by RFLP, RAPD, and isoenzyme analysis. The A. nidulans ribosomal repeat unit was assigned to chromosome V, while the benA and the pyrG genes were assigned to linkage groups VIII and I, respectively, of A. nidulans. None of the other cloned genes tested (gdhA, amdS and 25s rRNA) showed polymorphism between the two parents. The method was also used to assign RAPD markers and isoenzyme bands of -arylesterase, phosphatases, NAD-dependent malate dehydrogenase, and cellulase, to A. nidulans chromosomes and/or to their A. quadrilineatus equivalents. The isoenzyme and DNA sequences assigned to chromosomes could be used to saturate the genetic map of A. nidulans, or could serve as starting points for the construction of a genetic map of A. quadrilineatus. No method affording the same possibilities has been described so far in Aspergilli. This chromosome-assay method may be a useful alternative to pulsed-field-gel electrophoretic procedures for the assignment of molecular markers to chromosomes.     

Outcrossing in the homothallic oomycete,Pythium ultimum,detected with molecular markers

The oomycete Pythium ultimum is homothallic, thus a single isolate completes the sexual stage in pure culture. It has been generally assumed that homothallic oomycetes are predominantly inbreeding. In P. ultimum, antheridia occasionally develop from hyphae not directly connected to the oogonium and appear to participate in fertilization, suggesting a possible mechanism for outcrossing. We have used molecular markers to confirm that outcrossing can occur between isolates of P. ultimum. Genetic markers based on randomly amplified polymorphic DNA (RAPD) and restriction fragment length polymorphisms (RFLP) were used to distinguish isolates in a collection of P. ultimum. Two isolates displaying a high level of polymorphism were mixed and placed on media which allows the development of the sexual stage. RAPD markers were used to screen single oospore progeny to identify potential hybrids between the two parental isolates. Subsequent self-fertilization of one putative F₁ yielded a F₂ population which demonstrated segregation and independent assortment of RAPD and RFLP markers. A similar strategy was used to show that an isolate which is incapable of producing oospores in pure culture can outcross when mixed with a homothallic isolate. These results suggest that other homothallic oomycetes may be capable of outcrossing, and sexual reproduction may, therefore, play an important role in the generation of variation in homothallic oomycetes.     

Evidence of two genetic entities inBothriocephalus funiculus (Cestoda) detected by arbitrary-primer polymerase chain reaction random amplified polymorphic DNA fingerprinting

The genetic diversity of two samples of Cestoda (Bothriocephalus funiculus, Renaud and Gabrion, 1984) parasitizing two sympatric teleostean species was assessed using random amplified polymorphic DNA (RAPD). A total of 72Bothriocephalus were analyzed individually, and electrophoretic analysis of the amplification products of 65 primers among the 68 tested revealed monomorphic patterns, reflecting the close genetic relatedness within and between the parasites of the two samples. However, 3 primers showed polymorphic patterns at 6 RAPD sites. Analysis of the distribution of these genomic fragments, assuming random mating, showed strong linkage disequilibria (only 8 genetic combinations were observed among the 32 expected). Two genetic entities displaying a high degree of host specificity were evidence within our two samples ofB. funiculus. This powerful molecular technique can be used as a diagnostic tool in studies concerning the biodiversity of related genetic entities and could have broad applications in parasitology.     

Relationships among genotypes, virulence and clinical forms of Sporothrix schenckii infection

This study explored the relationships among genotypes, virulence and clinical forms of Sporothrix schenckii. Genomic DNA from isolates of S. schenckii, collected from different clinical forms of sporotrichosis, was amplified by randomly amplified polymorphic DNA (RAPD). Suspensions of different isolates of S. schenckii were inoculated into healthy BALB/c mice to compare their virulence, and the numbers and distribution of spores were determined by histological analysis. RAPD analysis indicated that the isolates from different clinical forms of sporotrichosis belonged to different genotypes. The mice inoculated with isolates from disseminated sporotrichosis showed an earlier onset of illness and more severe lesions than those inoculated with isolates from lymphocutaneous sporotrichosis, which, in turn, showed an earlier onset of illness and more severe lesions than those inoculated with isolates from fixed cutaneous sporotrichosis. Healthy BALB/c mice injected with isolates from disseminated sporotrichosis died within 10 days, whereas isolates from lymphocutaneous sporotrichosis and fixed cutaneous sporotrichosis failed to cause death. Histologically, mice inoculated with isolates from disseminated sporotrichosis had more spores than those inoculated with isolates from lymphocutaneous sporotrichosis and fixed cutaneous sporotrichosis. Thus, different genotypes may be associated closely with the virulence of different clinical forms of S. schenckii infection.     

Identification and typing of Streptomyces strains: evaluation of interspecific, intraspecific and intraclonal differences by RAPD fingerprinting

The suitability of random amplified polymorphic DNA PCR for the detection of differences between Streptomyces species and strains was evaluated. For this purpose, a protocol of RAPD specific for Streptomyces DNA, i.e. suitable for DNA presenting a high G+C content, was developed using S. ambofaciens ATCC23877. Among the 30 primers tested, all containing 80% G+C, 17 gave a pattern with this strain. Six oligonucleotides were chosen to compare 12 strains belonging to six species of Streptomyces. These oligonucleotides were then used to determine whether these strains could be differentiated at the DNA level with this method. All fingerprints obtained with six primers differed from one species to another. We showed that the RAPD method could be used to reveal intraspecific and intraclonal polymorphisms. Thus, RAPD allows for the rapid, sensitive and specific detection of genetic diversity among species and strains of Streptomyces.     

Randomly amplified polymorphic DNAs assess recombination following an induced parasexual cycle in Penicillium roqueforti

Random amplified polymorphic DNAs (RAPDs) were used as a genetic marker system to characterize recombinant strains following the parasexual cycle of Penicillium roqueforti. After protoplast fusion and haploidization of diploid hybrids, segregants characterized by a reassortment of the parental genetic markers displayed specific RAPD fingerprints. The appearance or the loss of RAPD fragments demonstrate that these markers provide an efficient method to analyze recombination and to characterize somatic hybrids.     

珍稀中草药金线莲的RAPD研究

为了鉴别金线莲的品种,探索同属植物之间的亲缘关系及同种植物不同产地的遗传变异,并制定正品金线莲的DNA指纹图谱,采用随机扩增多态DNA(RAPD)技术对兰科开唇兰属植物花叶开唇兰Anoectochilus roxburghii(Wall．)．Lindl．、台湾开唇兰A.formosanus Hay．进行了鉴定,结果选择的7个引物对3个品种共扩增出98个位点,其中3个引物为高特异性引物。结论 RAPD技术不仅能鉴别种间差异,而且能揭示同种不同产地植物的遗传变异。     

应用40条引物进行不同品系小鼠RAPD遗传检测的初步研究

目的：探讨运用ＲＡＰＤ－ＰＣＲ对小鼠进行遗传检测的可能性,筛选在各品系小鼠间具有多态性的引物。方法：运用４０条引物对ＴＡ１、ＢＡＬＢ／ｃ、ＮＩＨ、ＳＣＩＤ、Ｔ７３９、ＤＢＡ／２、６１５、ＢＡＬＢ／ｃ－ｎｕ／ｎｕ等９个品系小鼠的ＤＮＡ进行ＲＡＰＤ－ＰＣＲ扩增,以琼脂糖凝胶电泳观察结果。结果：４０条引物中只筛选出２条多态性引物,其它３８条引物扩增结果无品系间多态性。结论：ＲＡＰＤ－ＰＣＲ方法可以区分９