片形吸虫DNA随机扩增多态性分析

为区别从南京市江宁县采集的片形吸虫非典型形态虫体，应用随机扩增多态性DNA（RAPD）技术，对6株片形吸虫总DNA进行了扩增。结果，10条引物中有8条能产生扩增图谱，电泳图谱经聚类分析，与传统的分类结果一致，并表明来自江宁的片形吸虫既有形态典型的肝片形吸虫，也有形态不典型的大片形吸虫。     

我国片形吸虫线粒体NADH脱氢酶亚单位1基因(nad1)多态性的研究

用γ^33P对引物进行标记，首次对来自国内不同地区、不同宿主的片形吸虫线粒体烟酰胺腺嘌呤二核苷酸(NADH)脱氢酶亚单位Ⅰ基因部分序列(pnad1)进行PCR扩增及DNA单链构象多态性(PCR-SSCP)分析，结果76个虫体均成功地扩增出约200bp的基因片段；76个样品经过PCR-SSCP分析后，筛选出11个代表性样品进行测序。测序结果显示我国片形吸虫pnad1序列种间差异大于种内变异，表明nad1序列可以作为片形吸虫种内和种间遗传多态性研究的标记。     

片形吸虫第一内转录间隔区DNA多态性的研究

以不同地区的片形吸虫虫株为研究对象，经PCR扩增出了ITS-1部分基因片段，采用单链构象多态性(SSCP)方法，并结合序列分析研究了不同地区片形吸虫ITS-1 DNA的多态性。不同地区的样品经SSCP分析，显示3种带型，第1种为大片形吸虫的带型，第2种为肝片形吸虫的带型，第3种为2种带形的混合带型。广西区样品和大部分贵州省样品属于大片形吸虫带型；四川省、黑龙江省和部分贵州省样品为混合带型；南京市和甘肃省样品为肝片形吸虫带型或混合带型。测序结果表明，根据ITS-1基因的序列变异位点可区分2种片形吸虫；表现为混合带型的样品在变异位点具有多态性。本研究结果显示，ITS-1片段可作为遗传标记用以区分大片形吸虫和肝片形吸虫，同时也证实，在我国除了这2种片形吸虫外，还可能存在着“中间型”的片形吸虫。     

我国片形吸虫(Fasciola)线粒体细胞色素c氧化酶亚基I基因(cox1)部分序列的多态性

用γ^33P对引物进行标记，对来自国内不同地区不同宿主的片形吸虫(Fasciola)的线粒体基因组细胞色素氧化酶亚基I基因(pcox1)部分序列进行PCR扩增及DNA单链构象多态性(SSCP)分析，结果76个虫体均成功地扩增出约450bp的基因片段；SSCP分析显示，不同地区或同一地区的不同样品在pcox1的SSCP带型上存在多态性；代表性样品的测序结果表明，其碱基序列存在差异。试验结果显示，我国片形吸虫pcox1序列种内变异不明显，种间变异显著．表明cox1序列可以作为片形吸虫分类鉴定中一个可靠的遗传标记。     

片形吸虫Cold-SSCP鉴别方法的建立

以采自我国不同地区的片形吸虫虫体为研究对象，PCR扩增出核糖体DNA (rDNA)的第一内转录间隔区(ITS-1)序列，然后采用非同位素的单链构象多态性(Cold-SSCP)方法分析PCR产物，对不同地区片形吸虫进行分子鉴定。所有样品经Cold-SSCP分析显示2种带型。样品测序及序列分析结果表明，第1种为肝片形吸虫带型，另1种为大片形吸虫带型。本研究建立了区分大片形吸虫和肝片形吸虫的Cold SSCP方法，可用于这2种吸虫的分子流行病学调查，从而为片形吸虫分子生物学的进一步研究奠定了基础。     

青海省藏羊片形吸虫18S rRNA基因的扩增及虫种鉴定

为了进一步确定青海地区藏羊体内片形吸虫,并为青海省藏羊体内片形吸虫的分类研究提供科学的参考依据,取片形吸虫基因组DNA,利用保守引物,PCR扩增18S r RNA片段并测序。应用DNAMAN软件用对所测得的序列与Gen Bank中已经发布的大片吸虫(Fasciola gigantica)和肝片吸虫(Fasciola hepatica)的18S r RNA序列进行比对分析。结果发现测得目的片段长度为1 737 bp,测得序列与大片吸虫18S r RNA序列相似度为92.98%,与肝片吸虫的18S r RNA序列相似度为99.77%,从而进一步确定所采虫体为肝片吸虫。     

片形吸虫遗传变异及分子分类的研究

从染色体分析、同工酶多态性、可溶性蛋白图谱、分子遗传学分析等角度,综述了近年来国内外有关片形吸虫遗传变异、分类鉴定的研究进展.介绍了用于片形吸虫遗传变异及分子分类研究的几种分子遗传学方法.     

酶切图谱及序列分析虫体ITS2鉴别中国的片形吸虫

提取我国广西、四川、黑龙江、甘肃等地及法国的片形吸虫的总DNA,用PCR扩增完整的ITS-2.对得到的PCR产物用限制性内切酶Hsp 92Ⅱ,Rca I进行酶切和RFLP技术分析.并对ITS-2 PCR产物进行双向测序.以便确定国内片形吸虫种内及种间ITS-2的特点和序列变异的水平.结果显示:广西、四川、黑龙江、法国等地的样品通过PCR均扩增到约550 bp的ITS-2目标片段.Hsp92Ⅱ,Rcal可区别不同种的片形吸虫.对PCR产物的序列分析结果表明片形吸虫ITS-2全长均为362 bp,同种内ITS-2序列无变异,不同种间ITS-2序列存在5～6个碱基差异,变异率约为2.8%.对各地区片形吸虫ITS-2的PCR RFLP分析与对PCR产物的DNA序列分析一致证明,来自四川的样品和法国样品同属肝片形吸虫F.hepattca;广西样品属大片形吸虫F.gigantica;黑龙江的样品为中间型片形吸虫.本研究首次从分子水平上证实我国除有肝片形吸虫、大片形吸虫外,还存在中间型的片形吸虫.     

片形吸虫种类鉴定的分子生物学研究进展

片形吸虫是反刍动物的重要病原，主要有肝片吸虫和大片吸虫，两者形态极相似，故对其鉴定一直是研究的热点。近年来应用分子生物学技术，由蛋白质、染色体、特定基因组DNA等不同分子水平鉴定片形吸虫种类的研究报道较多，揭示了片形吸虫的种内差异，种间差异和亲缘关系，从而为片形吸虫（尤其是中间种和亲缘种）种类鉴定提供了新途径，就此进展作一概述。     

粪便中华支睾吸虫虫卵DNA的提取及PCR检测方法的优化

华支睾吸虫病又称肝吸虫病,是由华支睾吸虫寄生在人或多种动物胆管内所引起的一种人兽共患病.目前,华支睾吸虫的分离鉴定主要集中在成虫或囊蚴,对虫卵的研究甚少.采用分子生物学方法成功建立了感染人粪便中华支睾吸虫虫卵DNA的提取方法及华支睾吸虫基因片段PCR检测方法,同时对PCR反应条件进行优化,为从分子水平上检测华支睾吸虫虫卵提供科学依据.     

Genomic relatedness among Actinobacillus pleuropneumoniae field strains of sterotypes 1 and 5 isolated from healthy and diseased pigs.

Forty-four Actinobacillus pleuropneumoniae isolates recovered from both healthy and diseased pigs were characterized by random amplified polymorphic DNA analysis (RAPD), pulsed field gel electrophoresis (PFGE) and apx toxin gene typing. Nine RAPD types and 14 PFGE patterns were identified. No common RAPD or PFGE patterns were found between strains of serotype 1 and those of serotype 5. The RAPD analysis indicated that the 15 serotype 1 strains isolated from diseased pigs were assigned to 4 RAPD types, with 66% of strains characterized by the same RAPD type. By contrast, the 5 strains of serotype 1 isolated from healthy carriers were dispersed in 4 RAPD types. These data suggest that the diversity of strains isolated from healthy pigs could be higher than that of strains recovered from diseased pigs. In addition, all serotype 5 strains exhibited a unique RAPD type. Unlike RAPD, PFGE analysis allowed discrimination among isolates of serotype 1 and among those of serotype 5. All but 3 isolates showed the same apx genotype as their respective serotype reference strain. These data indicate that RAPD analysis is a valuable rapid tool for routine subtyping of strains of serotype 1. For strains of serotype 5, a combination of several typing methods, such as PFGE and apx gene typing, is needed to provide useful information on the molecular epidemiology of swine pleuropneumonia.     

Differentiation of avian pathogenic Escherichia coli (APEC) strains by random amplified polymorphic DNA (RAPD) analysis

Here we describe the application of a random amplified polymorphic DNA (RAPD) analysis for molecular genetic typing avian pathogenic Escherichia coli (APEC) strains. The RAPD technique was shown to be highly reproducible. Stable banding patterns with a high discriminatory capacity were obtained using two different primers. Overall, 55 E. coli strains were analyzed with a RAPD technique. The RAPD analysis showed that the E. coli strains isolated from poultry in Thailand and Sweden could be grouped into 50 of RAPD types by using these two different primer sets. Most of these different E. coli RAPD types were not geographically restricted. There was, as expected, a tendency of higher genetic relationship among E. coli strains isolated from the same farm. It is suggested that the RAPD technique may provide a rapid, low cost, simple and powerful tool to study the clonal epidemiology of avian E. coli infections.     

Identification of Escherichia coli recovered from milk of sows with coliform mastitis by random amplified polymorphic DNA (RAPD) using standardized reagents

A standardized-reagents commercial kit for random amplified polymorphic DNA (RAPD) analysis was used for typing 58 Escherichia coli strains that were recovered from the milk of sows, having coliform mastitis, within a single swineherd in Sweden. Previously, the 58 E. coli strains were characterized serologically and profiled biochemically. They were also evaluated for their serum resistance and their ability to adhere to fibronectin and bovine fetal fibroblasts. The RAPD analysis was fast, easily performed, and required only a nanogram of DNA. The indistinguishable banding patterns obtained with repeated analyses of 2 isolates from each strain demonstrated that RAPD analysis using standardized beads is a technique that provides reproducible results for typing E. coli strains that cause mastitis in sows. The results of the RAPD analyses demonstrated that E. coli sow mastitis strains are highly variable in serotype, biochemical profiles, virulence factors, and RAPD type, and that all 58 strains can be differentiated by means of the RAPD technique. The strains grouped into 24 RAPD types by combining the results of 2 primers, and into 38 groups by combining the results of serotype and RAPD type. No relationship between serotypes, virulence factors and RAPD types was found.     

Comparison of Mycobacterium avium complex (MAC) strains from pigs and humans in Sweden by random amplified polymorphic DNA (RAPD) using standardized reagents

Infections with atypical mycobacteria belonging to the Mycobacterium avium/intracellulare complex (MAC) can cause infection in both animals and humans. Using a standardized reagents commercial kit for random amplified polymorphic DNA (RAPD) analysis, 49 MAC strains isolated from 32 slaughter pigs and 17 humans in Sweden were identified and sorted out, yielding 6 RAPD types. By combining the results of RAPD primers 4 and 5 and the primer IS1245A, we found that pigs and humans may be infected with the same types of MAC strains, since 14 strains from humans and 8 strains from pigs were essentially identical and together, comprised RAPD type 2, the largest group of strains (44.8% of strains). With respect to grouping of strains, serotype and RAPD type were uncorrelated, except for serotype 20 and RAPD type 6. Using standardized beads, RAPD analysis is a reproducible technique for typing MAC strains, as the indistinguishable banding patterns obtained with repeated analyses of two isolates from each strain in this study demonstrate. However, primer selection and DNA purity were crucial for differentiating closely related strains.     

Genetic diversity among Escherichia coli O149:K91 strains isolated from pigs with diarrhoea determined by randomly amplified polymorphic DNA analysis.

The genetic relatedness among 72 Escherichia coli strains of serotype O149:K91 isolated from pigs with diarrhoea was investigated by randomly amplified polymorphic DNA (RAPD) analysis. Fimbrial and toxic virulence markers of the isolates were also tested. Amplification with primer 1254 resulted in three different RAPD types whereas primer 1290 generated one RAPD profile only. Based on the RAPD and fimbrial/toxin types the strains were classified into five distinct groups.     

Molecular Differentiation of Mycoplasma gallisepticum and Mycoplasma imitans Strains by Pulsed‐Field Gel Electrophoresis and Random Amplified Polymorphic DNA

Pulsed‐field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD) analysis were used to compare 21 Mycoplasma gallisepticum strains and five M. imitans strains. Each strain of M. gallisepticum typed by PFGE and RAPD methods was genetically quite unique and RAPD and PFGE fingerprinting enabled strain characterization. Relationships between the M. gallisepticum and M. imitans strains were established and dendrograms were drawn from PFGE and RAPD patterns. PFGE group A and RAPD group D were significantly associated with M. imitans strains (P < 0.05). Three M. imitans strains shared the same PFGE and RAPD patterns. The two M. gallisepticum vaccine strains had singular PFGE and RAPD patterns. Thus, PFGE and RAPD can be used to investigate disease outbreaks in vaccinated flocks or for epidemiological tracking. For M. gallisepticum, the RAPD and PFGE discriminatory powers were superior to 0.95 and the in vitro, in ovo and in vivo reproducibility of RAPD and PFGE was 100%. The RAPD drawback was the inconsistent band intensity complicating the interpretation of patterns, while the PFGE limit was its low typeability (86%). Thus, these two molecular typing methods seemed complementary for M. gallisepticum epidemiological studies.     

Genomic fingerprinting of pigeon Streptococcus gallolyticus strains of different virulence by randomly amplified polymorphic DNA (RAPD) analysis

Randomly amplified polymorphic DNA (RAPD) analysis was performed on 95 pigeon S. gallolyticus strains of different virulence and belonging to different biotypes and different culture supernatant phenotypes as determined by SDS-PAGE. Four distinct RAPD patterns, designated A, B, C and D, were distinguished using primer OPM6 (5'CTGGGCAACT). All 76 strains generating RAPD pattern A or B were designated highly virulent on the basis of their SDS-PAGE pattern. Five of seven strains generating RAPD pattern C and 11 of 12 strains generating RAPD pattern D belonged to the moderately virulent and low virulent culture supernatant phenotype groups, respectively. Only one RAPD group C strain belonged to a highly virulent culture supernatant phenotype group. There was a correlation between biotype and RAPD patterns. These findings indicate that there is a high correlation between RAPD pattern and virulence for pigeons. Therefore, RAPD typing seems a rapid, reliable method to distinguish pigeon S. gallolyticus strains of high, moderate and low virulence.     

山东省区保存家蚕品种的RAPD分析

采用RAPD标记技术分析山东省区保存的58个家蚕品种资源的DNA多态性。选用重复性较好的20个引物对58份家蚕品种资源材料扩增的总条带数为155条,多态率为93.73%,RAPD标记在家蚕品种间表现出丰富的多态性。根据58个家蚕品种的指纹图谱,采用UPGMA方法进行聚类分析,构建了供试家蚕品种资源的分子系统发育树,可为家蚕新品种选育提供基础信息。     

利用RAPD标记对不同秋眠级苜蓿种质的聚类和评价

利用RAPD标记对25份不同秋眠等级的苜蓿材料进行分析,构建了25份苜蓿材料的DNA指纹图谱,用两种方法(特异的谱带类型和不同引物谱带类型的组合)可以有效的鉴别苜蓿单株,说明RAPD标记是鉴定苜蓿的一种有效方法。通过计算25份苜蓿材料的遗传距离,进行聚类分析,探讨它们之间的亲缘关系。结果如下:25份苜蓿材料间的遗传距离介于4.69-8.14之间,说明材料间的遗传距离较大,亲缘关系较远,遗传基础较宽;从50条RAPD引物中筛选出19条引物,总共扩增出144条带,其中134条呈多态性,占93.05%,10条为单态性带,占6.94%;遗传距离为7.39时,试验材料可以分为差异明显的4类,苜蓿材料间有较大的遗传差异,这为苜蓿引种、亲本选配,分子标记辅助选择育种提供良好的理论基础和科学依据。     

内蒙古驼蹄瓣属植物亲缘关系的RAPD分析

应用RAPD技术对七种驼蹄瓣属植物及其近缘种霸王的亲缘关系进行了研究,16个引物共扩增出125条带,其中99条表现出多态性,占79.2%,表现出丰富的RAPD多态性;利用UPGMA构建了分子系统树,结果表明RAPD分析的亲缘关系聚类基本上与经典分类相一致,表明RAPD技术适用于驼蹄瓣属植物亲缘关系的研究.