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.     

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.     

Complementary randomly amplified polymorphic DNA (RAPD) analysis patterns and primer sets to differentiate Mycoplasma gallisepticum strains.

Randomly amplified polymorphic DNA (RAPD) analysis was used to differentiate 7 strains of Mycoplasma gallisepticum. Six commercially available primers or primer combinations were screened for their ability to differentiate vaccine and type strains. Although major and minor bands were produced with each primer, many of the primers were unsuitable for strain differentiation. The use of primer 6 and combined primers 3 and 4 resulted in complementary RAPD banding patterns for each M. gallisepticum strain. Eleven different isolates representing 7 different strains were segregated into 7 different patterns, corresponding to the 7 strains.     

Evaluation of canine Bordetella bronchiseptica isolates using randomly amplified polymorphic DNA fingerprinting and ribotyping

Bordetella bronchiseptica is a respiratory tract pathogen in a variety of species. Previous studies suggest little genetic variation among canine B. bronchiseptica isolates. The degree of genetic diversity in 26 canine B. bronchiseptica strains was evaluated using randomly amplified polymorphic DNA (RAPD) fingerprinting and ribotyping. Strains evaluated include historic reference strains (N=3). vaccine strains (N=5) and clinical isolates (N=18). RAPD fingerprinting with the 10-nucleotide primer OPA-4 resulted in four distinct fingerprint patterns. RAPD fingerprinting consistently separated four previously characterized electromorphotype (EMT) 6 strains into two fingerprint types. Ribotyping, using the restriction endonuclease PvuI, resulted in six distinct ribotypes. With the exception of vaccine strains, considerable genetic diversity exists in the canine B. bronchiseptica isolates examined. These findings indicate the genetic variability within canine strains of B. bronchiseptica is greater than appreciated previously. Additionally, OPA-4 RAPD fingerprinting and PvuI ribotyping will be useful tools in epidemiologic studies of canine B. bronchiseptica isolates.     

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.     

鸡柔嫩艾美耳球虫不同抗药性虫株的种内多态性研究

应用RAPD技术进行柔嫩艾美耳球虫4个单一抗药性虫株与1个敏感株的基因组DNA多态性分析，发现4个抗药性虫株及敏感株之间的相似值均大于99%；OPAO3引物对抗盐霉素株扩增出一条约500bp的特异条带，OPH02引物对抗克球粉株和抗盐霉素株均扩增出了约1kb的第三条主带，这些特异条带的出现很可能与球虫抗药性基因有关，有可能用于抗药性虫株的诊断与鉴定。     

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.     

Genomic characterization of lung flukes, Paragonimus heterotremus, P. siamensis, P. harinasutai, P. westermani and P. bangkokensis by RAPD markers

Random amplified polymorphic DNA (RAPD) markers were assayed in an attempt to discriminate among five species of Paragonimus. Genomic DNAs of two strains of Paragonimus heterotremus from two provinces in Thailand, Saraburi and Phitsanulok, as well as of P. siamensis, P. harinasutai, P. westermani and P. bangkokensis were extracted and amplified by an arbitrary primer, namely P2 (5-GTTTCGCTCC-3). RAPD patterns showed that those five species were genetically distinct, although they shared genomic DNA to some extent. This primer could also distinguish between two strains of P. heterotremus. The polymorphism observed allowed to construct a relationship dendrogram. The phylogenetic dendrogram showed that the P. heterotremus strains were closest to P. harinasutai, followed by P. siamensis, P. bangkokensis and P. westermani.     

嗜水气单胞菌分离株ERIC-PCR及RAPD分型比较

采用ERIC-PCR和RAPD 2种方法对嗜水气单胞菌2009年分离菌株及实验室保存菌株共83株进行分子分型。参考相关文献设计并合成引物,分别进行PCR扩增及琼脂糖凝胶电泳,电泳结果经Quantity One软件数值化后利用SPSS软件进行聚类分析。结果显示,除少数分离株外,使用2种方法对大多数菌株分型,均能得到多态性较好的指纹图谱。RAPD的AP12H引物能扩增出1～12个200～4 500bp之间的条带,可将嗜水气单胞菌分离株分为4群、12类。ERIC-PCR能扩增出4～16个100～5 800bp之间的条带,将嗜水气单胞菌分离株分为4群、17类,同一年代、同一地域分离株分别有聚成一类的趋势。2种分型方法均体现很好的分型能力。但ERIC-PCR分型与RAPD分型相比,重复性和稳定性更好,分辨力更高,且具备RAPD不要求模板序列已知而直接进行扩增的优点,更适合嗜水气单胞菌的分型研究。     

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.     

奶牛乳房炎克雷伯氏菌的基因分型

采用随机扩增多态性DNA（RAPD）技术对内蒙地区37株奶牛乳房炎克雷伯氏菌进行了基因分型。试验结果表明：利用引物（P1）将37株菌分7个基因型，37株的56．8％（21／37）是同一个基因型。本结果对克雷伯氏菌性乳房炎的防治研究有重要的参考意义。     

中国美利奴羊(新疆型)四个品系的RAPD分子标记研究

选用120条随机引物分别对中国美利奴羊（新疆型）毛质好、体格大、毛密和超细4个品系的混合DNA进行RAPD扩增，共筛选出3条特异性多态引物，占产生扩增产物引物总数的3，0％，说明各品系问的遗传变异程度较小。用其中的特异性引物OPF15分别对4个品系的部分个体样品进行分析，同样表现出多态性，并出现混合DNA样品RAPD分析结果中未出现的谱带。其中超细型有94．12％的个体（16／17）都产生一条特异的相同谱带，大小约为826bp，而其他3个品系混合DNA及个体的扩增产物均未获得此大小扩增片段，由此，可将该扩增片段作为中国美利奴羊（新疆型）超细型的一个特征性RAPD标记。     

奶牛乳房炎病例中金黄色葡萄球菌RAPD-PCR基因分型的研究

用随机扩增多态性DNA 技术（RAPD）对来自2个集约化奶牛场不同奶牛乳房炎病例分出的16株金黄色葡萄球菌进行了基因分型。分别用3个寡核苷酸引物扩增，仅OLP11引物可扩增出质量良好、具有RAPD特征的条带图谱。对反应体系的退火温度、模板浓度进行了筛选，模板原倍浓度、33℃的退火温度能使所有检测菌株产生清晰、可分辨、具有较好重复性的带谱。分型结果表明，16株菌可分为2个类群，其中14株位于同一类群，大多数菌株的相关系数差距仅为0.04，这一结果提示这些菌株可能具有相同的来源和相似的遗传背景。     

Randomly amplified polymorphic DNA (RAPD) analysis of Mycoplasma gallisepticum isolates from turkeys from the central valley of California.

Randomly amplified polymorphic DNA (RAPD) analysis was used to investigate the molecular epidemiology of 26 Mycoplasma gallisepticum (MG) isolates obtained from turkeys located in the central valley of California. The MG isolates were recovered from 5 different companies and 13 ranches. Each company had unique MG strains. No evidence of spread of MG between companies was detected. RAPD analysis of MG isolates within a ranch during an outbreak revealed only a single strain involved in each outbreak. RAPD analysis identified an isolate from 1 ranch with a banding pattern identical to that of the 6/85 vaccine strain, which had been used on that particular ranch. Similar RAPD banding patterns of isolates from different ranches within the same company suggested horizontal spread of MG between ranches. The use of 2 primer sets in RAPD analysis was critical to prevent misinterpretation of relationships between different isolates.     

Comparison of Closely Related Orthopoxvirus Isolates by Random Amplified Polymorphic DNA and Restriction Fragment Length Polymorphism Analysis

The reliability and reproducibility of random amplified polymorphic DNA analysis (RAPD) was compared with restriction fragment length polymorphism (RFLP) by analysing three virus strains isolated from zoo animals in Berlin and three isolates which were cultivated from pets from Northern Germany. The RAPD technique was evaluated as a reliable tool with good reproducibility of the patterns for each virus strain investigated. Problems of interpretation due to inconsistent intensity of bands in different polymerase chain reaction runs may arise for less experienced personnel. The RAPD analysis can be performed within one working day and needs less DNA compared with RFLP so costs will be reduced. The obvious advantage of RFLP is that the pattern can be traced to the recognition site of the restriction enzyme whereas the RAPD primer sequence is not present in the orthopoxvirus genome at all. To the authors knowledge, the RAPD technique has never been applied in DNA viruses before and they conclude that this technique is a useful tool for the discrimination of closely related cowpoxviruses.     

Comparison of methods for differentiation of Salmonella enterica serovar Enteritidis phage type 4 isolates

OBJECTIVE: To compare molecular typing methods for the differentiation of Salmonella enterica serovar Enteritidis phage type (PT) 4 isolates that allowed for the determination of their genetic relatedness. SAMPLE POPULATION: 27 Salmonella Enteritidis PT 4 strains isolated in the United States and Europe. PROCEDURE: Several molecular typing methods were performed to assess their ability to genetically differentiate among Salmonella Enteritidis PT 4 isolates. Results of pulse-field gel electrophoresis (PFGE), repetitive polymerase chain reaction (PCR) assay, 16S rRNA gene sequencing, random amplification of polymorphic DNA (RAPD), PCR-restriction fragment length polymorphism of 16S rRNA, and antimicrobial susceptibility were evaluated. RESULTS: Compared with results for other techniques, results for the RAPD typing method with the RAPD1 primer reveal that it was the most discriminatory fingerprinting technique, and it allowed us to cluster Salmonella Enteritidis PT 4 isolates on the basis of their genetic similarity. CONCLUSIONS AND CLINICAL RELEVANCE: This study revealed the value of RAPD with the RAPD1 primer as a tool for epidemiologic investigations of Salmonella Enteritidis PT 4. It can be used in conjunction with PFGE and phage typing to determine the genetic relatedness of Salmonella Enteritidis isolates involved in outbreaks of disease. A reliable and highly discriminatory method for epidemiologic investigations is critical to allow investigators to identify the source of infections and consequently prevent the spread of Salmonella Enteritidis PT 4.     

Improvement and validation of RAPD in combination with PFGE analysis of Salmonella enterica ssp. enterica serovar Senftenberg strains isolated from feed mills

In 1995 and 1996 a Swedish feed mill had problems due to a persistent contamination of Salmonella enterica spp. enterica serovar Senftenberg that was difficult to eliminate. Forty-eight strains isolated from the feed mill, together with unrelated strains included to evaluate the discriminatory power and reproducibility, were analysed by pulsed-field gel electrophoresis (PFGE). The source of contamination in the feed mill was identified and preventative measures were taken, that led to a resolution of the problem. A previously developed randomly amplified polymorphic DNA (RAPD) protocol was used, to evaluate a rapid and low-cost alternative to PFGE typing. The use of the alternative thermostable DNA polymerase Tth was shown to increase the reproducibility of the RAPD analysis. The reproducibility, in terms of Pearson's and Dice's similarity coefficients for duplicate runs, increased from 72.0 +/- 16.9% and 72.3 +/- 12.9% for Taq to 91.6 +/- 7.5% and 90.9 +/- 5.3% for the fingerprints obtained for the RAPD method employing Tth DNA polymerase. Simpson's index of diversity was calculated and found to be 0.580 for RAPD and 0.896 for PFGE. All of the seven RAPD types could be subdivided into one or more PFGE types, whereas none of the 22 PFGE types was divided into more than one RAPD type. RAPD provides a simple, rapid and powerful screening method that can be used to initially select isolates for further analysis by PFGE.     

Molecular typing by random amplification of polymorphic DNA (RAPD) and detection of virulence genes of Salmonella enterica subspecies enterica serovar Gallinarum biovar gallinarum

Salmonella enterica subspecies enterica serovar Gallinarum biovar Gallinarum is the causative agent of fowl typhoid in chickens, outbreaks of which have devastated poultry populations in Korea since 1992. In order to identify genetic differences among S. Gallinarum isolates, bacteria were examined using the random amplified polymorphic DNA (RAPD) method. Of 13 arbitrary primers screened initially, the primer designated as universal rice primer-6 (URP-6) was selected for subsequent typing assays because it produced a distinctive and reproducible DNA fingerprint for a S. Gallinarum reference strain. URP-6-based RAPD analysis assigned 30 S. Gallinarum isolates into 6 types, with 26 isolates (86.6%) belonging to 2 major RAPD types. The distribution of virulence genes in S. Gallinarum isolates was examined by Southern hybridization. All tested isolates had the invasion gene, invA, the virulence plasmid gene, spvB, and the S. Enteritidis fimbrial gene, sefC. The distribution of virulence genes among S. Gallinarum isolates did not correlate with any specific RAPD type.     

42份高粱与苏丹草及其2个杂交种DNA指纹图谱的构建

选用100个RAPD引物和95对SSR引物进行PCR扩增,旨在构建42份高粱和苏丹草品种资源及2份国审品种高粱-苏丹草杂交种的DNA指纹图谱。结果表明,从100个RAPD引物中筛选到9个多态性高、重复性好的引物,多态性条带比率为64.06%,利用4个核心RAPD引物可以为每份品种构建1张特定的数字指纹,并通过其中1个引物F-01构建了1张能鉴别2个杂交种的RAPD指纹图谱,不过该图谱不能区别皖草3号与其父本Sa。从95对SSR引物中筛选出多态性丰富的引物73对,多态性条带比率为86.06%,通过3对核心SSR引物就可以构建42份高粱和苏丹草的SSR数字指纹,同时利用其中1对SSR引物txp18,寻找到2个杂交种的互补带,从而构建了2个高粱-苏丹草杂交种的SSR指纹图谱,这张SSR指纹图谱不仅能鉴别皖草2号和3号,还可以把杂交种与其亲本区别开来。     

Typing Listeria monocytogenes by random amplified polymorphic DNA (RAPD) fingerprinting.

Twenty epidemiologically unrelated Listeria monocytogenes strains isolated from different animals, locations and on different dates in Japan were classified into 18 types by the random amplified polymorphic DNA (RAPD) fingerprinting technique with four primers. Further, seven epidemiologically related L. monocytogenes strains isolated from raw milk and a bulk tank on a dairy farm represented the same RAPD type suggesting that they were all of the same origin. Therefore, RAPD-polymerase chain reaction (PCR) analysis, which is rapid, simple and inexpensive to perform, can be used in surveys as a convenient epidemiological technique.