A random amplified polymorphic DNA polymerase chain reaction technique that differentiates between Neospora species

Neospora caninum is a recently described coccidial parasite that was first isolated from a dog in 1988 and has subsequently been shown to infect a wide range of mammals. Neospora hughesi, a new species of this genus, has recently been isolated from the spinal cord of horses showing clinical signs of equine protozoal myeloencephalitis. The random amplified polymorphic DNA polymerase chain reaction technique is capable of differentiating between N. caninum and N. hughesi.     

Differentiation of Aeromonas genomospecies using random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR)

Random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) was used to investigate the differentiation of the genus Aeromonas at the genomospecies level. Of 20 primers evaluated, six produced profiles which contained multiple bands capable of differentiating the genomospecies. These six primers were also used in RAPD-PCR analyses of clinical and environmental isolates of the different genomospecies. In most cases. each strain gave a unique fingerprint, illustrating genetic heterogeneity at the genomospecies level. However, some homogeneity in fragment sizes was seen among strains within a genomospecies which was not apparent in strains from different genomospecies. This study therefore complements and supports the current classification of Aeromonas into genomospecies. These results also show that RAPD-PCR has the potential to differentiate between the genomospecies of Aeromonas.     

鲍曼不动杆菌随机扩增多态性DNA法基因分型的研究

目的 利用随机扩增多态性DNA(RAPD)技术对鲍曼不动杆菌进行基因分型建立DNA指纹图谱,调查该菌在各临床科室的流行情况,并将其与药敏谱进行比较.方法 随机收集中南大学湘雅二医院2007年9月到2008年9月分离出的86株鲍曼不动杆菌,采用WHO推荐的K-B法对鲍曼不动杆菌进行药物敏感试验,建立药敏谱,同时利用随机扩增多态性DNA法(RAPD)技术进行基因分型,建立DNA指纹图谱,对二者进行比较,然后结合药敏谱和指纹图谱分析各临床科室鲍曼不动杆菌的感染情况.结果 药物敏感试验将86株鲍曼不动杆菌分为47型,RAPD技术将其分为14型,其中A、F、D、B和L型为5种优势型,其菌株数分别为22、15、11、9和7株.本院ICU,老年病科,神经内科,呼吸内科,神经外科鲍曼不动杆菌检出率较高.结论 RAPD分型方法优于药敏分型,其在流行病学研究上更能证实菌株的相关性,在早期发现和预防感染暴发流行中起重要作用.     

Suppression of randomly primed polymerase chain reaction products (random amplified polymorphic DNA) in heterozygous diploids

In a study of genetic polymorphism in the gypsy moth Lymantria dispar we observed the aberrant inheritance of a random amplified polymorphic DNA (RAPD) fragment designated H11-589. This fragment was present in amplification products of F1 progeny of different crosses although it was not amplified from either parental DNA. DNA-mixing experiments revealed that the presence of DNA containing a template for another product (H11-746), amplified with the same primer, suppressed the synthesis of H11-589. The templates for both RAPD products were highly repetitive and scattered throughout the L. dispar genome. Southern hybridization and sequence analysis of H11-746 and H11-589 revealed an extensive sequence homology and an internal repetitive motif of 17 nucleotides present in both products. Interactions between templates for H11-746 and H11-589 are expected to occur during the polymerase chain reaction (PCR), offering an explanation for the suppression of the amplification of H11-589. The role of the internal repetitive motif and of the copy number of both templates in the suppression effect are discussed. Our results corroborate doubts regarding the suitability of the RAPD technique for quantitative genetic analysis, in particular where mixed populations are concerned.     

Enhanced resolution of random amplified polymorphic DNA genotyping of Pseudomonas aeruginosa

AIMS: To develop a rapid, sensitive and reproducible screening test for the detection of nosocomial spreading of Pseudomonas aeruginosa. METHODS AND RESULTS: Ps. aeruginosa genomic DNA extraction, RAPD-PCR, electrophoresis on acrylamide gel and silver staining were performed by using standardized reagents and conditions. The results were compared with the agarose gel electrophoresis followed by ethidium bromide staining. CONCLUSIONS: The coupling of acrylamide gel electrophoresis and silver staining gave about 80% more DNA bands than the traditional method, allowing a finer discrimination among different Ps. aeruginosa strains. SIGNIFICANCE AND IMPACT OF THE STUDY: By enhancing the resolution of the electrophoretic separation and the sensitivity of the staining, random amplification could be easily applied to the surveillance and prevention of nosocomial infections by clinical microbiology laboratories.     

Analysis of ancient DNA from coprolites: a perspective with random amplified polymorphic DNA-polymerase chain reaction approach

The aim of this work was to determine approaches that would improve the quality of ancient DNA (aDNA) present in coprolites to enhance the possibility of success in retrieving specific sequence targets. We worked with coprolites from South American archaeological sites in Brazil and Chile dating up to 7,000 years ago. Using established protocols for aDNA extraction we obtained samples showing high degradation as usually happens with this kind of material. The reconstructive polymerization pretreatment was essential to overcome the DNA degradation and the serial dilutions helped with to prevent polymerase chain reaction (PCR) inhibitors. Moreover, the random amplified polymorphic DNA-PCR has been shown to be a reliable technique for further experiments to recover specific aDNA sequences.     

Differentiation of Brucella species by random amplified polymorphic DNA analysis

Random amplification of polymorphic DNA (RAPD) was used for discrimination between 46 Brucella strains and 14 representatives of the alpha-2 and alpha-1 subgroups of Proteobacteria. To evaluate a relatively quick and exact method for Brucella identification, the authors specified the most suitable conditions for RAPD amplification of Brucella DNA with two 10-mer primers, containing lower and higher percentages of G and C. The software package PHYLIP 3.1 was used for cluster analysis of the RAPD fingerprints. The optimization of RAPD conditions resulted in PCR mixes suitable for reliable typing of Brucellae. The distance-based methods (Fitch-Margoliash, UPGMA and Neighbour-joining) gave clear discrimination between Brucella species. The constructed dendrograms put Br. canis and Br. suis bv. 1 in the same cluster and differentiated Brucella strains according to their host preferences. RAPD can be useful method to distinguish related bacterial species, and under strictly established conditions the reaction appears to be a simple, quick and sensitive technique for the epidemiological investigation of brucellosis.     

Comparison between human and armadillo Paracoccidioides brasiliensis isolates by random amplified polymorphic DNA analysis

Sixty-three Paracoccidioides brasiliensis isolates obtained from three nine-banded armadillos ( Dasypus novemcinctus), one Amazonian armadillo's and 19 clinical isolates were compared by random amplified polymorphic DNA analysis with the primer OPG-19. The isolates were divided into three major clusters, I, II and III. Coincidences between human and armadillo isolates were observed in clusters I and II. Cluster III consisted only of armadillos' isolates. The results suggested that (I) humans may acquire P. brasiliensis infection by contact with armadillo's environment, (II) there may be P. brasiliensis genotypes peculiar to the animal, and (III) individual armadillos may be infected with P. brasiliensis cells with different genotypes. This revised version was published online in June 2006 with corrections to the Cover Date.     

The differentiation of Carnobacterium divergens using the random amplification of polymorphic DNA polymerase chain reaction technique

The potential of the randomly amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) technique to differentiate Carnobacterium divergens from other members of the genus Carnobacterium was examined. A numerical analysis of the genomic profiles obtained demonstrated that it was possible to differentiate the C. divergens strains from other Carnobacterium strains using this technique. The heterogeneity observed in the representatives of the species C. piscicola adds further weight to the suggestion in other taxonomic studies that subspecies of this species exist.     

Quantitative analysis of DNA aberrations amplified by competitive polymerase chain reaction using capillary electrophoresis

We compared the use of capillary electrophoresis (CE) in a polymer network with the use of slab gel electrophoresis for the quantitative analysis of polymerase chain reaction (PCR)-amplified DNA samples. We quantified residual lymphoma cells carrying a translocation between chromosomes 14 and 18, in consecutive patient peripheral blood samples that were amplified by competitive PCR. For CE analysis we used a 4% linear polyacrylamide network. Results show that the calculated number of translocations in patient samples using both analyses were comparable. We conclude that CE is a sensitive, non-radioactive, fast and accurate method for quantitation of competitive PCR products.     

Genetic diversity of potato determined by random amplified polymorphic DNA analysis

Summary The RAPD procedure was used to establish genetic diversity of 28 potato genotypes including siblings and genotypes with no immediate relationship. In addition amplified DNA from three parents and Solanum chacoense were compared with that from six progeny to determine the genetic relationships. Amplification of genomic DNA from the 28 genotypes using PCR and 12 decamer primers yielded 158 amplified DNA fragments, ranging in size from 490 to 3200 bp. A total of 128 unique RAPD fragments were observed among the 28 potato genotypes. Similarity measures and principal coordinate analysis generally reflected the expected trends in relationships of the full and half-sib potato genotypes. However there were important exceptions to this general trend and it appears that related varieties can be as genetically different as varieties with no immediate relationship. The data suggest that RAPD analysis used in conjunction with pedigree information can provide a superior measure of genetic divergence than analysis based solely on pedigree information.Abbreviations PCR polymerase chain reaction - RAPD random amplified polymorphic DNA - DNA deoxyribonucleic acid - CTAB cetyltrimethylammonium bromide - RNA ribonucleic acid - PCO principal coordinate analysis     

Isolation and random amplified polymorphic DNA analysis of genomic DNA from soybean embryos

Summary An isolation procedure was developed for the extraction of genomic DNA and Random Amplified Polymorphic DNA (RAPD) analysis using individual soybean embryos. This procedure can be used to quickly and efficiently isolate DNA from a large number of individuals. DNA isolations were analyzed for total yield, integrity, and usefulness as a template in RAPD analysis.     

Optimization of asymmetric polymerase chain reaction for rapid fluorescent DNA sequencing

A high-throughput method for the preparation of single-stranded template DNA, which is suitable for sequence analysis using fluorescent labeling chemistry, is described here. In this procedure, the asymmetric polymerase chain reaction is employed to amplify recombinant plasmid or bacteriophage DNA directly from colonies or plaques. The use of amplification primers located at least 200 base pairs 5' to the site of sequencing primer annealing removes the need for extensive purification of the asymmetric polymerase chain reaction product. Instead, the single-stranded product DNA is purified by a simple isopropanol precipitation step and then directly sequenced using fluorescent dye-labeled oligonucleotides. This method significantly reduces the time and labor required for template preparation and improves fluorescent DNA sequencing strategies by providing a much more uniform yield of single-stranded DNA.     

Correlation between polymerase chain reaction analysis of the histidine biosynthesis operon, randomly amplified polymorphic DNA analysis and phenotypic characterization of dairy Lactococcus isolates

A collection of 32 lactococcal strains isolated from raw milk in the Camembert RDO (registered designation of origin) area were phenotypically and genotypically characterized. As expected for environmental isolates, all strains had a Lactococcus lactis subsp. lactis phenotype. The strains were then genotypically identified by the randomly amplified polymorphic DNA (RAPD) technique, using reference strains of lactococci. Two major clusters were identified containing the two subspecies lactis and cremoris. The subspecies lactis cluster could be divided into five subgroups whereas there was a high coefficient of similarity between all strains in the subspecies cremoris cluster. This RAPD classification was then compared with that of a traditional PCR assay using L.lactis species-specific primers corresponding to part of the histidine biosynthesis operon. The two subspecies were differentiated by the size of the fragment amplified (about 200 bp longer for subspecies cremoris). Unlike preliminary phenotypic assignments, the results of PCR experiments corroborated the genotypic identification of the lactococcal strains by RAPD allowing the technique to be reconsidered on the basis of its taxonomic efficiency. Received: 14 May 1998 / Accepted: 3 September 1998     

Technical aspects of random amplified polymorphic DNA (RAPD) technique in genotyping of bacterial strains

Random Amplified Polymorphic DNA (RAPD) method became widely applied for sensitive, efficient and fast distinguishing of different isolates of a given species, if pure culture is available. Problems with reproducibility and discriminatory power, frequently cited in the literature, can be overcome by precise optimization procedure allowing to achieve reliable conditions for each species analysed. Basing on two examples of different species, H. pylori and E. faecium, particular parameters of RAPD fingerprinting were evaluated with respect to selection of best working primers generating medium-complex profiles, using only high quality DNA samples and evaluation optimum for every reaction reagent. Stable and informative amplification patterns were obtained with different best working primers which could discriminate between all H. pylori and E. faecium strains tested. For both analysed species different optima were found, suggesting species-specific need of precise RAPD conditions evaluation. This study proved high sensitivity and efficiency of optimized RAPD profiling applicable for searching the epidemiology traces for both species.     

Comparison of DNA extraction methods for multiplex polymerase chain reaction

We compared six DNA extraction methods for obtaining DNA from whole blood and saliva for use in multiplex polymerase chain reaction (PCR) assays. The aim was to evaluate saliva sampling as an alternative to blood sampling to obtain DNA for molecular diagnostics, genetic genealogy, and research purposes. The DNA quantity, DNA purity (A_260/280), PCR inhibition ratio, and mitochondrial DNA/genomic DNA ratio were measured to compare the extraction methods. The different extraction methods resulted in variable DNA quantity and purity, but there were no significant differences in the efficiency of multiplex PCR and oligomicroarray signals after single-base extension on the arrayed primer extension 2 (APEX-2).     

Characterization of Paracoccidioides brasiliensis atypical isolates by random amplified polymorphic DNA analysis

Two atypical Paracoccidioides brasiliensis strains (yeast form at room temperature) have been isolated from chronically infected patients living in Brazil. Different random primers were used to characterize these isolates and compare them to typical strains. The RAPD patterns allowed the differentiation of all the selected isolates. Their genetic distance ranged from 5% to 80% of non-shared bands depending on the strains and the primer used. The RAPD data were used to build a Wagner phenogram, which showed two major branched with more than 56% of genetic distance separating them. No significant difference was observed between the atypical isolates and the others suggesting that specific genes are involved in the dimorphism phenomenon.     

Detection of sequence variation and genotyping of polymorphic genes using polymerase chain reaction stem–loop conformational polymorphism analysis

Here we describe a technique we have named polymerase chain reaction stem–loop conformational polymorphism (PCR–SLCP) analysis for screening DNA sequence variation. This technique uses PCR primers with adapters at the 5′ ends to generate amplicons containing inverted terminal repeat sequences. These enable the formation of specific conformers for single-stranded molecules on denaturation, and sequence variation in the loop region may affect the structure of these and their mobilities during electrophoresis. The technique has the ability to resolve sequence variation in amplicons that cannot be resolved using PCR single-strand conformational polymorphism (PCR–SSCP). PCR–SLCP is simple and sensitive, and the results are highly repeatable.     

The application of random amplified polymorphic DNA for sandfly species identification

Abstract. We have applied the recently developed Random Amplified Polymorphic DNA (RAPD) method to produce species-specific, DNA profiles for two sympatric, Venezuelan sandfly species, thought to be the vectors responsible for recent outbreaks of cutaneous and mucocutaneous leishmaniasis in the Andean State of Tachira. Moreover, within the profile, it was possible to identify a diagnostic DNA band for Lu.youngi of 0.32 kb. Results showed that the size of this diagnostic DNA band remained constant and did not vary with sex or geographical distribution.