Use of arbitrarily primed polymerase chain reaction to differentiate Trichophyton dermatophytes

Abstract Dermatophytes such as Trichophyton species are common human pathogens, the infection of which results in dermatophytosis (also known as ringworm). Several laboratory tests are used routinely for the diagnosis of dermatophytosis, but they are either slow or lacking specificity. Through examination of genomic DNA from Trichophyton dermatophytes and other fungi in arbitrarily primed PCR, it was shown that a random primer 5'-ACCCGACCTG-3' produced bands of 4.3 kb, 1.9 kb, 1.7 kb and 0.7 kb in T. rubrum DNA, bands of 2.5 kb, 1.9 kb and 0.8 kb in T. mentagrophytes var. interdigitale and T. mentagrophytes var. mentagrophytes DNA, and bands of 2.5 kb, 1.9 kb, 1.5 kb and 0.9 kb in T. tonsurans DNA. This primer amplified bands of different sizes in other fungal DNA. Therefore, based on the distinct band patterns observed in arbitrarily primed PCR using this primer, T. rubrum , T. mentagrophytes and T. tonsurans dermatophytes could be rapidly differentiated.     

Genetic speciation of Candida isolates by arbitrarily primed polymerase chain reaction

Abstract Candida species are opportunistic human pathogens capable of causing a variety of clinical diseases. Rapid and precise identification of Candida to species level is essential for effective treatment and management strategies. Conventional diagnosis of candidiasis is sometimes slow and variable. By using a random primer 5'-ACGGGCCAGT-3' in arbitrarily primed polymerase chain reaction, seven common Candida species (i.e. C. albicans, C. guilliermondii, C. parapsilosis, C. krusei, C. lipolytica, C. tropicalis and C. (Torulopsis) glabrata ) produced characteristic DNA band patterns, which enabled rapid determination to species level among them. Further analysis of the nucleotide sequences of Candida specific fragments should improve our understanding of the molecular structures and possible functions of the gene regions involved.     

Analysis of Vibrio mimicus clinical strains by arbitrarily primed polymerase chain reaction

A total of 51 Vibrio mimicus clinical strains from different geographic locations were examined by arbitrarily primed polymerase chain reaction (AP-PCR). The primer VMH-3 divided them into 28 groups, although 18 groups consisted of a single strain at present. All groups had a common 1.0-kb amplification fragment. Most of the groups consisted of strains from same region, although two exceptional groups showed a few amplification fragments including strains from different regions. AP-PCR groups were not consistently associated with serogroups. AP-PCR is thought to be a valuable and easy method for the epidemiological study of V. mimicus.     

Identification of DNA methylation differences during tumorigenesis by methylation-sensitive arbitrarily primed polymerase chain reaction

The ability to detect methylation changes associated with oncogenic transformation is of critical importance in understanding how DNA methylation may contribute to tumorigenesis. We have developed a simple and reproducible fingerprinting method called methylation-sensitive arbitrarily primed polymerase chain reaction (AP-PCR) to screen for DNA methylation changes. This technique relies on digesting genomic DNA with methylation-sensitive and -insensitive restriction enzymes (e.g., HpaII and MspI) prior to AP-PCR amplification. Matched normal and tumor DNAs were compared to identify differential methylation. After the PCR products were resolved on high-resolution polyacrylamide gels, regions of genomic DNA that showed hypo- and hypermethylation associated with tumors were detected. These fragments were then isolated, cloned, and sequenced. Novel CpG islands were found to be frequently hypermethylated in bladder and colon tumors. We have demonstrated that this technique is a rapid and efficient method that can be used to screen for altered methylation patterns in genomic DNA and to isolate specific sequences associated with these changes.     

Differentiation of aphid clones by arbitrarily primed polymerase chain reaction (AP-PCR) DNA fingerprinting

Differentiation of aphid clones was attempted using AP-PCR which is a simple and rapid method to obtain DNA fingerprints of complex genomes. To establish optimal reaction conditions and examine reproducibility of the method, a laboratory-maintained clone of the pea aphid, Acyrthosiphon pisum , was used as test material. Under the reaction conditions employed, identical fingerprint patterns were obtained throughout a wide range of template DNA amount, from 5 to 800 ng, and irrespective of aphid instar. No changes in the patterns were seen throughout five parthenogenetic generations. When this method was applied to a wild population of the gall-forming aphid, Ceratovacuna nekoashi , five groups of insects originating from different galls formed on the same twig were successfully differentiated from one another by means of polymorphic fingerprint bands. In contrast, the fingerprints of the insects derived from the subgalls of the same gall were identical. These results indicated that in C. nekoashi : (i) members of a gall constitute a clonal population; (ii) a gall is founded by a single fundatrix; and (iii) intergall migration is absent or at least not frequent.     

Parentage determination in maize hybrids using the arbitrarily primed polymerase chain reaction (AP-PCR)

Summary Using a novel procedure based on the polymerase chain reaction, we have developed a rapid, efficient, and economical method for identifying plant genotypes. The arbitrarily primed polymerase chain reaction (AP-PCR) generates reproducible fingerprints from any organism, without the need for DNA sequence information. These fingerprints include DNA fragment polymorphisms that can be (1) used for varietal identification and parentage determination, (2) followed in segregating populations produced by crosses, (3) used as markers for the construction of genetic maps, and (4) used to generate dendograms of phylogenetic relationships, especially at the intraspecific level. AP-PCR requires only minute quantities of DNA (10–25 ng per reaction) and therefore can be used in situations in which DNA is limiting. We demonstrate the use of AP-PCR to identify inbred parents of hybrid maize plants in double-blind experiments.     

Actinobacillus actinomycetemcomitans genetic heterogeneity: amplification of JP2-like ltx promoter pattern correlated with specific arbitrarily primed polymerase chain reaction (AP-PCR) genotypes from human but not marmoset Brazilian isolates

Specific clonal types of Actinobacillus actinomycetemcomitans, a major human periodontal pathogen, may be responsible for clinical manifestations and the production of leukotoxin virulence factors. Leukotoxicity is associated with genetic polymorphism at the promoter region of the leukotoxin (lItx) gene. Here, we describe the use of arbitrarily primed polymerase chain reaction (AP-PCR) and ltx promoter PCR to molecularly characterise 35 A. actinomycetemcomitans Brazilian isolates: 21 of human origin and 14 from captive marmosets (Callitrix spp., primates commonly used as animal models for periodontal research). The discriminative capacity of each of 12 arbitrary primers was found to be variable, yielding between 3 and 24 PCR amplitypes. Combination of the results for all primers led to characterisation of 14 genotypes that grouped into four major clusters based on genetic similarity. Clusters 2, 3, and 4 were discriminative to host origin. A correlation with periodontal disease was suggested for strains belonging to clusters 3 and 4. The JP2-like PCR amplification pattern, associated with highly leukotoxic strains, was exclusive to human isolates and present in 29% of human isolates where it occurred in close relationship with AP genotypes L and J (cluster 3).     

任意引物PCR及其应用研究进展

任意引物PCR技术又称为随机扩增多态性DNA技术,它是在PCR技术基础上发展起来的一项分子检测技术。它具有简便、快速,一套引物可用于多个物种的分析,不需预知分析对象的核酸序列,可以显示差异表达基因等特点,已广泛应用于病原微生物的分型鉴定、物种亲源关系分析、遗传育种研究和特异表达基因的克隆与鉴定等方面。     

Construction of a phylogenetic tree for inbred strains of rat by arbitrarily primed polymerase chain reaction (AP-PCR)

We constructed a tentative genealogic tree of 13 inbred rat strains using genetic markers identified by the AP-PCR (Arbitrarily Primed Polymerase Chain Reaction) technique, which consists of PCR amplification under low stringency conditions, with only one oligonucleotide as both forward and reverse primers. We obtained discrete-state genotypes of 264 loci, presumably covering the whole rat genome. Computational analysis of this panel allowed the construction of a genealogic tree representing the relations among the 13 strains. The accordance of the present results with available data on the histories of some of these strains validates the use of the AP-PCR technique for studies on phylogeny.     

Genomic fingerprinting by arbitrarily primed polymerase chain reaction resolves Borrelia burgdorferi into three distinct phyletic groups.

The causative agent of Lyme disease, Borrelia burgdorferi, was first identified by Burgdorfer et al. in 1982 (W. Burgdorfer, A. G. Barbour, S. F. Hayes, J. L. Benach, E. Grunwaldt, and J. P. Davis, Science 216:1317-1319, 1982) and was isolated by Barbour et al. in 1983 (A. G. Barbour, W. Burgdorfer, S. E. Hayes, O. Peter, and A. Aeschlimann, Curr. Microbiol. 8:123-126, 1983). Since then, a large number of isolates have been collected, and there have been questions regarding the relationships among the various strains. Using genomic fingerprinting by an arbitrarily primed polymerase chain reaction, we resolved into three groups a collection of Eurasian and North American isolates of spirochetes that are generally categorized as B. burgdorferi. Group I strains have been identified in both North America and Eurasia, while strains belonging to Borrelia groups II and III have been found only in Eurasia. These same three groups have also been delineated by Baranton et al. (G. Baranton, D. Postic, I. Saint Girons, P. Boerlin, J.-C. Piffaretti, M. Assous, and P. A. D. Grimont, Int. J. Syst. Bacteriol. 42:370-375, 1992) by independent methods. Two isolates are distinct from all of the other strains in our collection but are clearly members of the genus Borrelia.     

Rapid genomic fingerprinting of Lactococcus lactis strains by arbitrarily primed polymerase chain reaction with 32P and fluorescent labels.

Arbitrarily primed polymerase chain reaction, with incorporation of either radioactive or fluorescent labels, was used as a rapid and sensitive method for obtaining genomic fingerprints of strains of Lactococcus lactis. Closely related strains produced almost identical fingerprints. Fingerprints of other strains showed only some similarities.     

Rapid genomic fingerprinting of Lactococcus lactis strains by arbitrarily primed polymerase chain reaction with 32P and fluorescent labels.

Arbitrarily primed polymerase chain reaction, with incorporation of either radioactive or fluorescent labels, was used as a rapid and sensitive method for obtaining genomic fingerprints of strains of Lactococcus lactis. Closely related strains produced almost identical fingerprints. Fingerprints of other strains showed only some similarities.     

Investigations of genetic variation between olive (Olea europaea L.) cultivars using arbitrarily primed polymerase chain reaction (AP-PCR)

Characterization and selection of olive clones for the production of olive oil is essential in Turkey because of its profitable exploitation. AP-PCR (Arbitrarily-Primed PCR) is a technique that can distinguish the genetic relationship among plant species and other organisms. In this study, AP-PCR approach was used in order to determine the genetic relationship of different six olive clones. The purity of DNA is one of the most important factors affecting the product of the AP-PCR method. In this respect, modified genomic DNA isolation procedure from Oleae europaea clones was developed so that this procedure can be used to obtain plant genomic DNA from diverse aromatic plants, which produce essential oils and secondary metabolites. By following the optimized AP-PCR amplification protocol, unique DNA fingerprint profiles for each olive clone were produced. AP-PCR-generated unique DNA fingerprint profiles can be used in the identification, distribution and diversity of various olive cultivars.     

Leptospira species categorized by arbitrarily primed polymerase chain reaction (PCR) and by mapped restriction polymorphisms in PCR-amplified rRNA genes.

Reference strains from 48 selected serovars representing eight species of Leptospira were examined by two polymerase chain reaction (PCR)-based strategies. First, mapped restriction site polymorphisms (MRSP) were examined in PCR products from portions of rrs (16S rRNA gene) and rrl (23S rRNA gene). Twenty MRSP and 2 length polymorphisms were used to group reference strains into 16 MRSP profiles. Species assignments were consistent with those obtained by a second method, genomic fingerprinting with arbitrarily primed PCR, in which strains within a species were characterized by many shared arbitrarily primed PCR products. The results of both of these methods were in general agreement with those of previous studies that used DNA-DNA relatedness and confirmed the high level of divergence among the recognized species of Leptospira. However, Leptospira meyeri serovar ranarum and evansi strains were indistinguishable from some strains of Leptospira interrogans sensu stricto. Intervening sequences of about 485 to 740 bp were located near base 1230 in rrl of some strains.     

Comparison of automated ribotyping and pulsed-field gel electrophoresis for subtyping of Vibrio cholerae

Aims:  To compare the discriminatory power of an automated ribotyping method for Vibrio cholerae subtyping with the pulsed-field gel electrophoresis (PFGE), to evaluate the possibility of automated ribotyping in use of outbreak investigations and surveillance of cholera.
Methods and Results:  Eight-one epidemiologically unrelated isolates of V. cholerae , and 19 isolates from seven cholera outbreaks were used as the panels. When comparing the two methods using the epidemiologically unrelated isolates, automated ribotyping using Pvu II distinguished 38 different ribotypes with a D -value of 0·8956. When combined with serotyping, the D -value is 0·9466. However, PFGE with Not I and Sfi I digestions had higher D -values of 0·9951 and 0·9948, respectively. PFGE could cluster the isolates from each outbreak into the same pattern, and distinguish different patterns from different outbreaks, whereas automated ribotyping had lower discriminatory ability.
Conclusions:  The automated ribotyping has lower discriminatory ability compared to PFGE, and is limited to application in V. cholerae subtyping and outbreak investigation.
Significance and Impact of the Study:  The study evaluated the limitation in subtyping of automated ribotyping for V. cholerae , and raise the question of improvement for the automated ribotyping in subtyping.     

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).     

In situ polymerase chain reaction and cycling primed in situ amplification: improvements and adaptations

 Ethanol fixation combined with microwave pretreatment allows rapid and simple detection of signals produced by cycling primed in situ (PRINS) amplification, which uses a single primer, and in situ polymerase chain reaction (ISPCR) in intact cells. After thermal cycling, signals remain as discrete subnuclear spots in the region of amplification and are clearly distinguishable from non-specific background labelling. These methods are applicable to routine blood smears, even after Giemsa staining or immunocytochemistry, and cellular morphology is retained. Chromosome enumeration by cycling PRINS is demonstrated using primers for repeat DNA sequences, whilst single copy sequence detection is demonstrated using bcl-2, CFTR and chromosome 21 specific primer pairs in ISPCR. We show that ethanol fixation supports efficient extension of cycling PRINS products to approximately 550 bp using up to 70 rounds of thermal cycling. Accepted: 15 February 1999     

Comparison of arbitrarily primed-polymerase chain reaction and pulse-field gel electrophoresis for characterizing methicillin-resistant Staphylococcus aureus

AIMS: The aim of this study was to analyse genotypes for clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA), including hetero-vancomycin-resistant Staph. aureus (VRSA), at a Japanese university hospital. METHODS AND RESULTS: Seventy-eight clinical isolates of MRSA were analysed by arbitrarily primed-polymerase chain reaction (AP-PCR) using ERIC2 primer and by pulse-field gel electrophoresis (PFGE) following SmaI digestion. Analyses of the nine genotypes and 28 subtypes defined by PFGE, and of the three genotypes and 22 subtypes defined by AP-PCR, both facilitated epidemiological tracing. Used in combination, AP-PCR and PFGE provided more precise classification than the use of a single genotyping method. The six hetero-VRSA isolates were classified into four genotypes defined by the combination of both methods, but these genotypes contained non-VRSA isolates. CONCLUSIONS: The results suggest that both PFGE and AP-PCR are useful in discriminating MRSA, but not hetero-VRSA, isolates for epidemiological analysis. SIGNIFICANCE AND IMPACT OF THE STUDY: Combining the results of PFGE with the results of AP-PCR can provide more detail differentiation of MRSA and hetero-MRSA isolates than either method alone.