Arbitrarily Primed Polymerase Chain Reaction Research Articles

Arbitrary primed PCR fingerprinting and serotyping of clinical Pseudomonas aeruginosa strains

Arbitrary primed PCR (AP-PCR) analysis was compared with serotyping as a means of high-resolution typing of Pseudomonas aeruginosa. Seventy-four isolates from 3 different hospitals and 18 reference strains were studied. Serotyping provided good index of discrimination, although eleven isolates could not be serotyped. Genomic DNA was amplified with a single 10 nucleotide primer (sequence 5′-AGG GGT CTT G-3′). The strains were genetically diverse and 61 different AP-PCR profiles of 2–7 bands between 0.3 and 2.4 kb were obtained. AP-PCR profiles were not consistently associated with serotypes, but they clearly subtyped strains of the same serotype. Numerical analysis of AP-PCR patterns defined 7 groups at the 55% similarity level, and identified predominant strains in each hospital. The results show that AP-PCR analysis provides a simple and practical approach to typing P. aeruginosa that is more discriminatory than traditional serotyping scheme. We suggest that maximum discrimination can be achieved by a combination of both methods.

Rapid identification of pathogenic Leptospira species (Leptospira interrogans, L. borgpetersenii, and L. kirschneri) with species-specific DNA probes produced by arbitrarily primed PCR.

Arbitrarily primed PCR (AP-PCR) assays can be used to discriminate between species of Leptospira. Comparative analysis of the fingerprints obtained from representative sets of serovar reference strains of Leptospira interrogans sensu stricto, L. borgpetersenii, and L. kirschneri and the reference strains of the other Leptospira spp. revealed species-specific DNA fragments. These species-specific sequences were reamplified in order to produce digoxigenin-11-dUTP-labeled genomic DNA probes that could be used to identify Leptospira species. Three probes (specific for L. interrogans sensu stricto, L. borgpetersenii, and L. kirschneri) were selected and tested with 72 representative serovar reference strains, all of which had previously been studied by DNA-DNA hybridization. The two techniques were in general agreement, and hybridization with AP-PCR-derived probes was shown to be a useful approach for rapid species determination of leptospires, without the prior need for DNA sequence information. These nonradioactive probes can be used to identify Leptospira species in nonspecialized laboratories, and this should contribute to a better knowledge of the molecular epidemiology of leptospirosis.

Molecular epidemiology of an outbreak of multidrug-resistant Enterobacter aerogenes infections and in vivo emergence of imipenem resistance.

Molecular typing was used to investigate an outbreak of infection caused by multidrug-resistant Enterobacter aerogenes (MREA) susceptible only to gentamicin and imipenem in an intensive care unit (ICU). Over a 9-month period, ciprofloxacin-resistant E. aerogenes isolates were isolated from 34 patients, or 4.1% of ICU admissions, compared with a baseline rate of 0.1% in the previous period (P < 0.001). Infection developed in 15 (44%) patients. In vivo emergence of imipenem resistance (MIC, 32 micrograms/ml) of organisms causing deep-seated infection was observed in two (13%) of these patients following prolonged therapy with imipenem and gentamicin. Arbitrarily primed PCR (AP-PCR) analysis with ERIC1R and ERIC2 primers and pulsed-field gel electrophoresis (PFGE) analysis of XbaI macrorestriction patterns concordantly showed that outbreak-associated MREA isolates were clonally related and distinct from epidemiologically unrelated strains. AP-PCR and PFGE showed discrimination indices of 0.88 and 0.98, respectively. Space-time clustering of cases within units suggests that the epidemic-related MREA isolates were transmitted on the hands of the health care personnel. A case-control study and repeated environmental culture surveys failed to identify a common source or procedure associated with transmission. In spite of the early implementation of isolation measures, the incidence of MREA colonization remained stable until all colonized patients were discharged. This study confirms the usefulness of AP-PCR and PFGE analyses for the epidemiological study of E. aerogenes and underscores the difficulty of controlling the spread of multiresistant clones of this organism in the ICU setting. The emergence of imipenem resistance represents a threat because virtually no therapeutic option is available for such strains.

Application of arbitrarily primed polymerase chain reaction analysis to epidemiological study of Salmonella enterica serovar enteritidis

An arbitrarily primed polymerase chain reaction (AP-PCR) DNA profile was applied to epidemiological analysis of Salmonella enterica subsp. enterica serovar Enteritidis. A total 21 strains of S. Enteritidis isolated from 21 cases (10 cases of healthy persons, 7 cases of food poisoning outbreaks and 4 sporadic diarrhea cases), during the period between December 1991 and August 1996 in Wakayama City, were used. A total of 60 arbitrary primers (DNA oligomer (12) set, Wako) were screened with 4 S. Enteritidis strains of different cases. A-11, B-32, C-42 and C-45 primers were chosen. Plasmid DNA profiles, antimicrobial susceptibility patterns and phage types were also examined. The combination of these three methods resolved the collection into five groups (A to E). And type C strains were found in 17 cases (81%) out of 21 cases. However, according to AP-PCR DNA profile, all 21 strains were classified into six groups (I to VI), and 17 type C strains were classified into three groups (III, IV and V). Type IV was predominant in Wakayama City, and type C.IV was found in 15 cases (71%). In conclusion, we considered that AP-PCR DNA profile using appropriate primers was an effective epidemiological marker.

Interspecific hybridization between Volvariella volvacea and V. bombycina by PEG-induced protoplast fusion

Interspecific hybridization between Volvariella volvacea and V. bombycina was studied using the protoplast fusion technique. The fusion frequency was found to be in the range of 0.032 to 0.333%. Protoplasts from various hybrids were released and regenerated to determine whether they were heterokaryons. In all regenerated colonies, both parental types could not be recovered at the same time. The nuclear DNA contents of hybrids were compared with their parents, and no diploid (parent 1 genome plus parent 2 genome) was found. Some hybrids revealed novel fragments in mitochondrial rDNA PCR profiles, which indicated that rearrangement of mtDNA could have occurred after fusion. Results from arbitrarily-primed polymerase chain reaction (AP-PCR) fingerprints also revealed that the majority of hybrids were similar to one parental type, but heterologous fragments were found in some hybrids.

Norepinephrine-induced growth and alteration of molecular fingerprints in Escherichia coli O157:H7.

The small intestine is richly innervated with adrenergic neurons that elaborate high levels of monoamines, especially norepinephrine (Meirieu et al., 1986). Previous reports from our laboratory have demonstrated the ability of norepinephrine to increase the in vi­tro growth of gram-negative bacteria (Lyte and Ernst, 1992, 1993). To determine whether norepinephrine-induced alterations in growth are accompanied by bacterial genetic rear­rangements, arbitrarily-primed polymerase chain reaction (AP-PCR) was performed on Escherichia coli 0157:H7. It is generally assumed that the molecular fingerprint of any one bacterial strain cannot be altered. The finding of norepinephrine-induced alterations in the molecular fingerprint would indicate that the neurohormonal environment of the small intestine may play a crucial role in determining subsequent pathogenicity.

Molecular Diagnosis of Helicobacter Pylori

H. pylori infection can be diagnosed with many different tests. If the patient is undergoing endoscopy with gastric biopsies, culture and histology remain the diagnostic methods of choice. Indirect tests include rapid urease tests, urea breath tests, and serology. Molecular methods such as PCR offer marginal improvements when done on biopsy material, but has the advantage of being able to accurately identify H. pylori in areas outside the stomach where cultures usually fail. PCR can detect low numbers of organisms in gastric juice, bile, stool and oral secretions. Because of its high sensitivity it can also be used for epidemiologic investigations of environmental sources. However, the largest role for PCR may be in molecular fingerprinting. Arbitrary Primer PCR (RAPD) on the whole bacterial genome can reliably and accurately distinguish between isolates. PCR-based RFLP analysis can separate isolates based on restriction fragment sizes in a smaller amplified genome segment. REP-PCR can group isolates into clusters that appear to have different clinical expressions. These methods promise to shed new light on the transmission and pathogenicity of H. pylori

A simple method for isolation of PCR fragments from silver-stained polyacrylamide gels by scratching with a fine needle

▼PCR-based DNA and RNA fingerprinting techniques, including arbitrarily primed PCR (AP-PCR) and differential display reverse transcriptase PCR (ddRT-PCR), represent comprehensive genetic screening methods (Ref. 1). Recently, sensitive nonradioactive detection techniques have been introduced for visualization of fingerprints using silver staining of the PCR products after polyacrylamide gel electrophoresis (Ref. 2, 3). Characterization of polymorphic PCR fragments depends on prior isolation of the corresponding bands from stained and dried gels. In most protocols, PCR products of interest have been cut out of the dried gel complete with the paper support, and DNA eluted by boiling in elution buffer (Ref. 4). Reamplification of the eluted PCR fragments has been successful in up to 75% of the experiments. However, additional bands neighbouring the fragment of interest were frequently coamplified. To avoid coamplification of contaminating bands, the whole procedure of cutting, boiling and reamplification was repeated up to five times by some protocols (Ref. 5). Here we demonstrate simple and rapid reamplification of PCR products after isolation by gently scratching over the silver-stained band using a sterile disposable needle [Microlance 3 (Becton-Dickinson; 0.7 mm)] prewetted with PCR mastermix, without destroying the gel. After scratching the band from the gel, the needle is placed into 20 μl standard PCR reaction mix for 1 min and then discarded. The scratched PCR products are reamplified by 30 PCR cycles (Fig. 1). Using this scratching technique, we are able to reamplify PCR products of interest in more than 90% of the experiments. In addition to the simplicity and speed of

Three Predominant Clones Identified Within Penicillin-Resistant South African Isolates of Streptococcus pneumoniae

The worldwide spread of antibiotic-resistant pneumococcal clones is cause for concern. In South Africa, penicillin-resistant pneumococci are mostly associated with serogroups 6 and 19, therefore, we have analyzed the clonal relationships between resistant isolates of these two serogroups. DNA fingerprinting of penicillin-binding protein genes and arbitrarily-primed PCR, have identified two penicillin-resistant pneumococcal clones of serotype 19A and serotype 6B, and a third multi-resistant serotype 19A clone, of which all three clones are predominant within South Africa's penicillin-resistant pneumococcal population. These clones are represented by strains isolated from hospitals that are widely separated in South Africa. Further studies are required to establish whether these clones have expanded in the rest of Africa or spread to other continents.

Spread of the Spanish multi-resistant serotype 23F clone of Streptococcus pneumoniae to Seoul, Korea.

Multi-resistant serotype 23F pneumococcal strains from an outbreak in a day-care centre in South Africa and strains from sporadic cases in Korea were analysed for their relatedness, by means of arbitrarily-primed PCR (AP-PCR), repetitive extragenic palindromic PCR (rep-PCR) and penicillin-binding protein (PBP) gene profiles. The South African strains previously shown to be identical to the 23F Spanish clone had identical AP-PCR and rep-PCR patterns to 13 Korean isolates. PBP gene analysis of strains from this cluster showed identical fingerprints, suggesting clonality. Isolates, identical genotypically to the 23F clone appeared to have acquired the type 19F and 14 capsular serotypes, respectively, a result that suggests horizontal transfer of capsular biosynthetic genes. These same techniques revealed three serotype 9V isolates not closely related to the 23F clone but which harbour PBP genes identical to the 23F clone. The data presented suggests that a clone of S. pneumoniae serotype 23F related to isolates from Spain and South Africa has become disseminated in Korea.

Identification and cloning of differentially expressed genes by DDRT-PCR.

The invention of polymerase chain reaction (PCR) and its application to amplification of reverse transcribed cDNA copies of mRNA has opened new possibilities for the development of techniques for identification of changes in gene expression patterns. Two very similar variants of the same strategy were published in 1992 that were named mRNA differential display () and RNA arbitrary primed PCR (). In contrast to the standard RT-PCR technique, where individual mRNAs are detected in a qualitative and sometimes also quantitative manner using gene-specific primers, in these new techniques a set of short primers with arbitrary sequences is used to generate amplified cDNA fragments from a large number of mRNAs used before for DNA fingerprinting (). Basically, the identification of every individual mRNA species expressed should be possible provided a sufficiently large number of primer pairs is used (). The method of differential display (DDRT-PCR) has already been described in detail in a number of reviews (, , , , , ). Therefore, we give only a very brief description of the principle. First, RNA is prepared from the different cell types of interest. Second, RNA is reverse transcribed in 12 (,), 4 (), or 3 () fractions using anchored oligo-dT primers (e.g., T11VV in Fig. 1) to generate single-stranded cDNA. Third, every cDNA fraction is amplified in a number of independent PCR reactions using various 10-mer upstream primers. Fourth, products of individual PCR reactions are separated on standard sequencing gels (,) or on nondenaturing polyacrylamide gels (,,) and visualized, normally by autoradiography. Fifth, fragments differing in their intensities between the parallel samples from different cells are cut out of the gel, reamplified, cloned into a suitable vector, and sequenced. Sixth, differential expression of the genes corresponding to the identified sequence tags is confirmed by an independent analysis. We have previously recommended nuclear run-on analysis (,). However, this method is not easy to apply to small amounts of cells and tissues. Therefore, we have recently optimized conditions for RNase protection analysis that we will describe here. Finally, if cloning of full-length cDNAs is the ultimate goal, simultaneous isolation of a larger number of cDNA clones from a suitable library can be carried out using a biotinylated fragment mix as probes and streptavidin-coated magnetic beads to purify specifically hybridizing cDNA clones. The cloning step is not an essential part of DDRT-PCR and it is usually omitted if only diagnosis of gene expression patterns is required. Open image in new window Fig. 1. Scheme of DDRT-PCR.

Likelihood of transmitting Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis in families with periodontitis

This study examined the frequency of spouse-to-spouse and parent-child transmission of the periodontal pathogens Actinobacillus actinomycetemcomitans (124 subjects in 47 families) and Porphyromonas gingivalis (78 subjects in 31 families). The two test organisms were recovered from subgingival and tongue surface specimens using established microbiological techniques. Arbitrarily primed polymerase chain reaction (AP-PCR) was used to genetically characterize isolates of the test species. The probability of isolating identical AP-PCR types of A. actinomycetemcomitans and P. gingivalis in family members by chance was estimated from the AP-PCR genotype distribution of the two species among unrelated individuals. A probability of 5% or less for occurrence by chance alone suggests intra-familial transmission. With a bacterium-positive spouse, A. actinomycetemcomitans revealed inter-spousal transmission in 4/11 (36%) married couples and P. gingivalis in 2/10 (20%) married couples. Parent-child transmission of A. actinomycetemcomitans took place in 6/19 (32%) families. P. gingivalis was not transmitted from parent to child in any of the study families. The intra-familial transmission of A. actinomycetemcomitans and P. gingivalis may in part explain a familial pattern of periodontitis and may have important prophylactic and treatment implications.

Arbitrary Primer PCR of Dog DNA With Estimates of Average Heterozygosity

Arbitrary primer polymerase chain reaction (AP-PCR) detected anonymous DNA polymorphisms between individual representatives of seven breeds of dogs, between members of a single breed, and between members of a single family of dogs. Short arbitrary sequence PCR primers and promoter/protein motif sequence primers typically amplified 15-30 bands ranging from 200-2000 bp in length. The average heterozygosity of 10 line bred Belgian sheepdogs (H = 0.024 +/- 0.011, mean +/- 95% CI) was significantly lower than that of a panel of unrelated beagles (H = 0.057 +/- 0.018, mean +/- 95% CI, P < .01). The average heterozygosity of a seven-breed panel was intermediate between the two (H = 0.043 +/- 0.011, mean +/- 95% CI) and not significantly different than either panel. One motif sequence PCR primer pair yielded products that strongly differentiated between Belgian sheepdogs, beagles, and members of five other breeds. AP-PCR could be used to generate DNA fingerprints that distinguish between dog breeds, as well as to provide markers in this sparsely mapped genome.

Changes in major populations of methicillin-resistant Staphylococcus aureus in Belgium

A total of 102 epidemic methicillin-resistant Staphylococcus aureus (MRSA) isolates collected in 13 Belgian hospitals during two periods (1981–1985 and 1991–1992) were tested for phage-type, for the presence of aminoglycoside-modifying enzymes (AME), and examined by arbitrarily primed polymerase chain reaction (AP-PCR). All isolates, but five, belonged to a few distinct phage-types of group III. Most isolates expressed a combination of AAC(6′)-APH(2″) with APH(3′)III, and ANT(4′,4″) or both. Both phage-typing and AME suggested a change in the MRSA population between the two periods but the AP-PCR method revealed only slight differences.

Genetic homogeneity of cultivated strains of shiitake ( Lentinula edodes) used in China as revealed by the polymerase chain reaction

Nineteen strains of shiitake (Lentinula edodes) which are used for spawn production for farms throughout mainland China were characterized by three arbitrarily-primed polymerase chain reaction (AP-PCR) profiles seven random amplified polymorphic DNA marker (RAPD) profiles and five restriction patterns (restriction fragment length polymorphism patterns of the PCR-amplified ribosomal DNAs; rDNA-RFLPs). For AP-PCR, 4–14 DNA bands were amplified for a particular strain while 1–9 DNA bands were amplified using RAPD. Among them only three of the strains tested showed different amplification profiles with most of the primers used. The others showed small differences in three or fewer DNA amplification profiles. All strains showed identical rDNA-RFLPs. The study indicates that cultivated strains of shiitake in China are genetically very homogeneous very like cultivated strains of Agaricus bisporus and Volvariella volvacea. However, our collection of L. edodes covers an enormous geographical area (approx. 1700 km N to S, 700 km E to W) and our results demonstrate that the shiitake industry in China depends on an extremely small gene pool. In comparison three wild strains collected from the same ecosystem in Fujian Province (SE China) showed more variable amplification profiles. This emphasises the importance of conservation of wild shiitake strains as a reserve of biodiversity for this important industry.

Intergeneric hybridization between Pleurotus ostreatus and Schizophyllum commune by PEG-induced protoplast fusion

Intergeneric hybridization between Pleurotus ostreatus and Schizophyllum commune was studied using PEG-induced fusion. The fusion of protoplasts from auxotrophic mutant strains resulted in the formation of fusion hybrids in the frequencies of 3.6 to 7.3×10(-5). Most of these fusion hybrids were monokaryotic and sterile and no heterokaryosis occurred. Most fusants showed a significantly higher nuclear DNA content when compared to parental strains and no diploids (parent 1 genome plus parent 2 genome) were found. Some fusion hybrids revealed both parental fragments in nuclear and mitochondrial rDNA PCR profiles. AP-PCR (Arbitrarily-primed Polymerase Chain Reaction) fingerprints also indicated that most of the fusion products were recombinant hybrids.

Monitoring Persistence of Coagulase-Negative Staphylococci in a Hematology Department Using Phenotypic and Genotypic Strategies

To determine persistence of coagulase-negative staphylococci (CNS) on a hematology-oncology ward and to determine the value of phenotypic and genotypic procedures for establishing clonality among CNS isolates. Strains of CNS isolated from bacteremic patients (n = 139) were typed by biochemical reactivity, antibiotic susceptibility, DNA macrorestriction analysis by pulsed-field gel electrophoresis (PFGE), and arbitrary primed polymerase chain reaction (AP PCR). Coagulase-negative staphylococci were subgrouped in a random collection (n = 20) used for the evaluation of the typing procedures and a collection of 119 CNS isolates from hematologic patients displaying multiple bacteremic episodes. Analysis of the reference collection demonstrated the usefulness of the DNA typing procedures, indicating that AP PCR and PFGE can be used for epidemiologic typing of CNS in a concordant fashion. Certain strains appeared to be permanent colonizers of the hematology ward or ward-related personnel. In individual patients, persistent colonization by a single type was demonstrated. However, a number of patients also experienced bacteremic episodes caused by CNS belonging to different types. We conclude that monitoring of CNS infections on a hematology ward by various genotypic techniques provides insight into nosocomial epidemiology and elucidates the complexity of the infections taking place. DNA typing is preferred over phenotypic procedures and can identify persistent CNS strains in a given location.

Monitoring Persistence of Coagulase-Negative Staphylococci in a Hematology Department Using Phenotypic and Genotypic Strategies

AbstractObjective:To determine persistence of coagulase-negative staphylococci (CNS) on a hematology-oncology ward and to determine the value of phenotypic and genotypic procedures for establishing clonality among CNS isolates.Design:Strains of CNS isolated from bacteremic patients (n=139) were typed by biochemical reactivity, antibiotic susceptibility, DNA macrorestriction analysis by pulsed-field gel electrophoresis (PFGE), and arbitrary primed polymerase chain reaction (AP PCR). Coagulase-negative staphylococci were subgrouped in a random collection (n=20) used for the evaluation of the typing procedures and a collection of 119 CNS isolates from hematologic patients displaying multiple bacteremic episodes.Results:Analysis of the reference collection demonstrated the usefulness of the DNA typing procedures, indicating that AP PCR and PFGE can be used for epidemiologic typing of CNS in a concordant fashion. Certain strains appeared to be permanent colonizers of the hematology ward or ward-related personnel. In individual patients, persistent colonization by a single type was demonstrated. However, a number of patients also experienced bacteremic episodes caused by CNS belonging to different types.Conclusion:We conclude that monitoring of CNS infections on a hematology ward by various genotypic techniques provides insight into nosocomial epidemiology and elucidates the complexity of the infections taking place. DNA typing is preferred over phenotypic procedures and can identify persistent CNS strains in a given location.

An outbreak of pseudobacteraemia caused by Burkholderia pickettii: the critical role of an epidemiological link

A protracted outbreak of pseudobacteraemia caused by Burkholderia pickettii is reported. Initial investigation did not reveal the source of the outbreak. Subsequently, careful examination of patient records provided an insight to the probable source, which was further confirmed by a case control study. It was found that 48% of cases had either hepatobiliary diseases or haematological malignancies, whereas only 15% of the controls had similar problems. Furthermore, a coagulation study was performed on all of the cases on the day the blood culture was taken, but was performed on only 17% of the controls. With the establishment of this epidemiological link, a breakdown in aseptic blood culture technique was suspected. B. pickettii was isolated from citrated bottles used in the wards and from the citrate solution stocked in the haematology laboratory. The clonality of these strains was established by the pulsed-field gel electrophoresis (PFGE) and the arbitrary primed-polymerase chain reaction (AP-PCR). The phlebotomists were notified of the findings of the outbreak investigation, and the importance of strict adherence to the aseptic blood culture technique was re-emphasized.

Sequence analysis of PG10.2, a gene expressed in the pineal gland and the outer nuclear layer of the retina

A cDNA (PG10.2) was cloned from rat using RNA fingerprinting by arbitrarily primed PCR (AP-PCR). Initially, a 145-bp DNA fragment was isolated from a pineal-specific band revealed on a sequencing gel. Riboprobes generated from the DNA fragment were used for hybridization histochemical and Northern analyses. Both techniques indicate a gene (8 kb mRNA) expressed only in the pineal gland and the outer nuclear layer of the retina. Two 4-kb cDNA fragments generated by 5′-RACE (rapid amplification of cDNA ends) and 3′-RACE were obtained using a long template PCR-based RACE technique. DNA sequencing revealed a single long open reading frame (ORF) encoding a predicted protein of 1239 amino acids, containing multiple motifs: a typical signal sequence of 20 amino acids at the NH 2-terminus, a long extracellular domain with multiple potential glycosylation sites, a 31-amino-acid transmembrane domain near the COOH-terminus and a 109-amino-acid cytoplasmic tail. There are also two domains similar, but not identical, to EGF-like (epidermal growth factor-like) domains located just upstream of the transmembrane domain. These features suggest that this gene may encode a glycoprotein involved in cell adhesion, growth, and differentiation of pineal and retinal photoreceptor cells.