Identification of Zygowillopsis californica strains of various origin by polymerase chain reaction with universal primers]

After reevaluation of the taxonomic position of 27 yeast collection strains of different origin by UP-PCR followed by dot-hybridization, only 22 strains were assigned to the biological species Zygowilliopsis californica (Lodder) Kudriavzev. Four strains were identified as Williopsis suaveolens (Kl?cker) Naumov et al. Universal primers L45 and N21 are recommended for identification of the Z. californica yeasts.     

Deletion mutagenesis in M13 by polymerase chain reaction using universal sequencing primers

A simple procedure is described for the efficient deletion of large DNA sequences. The method involves a combination of oligonucleotide-directed mutagenesis in bacteriophage M13 and amplification of the mutagenized product by polymerase chain reaction. In contrast to other protocols employing polymerase chain reaction, synthesis of only one specific primer is required. The efficiency of heteroduplex formation between mutagenic primers directing large deletions and single-stranded template is discussed.     

Amplification fragment length polymorphism in Brucella strains by use of polymerase chain reaction with arbitrary primers.

DNA heterogeneity among members of the genus Brucella was demonstrated with the arbitrarily primed polymerase chain reaction (AP-PCR). Simple, reproducible genomic fingerprints from DNA of 25 different Brucella strains were generated with five arbitrarily chosen primers, alone and in pairs, with the PCR. Reaction conditions were optimized for each primer. Several DNA segments were amplified in each sample with all of the primers. PCR products that are not shared among all strains act as polymorphic markers. Polymorphism was apparent for each primer. The Brucella strains can be distinguished according to the banding patterns of their amplified DNA on agarose gels, and the differences can be diagnostic of specific strains. To determine genetic relatedness among the Brucella strains, similarity coefficients were calculated. Statistical analysis of the similarity coefficients revealed the degrees of relatedness among strains of the genus Brucella.     

Improved single-strand conformation polymorphism analysis by asymmetric polymerase chain reaction with end-labeled primers

An improved form of single-strand conformation polymorphism (SSCP) assay has been developed for the analysis of bovine major histocompatibility complex class II alleles. The method uses asymmetric polymerase chain reaction (PCR) amplification from each of two end-labeled primers to generate individual single-stranded products that are analyzed by electrophoresis in nondenaturing polyacrylamide gels. This technique gives good resolution of labeled single strands derived from 392-bp bovine DRB exon-2 PCR products, without interference from double-stranded products, and enables assignment of SSCP bands to the individual strands of the template DNA. The allelic groupings defined by this method in a panel of test animals were confirmed by independent typing by restriction fragment-length polymorphism.     

Typing of toxic strains of Clostridium difficile using DNA fingerprints generated with arbitrary polymerase chain reaction primers

Clostridium difficile is the causative agent for pseudomembranous colitis in humans. Toxic strains of C. difficile produce two toxins, toxin A and toxin B. A reliable and definitive method of typing the toxic strains of C. difficile is needed since nosocomial cross infection is a primary concern in hospitals and other health care facilities. A method for typing toxic strains of Clostridium difficile using arbitrary polymerase chain reaction (PCR) primers is presented in this study. The C. difficile strains were initially characterized for the toxin A genetic determinant using specific PCR primers which differentiate toxin positive from toxin negative strains. These toxic strains were then PCR typed using six arbitrary primers which generated DNA patterns that were unique for all toxic strains examined. The use of this typing scheme in clinical applications is discussed.     

Apolipoprotein E genotyping using the polymerase chain reaction and allele-specific oligonucleotide primers

A method for apolipoprotein (apo) E genotyping was developed using the polymerase chain reaction (PCR) with allele-specific oligonucleotide primers (ASP). Synthetic oligonucleotides with base-pair mismatches at the 3' terminus were used as primers to amplify the apoE gene in subjects previously phenotyped using isoelectric focusing (IEF). Complementary primer-allele combinations were specifically amplified by PCR, together with a control pair of primers specific to the human prothrombin gene. Identification of genotype by PCR using ASP was consistent with the phenotypes that were determined by IEF for 14 healthy normolipidemic subjects. These results were achieved using DNA isolated from buccal epithelial cells obtained from a mouthwash or DNA extracted from leukocytes. Genotype identification required analysis of the PCR products on an ethidium-stained agarose gel, yielding results 3 h after DNA extraction. In comparison with other current methods, PCR using ASP is suggested as a rapid and simple noninvasive technique for determining population apoE allelic distribution.     

Detection of somatic mosaicism in humans using polymerase chain reaction with random primers

Using the method PCR amplification with random primers, DNA samples from human embryonic organs and tissues were examined. Among 27 oligonucleotide primers tested, 10 primers, producing stable, well-reproducible profiles of amplification products, were chosen for further analysis. With the help of two primers (447 and R45), the differences in RAPD PCR profiles obtained from the tissues of one embryo, were revealed. These differences were associated with the change of mobility, or with the fragment gain/loss in the RAPD profile, and could be caused either by genomic rearrangements, or mutations involving the regions of the DNA-primer pairing. Different epigenetic factors, like methylation, can also play the role in this process.     

Detection of somatic mosaicism in humans using polymerase chain reaction with random primers

Using the method PCR amplification with random primers, DNA samples from human embryonic organs and tissues were examined. Among 27 oligonucleotide primers tested, 10 primers, producing stable, well-reproducible profiles of amplification products, were chosen for further analysis. With the help of two primers (447 and R45), the differences in RAPD PCR profiles obtained from the tissues of one embryo, were revealed. These differences were associated with the change of mobility, or with the fragment gain/loss in the RAPD profile, and could be caused either by genomic rearrangements, or mutations involving the regions of the DNA-primer pairing. Different epigenetic factors, like methylation, can also play the role in this process.     

Polymerase chain reaction with universal primers for studying genomes]

Universal primer ability of generating conservative and variable UP-PCR (universally primed polymerase chain reaction) species-specific patterns was analysed on bacteria to serve as an example. Also, two important properties of the UP-PCR patterns (species/primer DNA hybridization specificity) are characterized.     

Genetic diversity among dairy lactococcal strains investigated by polymerase chain reaction with three arbitrary primers

Randomly amplified polymorphic DNA (RAPD) has been used for the rapid typing of Lactococcus lactis strains isolated from raw milk from the Camembert region of Normandy. It is thought that the diversity and perhaps the area strain specificity due to climatic and geographical factors of such wild-type lactococcal strains could contribute to the flavour differences and specific features detected for the same product in different areas. The patterns from 58 isolates were analysed by UPGMA dendrograms. At a similarity level of 50%, four RAPD clusters were distinguished. Clusters 1 and 2 contained strains of subspecies lactis and cluster 3 contained strains related to the C2 strain which is genetically cremoris but phenotypically lactis. The type strain of cremoris subspecies was significantly differentiated from these strains with primers P2 and P3. Thus, there was a real genetic diversity in pattern, making it possible to detect potential typical RAPD fragments.     

Alumorphs--human DNA polymorphisms detected by polymerase chain reaction using Alu-specific primers

The simultaneous analysis of multiple loci could substantially increase the efficiency of mapping studies. Toward this goal, we used the polymerase chain reaction to amplify multiple DNA fragments originating from dispersed genomic segments that are flanked by Alu repeats. Analysis of different human DNA samples revealed numerous amplification products distinguishable by size, some of which vary between individuals. A family study demonstrated that these polymorphic fragments are inherited in a Mendelian fashion. Because of the ubiquitous distribution of Alu repeats, these markers, called "alumorphs," could be useful for linkage mapping of the human genome. A major advantage of alumorphs is that no prior knowledge of DNA sequence of marker loci is required. This approach may find general application for any genome where interspersed repetitive sequences are found.     

Detection of dairy Leuconostoc strains using the polymerase chain reaction

This paper reports the design of a Leuconostoc -specific oligonucleotide based on 16S rRNA sequence data. When this oligonucleotide was used in a polymerase chain reaction (PCR) in conjunction with an oligonucleotide to a conserved region of the 16S rRNA sequence, a Leuconostoc -specific PCR product of approximately 470 bp was produced. The use of a second oligonucleotide to a conserved region allowed the production of an approximately 350 bp product in all PCRs, acting as a positive control. The PCR procedure described was particularly useful for detecting the presence of Leuconostoc in mixed mesophilic starter cultures. The Leuconostoc -specific oligonucleotide was used also as a specific hybridization probe.     

Differentiation of Trichinella genotypes by polymerase chain reaction using sequence-specific primers.

A method was developed to identify species and genotypes within the genus Trichinella using polymerase chain reaction (PCR) and specific primers. Enzymatic amplification of 2 partially conserved and repetitive genomic DNA sequences that have been shown to be variable in length within the different Trichinella genotypes form the basis of this test. Within these regions of the genome, 4 sets of primers were evaluated from which 2 were chosen for their ability to differentiate among the genotypes under stringent primer annealing conditions while maintaining high yields of amplification product. Differences in the size of PCR products from multiple isolates of each genotype indicate sufficient variation to identify 7 of the 8 parasite groups within this genus. One primer set can differentiate among some genotypes working from a single larva. Identification of Trichinella genotypes will assist in distinguishing between sylvatic and synanthropic life cycles. Such information will be critical in tracing sources of trichinellosis by easily and unambiguously identifying likely host reservoirs and will provide valuable information for instituting methods of control.     

Analysis of borderline-resistant strains of methicillin-resistant Staphylococcus aureus using polymerase chain reaction.

Identification of methicillin-resistant Staphylococcus aureus by drug-susceptibility tests alone poses a serious problem, because a considerable number of clinical S. aureus isolates are borderline resistant to methicillin. To circumvent this problem, we have developed a quick and sensitive method of PCR amplification for the detection of mecA gene, which, coding for PBP2', is the specific genetic element of methicillin-resistant Staphylococcus aureus. This method made it possible to identify MRSA strains in a short time using as few as 30 cells as a starting material for template DNA. Using this method, we found that the strains of borderline methicillin-resistance could be accurately identified. We also found one S. aureus clinical strain, T3, which lacked mecA gene in spite of its resistance to methicillin.     

Comparison of different primers for rapid detection of Vibrio parahaemolyticus using the polymerase chain reaction

AIMS: The aim of this study was to compare different primers for rapid and effective detection of Vibrio parahaemolyticus by polymerase chain reaction (PCR). METHODS AND RESULTS: A total of four pairs of primers, three previously published and one based on a newly developed V. parahaemolyticus metalloprotease (vpm) gene, have been assayed for PCR detection of V. parahaemolyticus. They have been tested for specificity and sensitivity on a total of 101 strains including reference and environment isolates belonging to V. parahaemolyticus and other species in Vibrio. Of the four sets of primers tested, the one designed on the basis of the metalloprotease gene (675 bp) gave optimal results with bacterial strains examined as they only amplified the specific fragment in strains that had been genetically and biochemically assessed as V. parahaemolyticus and the limit of detection was 4 pg of purified target DNA. CONCLUSIONS: The primers designed on the metalloprotease gene gave optimal results for specific, sensitive and rapid detection of V. parahaemolyticus by PCR. SIGNIFICANCE AND IMPACT OF THE STUDY: PCR amplification with the optimal primer set VPM1/VPM2 could facilitate the rapid diagnosis and surveillance of potentially pathogenic strains of V. parahaemolyticus and reduce food-borne illness.     

Leishmania (Viannia) braziliensis: New primers for identification using polymerase chain reaction

The objective of this study was to develop specific primers for Leishmania (Viannia) braziliensis species identification using PCR. The designed primers (LBF1 and LBR1) were evaluated for sensitivity and specificity using various L. (V.) braziliensis serodemes and various Leishmania species and also using Trypanosoma cruzi. A specific fragment of 536 bp was detected from 50 ng of DNA in a crude extract derived from L. (V.) braziliensis. The DNA fragment was not detected when DNA from other Leishmania species or from T. cruzi was used as template in the PCR. Furthermore, when tested with DNA from cutaneous leishmaniasis the designed primers and reaction gave positive results. Taking into consideration that the primers LBF1 and LBR1 could specifically identify L. (V.) braziliensis, they could be considered for use in L. (V.) braziliensis diagnosis and epidemiological studies.     

Cloning of Baeyer-Villiger monooxygenases from Comamonas,Xanthobacter and Rhodococcus using polymerase chain reaction with highly degenerate primers

To clone novel type 1 Baeyer-Villiger monooxygenase (BVMO) genes, we isolated or collected 25 bacterial strains able to grow on alicyclic compounds. Twelve of the bacterial strains yielded polymerase chain reaction (PCR) fragments with highly degenerate primers based on the sequences of known and putative BVMOs. All these fragments were found to encode peptides homologous to published BVMO sequences. The complete BVMO genes and flanking DNA were cloned from a Comamonas, a Xanthobacter and a Rhodococcus strain using the PCR fragments as probes. BVMO genes cloned from the first two strains could be expressed to high levels in Escherichia coli using standard expression vectors, and the recombinants converted cyclopentanone and cyclohexanone to the corresponding lactones. The Rhodococcus BVMO, a putative steroid monooxygenase, could be expressed after modification of the N-terminal sequence. However, recombinants expressing this protein did not show activity towards progesterone. An esterase homologue located directly upstream of the Xanthobacter BVMO gene and a dehydrogenase homologue encoded directly downstream of the Comamonas sp. NCIMB 9872 BVMO gene were also expressed in E. coli and shown to specify lactone hydrolase and cyclohexanol dehydrogenase activity respectively.