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.     

Nested polymerase chain reaction for detection of pathogenic leptospires

Leptospirosis is a widespread zoonosis caused by pathogenic members of the genus Leptospira that has a great impact on human and veterinary public health. Early diagnosis of leptospirosis is important because severe lepto spiral infection can have a fulminant course. The available serological techniques for the diagnosis of leptospirosis have low sensitivity during the early stage of the disease. Efforts are being made to develop simpler, effective, efficient, and inexpensive diagnostic methods. In this work, we first evaluate a polymerase chain reaction (PCR) based method for diagnosis of leptospirosis. Primers were designed to amplify a 264 bp region within the lipL32 gene that is conserved among pathogenic Leptospira and absent in nonpathogenic species. The sensitivity and specificity of the assay were evaluated using 7 saprophytic serovars, 37 pathogenic serovars, and 15 other microorganisms. The method was very specific for pathogenic serovars, however, it lacked sensitivity. To enhance the sensitivity, another primer pair was designed to amplify a 183 bp region within the 264 bp region of the lipL32 gene and was used in a nested PCR assay. This approach was much more sensitive than conventional PCR.     

Examination of two years of community dynamics in an anaerobic bioreactor using fluorescence polymerase chain reaction (PCR) single-strand conformation polymorphism analysis

The structures of the bacterial and archaeal communities in an anaerobic digester were monitored over a 2 year period. The study was performed on a fluidized bed reactor fed with vinasse. The objective was to characterize the population dynamics over a long time period under constant environmental parameters. Total bacterial and archaeal populations were measured independently by fluorescence-based polymerase chain reaction (PCR) single-strand conformation polymorphism (SSCP) analysis using an automated DNA sequencer. With the current level of accuracy, the technique was able to monitor 45 bacterial and seven archaeal 16S rDNA molecules. The community dynamics were compared with molecular inventories of the microbial community based on 16S rDNA sequences done at the beginning of the study. The six archaeal and the 22 most frequent bacterial operational taxonomic units (OTUs) identified were associated with their SSCP peak counterparts. Overall, the data indicated that, throughout the period of the study, rapid significant shifts in the species composition of the bacterial community occurred, whereas the archaeal community remained relatively stable.     

Capillary electrophoresis single-strand conformation polymorphism analysis for monitoring soil bacteria

The ability to effectively monitor a microbial community is necessary to design and implement remediation strategies for contaminated soil. Single-strand conformation polymorphism (SSCP), a technique which separates DNA fragments based on their sequence, was used to analyze amplified 16S rRNA gene fragments of 12 common soil bacteria. Separation was performed using capillary electrophoresis (CE), as opposed to other common gel techniques, to eliminate the need for band analysis on gel matrices. Four different universal bacterial primer sets were used for DNA amplification: 341-534, P11-P13, Er10-Er11, and Er14-Er15 corresponding to the V3, V8, V2, and V4 regions, respectively. The forward strand of each primer was labeled with 6-carboxy fluorescein fluorescent dye. Analyses were performed on the Applied Biosystems 310 genetic analyzer using GeneScan Analysis Software version 3.5. The best results were obtained using primer 341-534, in which 6 of the 12 bacteria could be distinguished. By combining primer sets 341-534 and Er10-Er11, all 12 of the bacteria could be separated, indicating various degrees of polymorphism within the selected primer regions. When performing simultaneous amplification and analysis of all 12 species some preferential amplification occurred, as not all peaks could be observed. However, SSCP profiles obtained for pure bacterial cultures show the potential of CE-SSCP for bacterial community analysis.     

Aeromonas detection and characterization using genus-specific PCR and single-strand conformation polymorphism (SSCP)

Based on sequence alignment, oligonucleotide primers targeting the Aeromonas extracellular lipase gene were developed for PCR detection of member of the genus. A pair of primers designed for conserved regions of the gene amplified a 276?bp sequence in all Aeromonas species and tested strains, but did not have a positive result with other Gram-positive and Gram-negative bacteria, showing high specificity and sensitivity. Selective enrichment in alkaline peptone water, followed by centrifugation, and direct usage of cells suspension as template, detected initial populations of 10?c.f.u.?ml(-1). Single-strand conformation polymorphism analysis of the PCR products allowed the characterization of Aeromonas strains with a high discriminatory power (Simpson's index?=?0.988). The method presented here provides a useful tool for the rapid detection of Aeromonas and the characterization of Aeromonas isolates.     

Rapid molecular identification of Neisseria meningitidis isolates using the polymerase chain reaction followed by single-stranded conformation polymorphism analysis

Typing of Neisseria meningitidis strains is currently performed with conventional and molecular methods. Our objectives were: first, to develop a polymerase chain reaction (PCR) followed by single-stranded conformation polymorphism (SSCP) analysis of the PorA gene (VR1 region) to distinguish N. meningitidis subtypes and second, to evaluate the method for the identification and characterization of N. meningitidis in patient specimens. SSCP analysis of the VR1 region of the PorA1/2 gene from 126 N. meningitidis strains and 29 clinical samples identified seven SSCP types (SP-1 to SP-7); four strains were not typeable by the method. Classification according to the SSCP methods and serosubtype agreed for 122 of the 126 typeable strains (96.8%). For the 24-culture positive clinical samples, serosubtype and SSCP agreed in all cases. Five samples, which were culture-negative but obtained from children during an apparent outbreak of meningococcal disease in a primary school, presented identical SSCP classification for each sample (SP-2). PCR-SSCP is a rapid and cost-effective method for typing N. meningitidis strains that could provide important early information in the surveillance of suspected meningococcal outbreaks, particularly when culture-negative specimens constitutes the main source of material to analyze.     

Molecular karyotyping and aneuploidy detection in Arabidopsis thaliana using quantitative fluorescent polymerase chain reaction

Certain cellular processes are sensitive to changes in gene dosage. Aneuploidy is deleterious because of an imbalance of gene dosage on a chromosomal scale. Identification, classification and characterization of aneuploidy are therefore important for molecular, population and medical genetics and for a deeper understanding of the mechanisms underlying dosage sensitivity. Notwithstanding recent progress in genomic technologies, limited means are available for detecting and classifying changes in chromosome dose. The development of an inexpensive and scalable karyotyping method would allow rapid detection and characterization of both simple and complex aneuploid types. In addition to the problem of karyotyping, genomic and molecular genetic studies of aneuploids and polyploids are complicated by multiple heterozygous combinations possible at loci present in more than two copies. Quantitative scoring of allele genotypes would enable large-scale population genetic experiments in polyploids, and permit genetic analyses on bulked populations in diploid species. Here, we demonstrate that quantitative fluorescent-polymerase chain reaction (QF-PCR) can be used to simultaneously genotype and karyotype aneuploid and polyploid Arabidopsis thaliana. Comparison of QF-PCR with flow cytometric determination of nuclear DNA content indicated near perfect agreement between the methods, but complete karyotype resolution was only possible using QF-PCR. A complex karyotype, determined by QF-PCR, was validated by comparative genomic hybridization to microarrays. Finally, we screened the progeny of tetraploid individuals and found that more than 25% were aneuploid and that our artificially induced tetraploid strain produced fewer aneuploid individuals than a tetraploid strain isolated from nature.     

Automated DNA mutation analysis by single-strand conformation polymorphism using capillary electrophoresis with laser-induced fluorescence detection

Automation is essential for rapid genetic-based mutation analysis in clinical laboratory to screen a large number of DNA samples. We propose in this report an automatic process using Beckman Coulter P/ACE™ capillary electrophoresis (CE) with laser-induced fluorescence (LIF) system to detect a single-point mutation in the codon 12 of human K-ras gene. Polymerase chain reaction (PCR) using a fluorescently labeled reverse primer and a plain forward primer to specifically amplify a selected 50 bp DNA fragment in human K-ras gene. The amplified DNA is placed on the sample tray of the CE system with a pre-programmed step for single-strand conformation polymorphism (SSCP) analysis. Sample injection and denaturation processes are performed online along with separation and real-time data analysis. The concept of automation for rapid DNA mutation analysis using CE-LIF system for SSCP is presented.     

Flow cytometric detection of specific genes in genetically modified bacteria using in situ polymerase chain reaction

Abstract Mycelium of Pleurotus ostreatus var. florida with a decreased growth rate contained seven double-stranded RNA segments and isometrical virus particles with diameters of 24 and 30 nm. Mycelium with a normal growth rate lacked dsRNA. Protoclones from virus-containing mycelium contained one to seven of these dsRNA segments in varying concentrations. The exact correlation between slow growth and the presence of dsRNA molecules could not be established. Infection of virus-free protoplasts with PEG-precipitated virus particles resulted in mycelium that stably maintained the 2.4 kbp dsRNA.     

Rapid detection of multiple nucleopolyhedroviruses using polymerase chain reaction.

A technique using the polymerase chain reaction (PCR) was developed for detection of the nucleopolyhedrovirus (NPV) polyhedrin gene. The amino acid sequences of the polyhedrin gene were compared in twenty-six NPVs. A highly conserved DNA sequence within the coding region of the polyhedrin gene was targeted for amplification. One pair of degenerate PCR primers was designed to produce fragments of about 430 bp. The NPVs detected by this technique were Autographa californica NPV, Bombyx mori NPV, Hyphantria cunea NPV, Spodoptera exigua NPV, S. litura NPV, and Lymantria dispar NPV. This technique would be useful in monitoring the distribution of NPVs and release of the wild type and recombinant NPVs.     

Molecular cloning of human cardiac troponin I using polymerase chain reaction

We have used the polymerase chain reaction (PCR) to synthesise a cDNA encoding part of human cardiac troponin I. Amplification was achieved using fully degenerate sets of oligonucleotides corresponding to conserved regions of amino acid sequence identified in other troponin I isoforms. The cloned PCR fragment was subsequently used to isolate full-length cDNAs from a cardiac cDNA library. We describe the approach, as a general cloning strategy starting from limited amino-acid sequence data and report the cloning, and complete amino acid sequence of human cardiac troponin I. Analysis of human development using these clones demonstrates early expression of this gene in the heart.     

Polymerase chain reaction detection of two novel human N-acetylgalactosamine-6-sulfate sulfatase gene polymorphisms by single-strand conformation polymorphism analysis or by StyI and StuI cleavages

A novel polymorphism (6376 G/T) in intron 7 (I7) of the human PROC gene has been identified by direct DNA sequencing. Restriction analysis with the use of mutagenic primers indicate that the allele frequencies are 0.17 (allele T) and 0.83 (allele G), with a calculated heterozygosity of 28%.     

Species-specific detection of Legionella using polymerase chain reaction and reverse dot-blotting

Abstract Legionella pneumophila and some other Legionella species are capable of causing Legionnaire's disease, a potentially fatal pneumonia. The identification of legionellae by standard laboratory techniques such as culture is difficult and time-consuming. In the present work, the DNA sequence of the 23S-5S spacer region was determined for 43 Legionella isolates, and the sequence information was used to develop a species-specific detection system using PCR and reverse dot-blotting which employs just one PCR amplicon to perform genus- and species-specific detection. L. pneumophila serogroups 1–16 as well as 21 non- pneumophila isolates could be identified and differentiated at the species level using this system.     

Sensitive detection of Mycoplasma pulmonis by using the polymerase chain reaction

Detection of Mycoplasma pulmonis was examined by using the polymerase chain reaction (PCR) for amplifying a specific DNA sequence. In gel electrophoresis which was conducted to detect the amplified products, only 1 pg of M. pulmonis DNA could be detected following 30 cycles of amplification, while no amplified product was detected even from 1 microgram of M. arthritidis or M. neurolyticum DNA. Furthermore, 10 colony-forming units of M. pulmonis could be detected by direct amplification from the mycoplasma suspension. These results suggest the usefulness of the PCR as a highly sensitive, specific, and rapid method for direct detection of M. pulmonis.     

Analysis of specific bacteria from environmental samples using a quantitative polymerase chain reaction

This article describes the use of quantitative PCR for measuring bacterial abundance in environmental samples. The two approaches discussed are: 1) The use of an internal PCR standard constructed to be the same size and have the same sequence as the primary amplification target, but differing from the primary target by 2-3 bases, corresponding to a unique restriction site. This allows the amount of target amplicon to be compared with the internal standard and circumvents the problem of differential amplification efficiencies when using dissimilar targets and standard amplicons. 2) The use of Taqman technology (Applied Biosystems, Foster City, California) with a dual labeled oligonucleotide probe which binds internal to the PCR primers. The detection of Bacteroides is used as an example for both approaches.     

Identification of endomycorrhizal fungi colonizing roots by fluorescent single-strand conformation polymorphism-polymerase chain reaction.

A method to identify arbuscular endomycorrhizal fungi based on the amplification of portions of the nuclear gene coding for the small subunit rRNA is presented. By coupling the sensitivity of the polymerase chain reaction and the specificity afforded by taxon-specific primers, a variety of samples can be analyzed, including small amounts of colonized roots. Family-specific primers as well as generic primers are described and can be used to amplify small subunit rRNA fragments from endomycorrhizal fungi by polymerase chain reaction. The amplified products are then subjected to single-strand conformation polymorphism analysis to detect sequence differences. Among the advantages of this approach is the possibility of directly identifying the fungi inside field-collected roots, without having to rely on the fortuitous presence of spores. This technique should have obvious applications in the study of arbuscular endomycorrhizal fungi populations and allow closer examination of their host specificity.