Microsatellite mutations during the polymerase chain reaction: mean field approximations and their applications

We develop a novel mathematical model for microsatellite mutations during polymerase chain reaction (PCR). Based on the model, we study the first- and second-order moments of the number of repeat units in a randomly chosen molecule after n PCR cycles and their corresponding mean field approximations. We give upper bounds for the approximation errors and show that the approximation errors are small when the mutation rate is low. Based on the theoretical results, we develop a moment estimation method to estimate the mutation rate per-repeat-unit per PCR cycle and the probability of expansion when mutations occur. Simulation studies show that the moment estimation method can accurately recover the true mutation rate and probability of expansion. Finally, the method is applied to experimental data from single-molecule PCR experiments.     

Detection of Norwalk virus in the UK by the polymerase chain reaction

Abstract We have developed a polymerase chain reaction for the detection of Norwalk virus using the published sequence of the virus RNA dependent RNA polymerase gene and have used this to clone and sequence this region of a virus from a UK outbreak. We have applied this method to a panel of UK Norwalk-like viruses using both Tet-z and Taq DNA polymerases and found that amplification produces a multiplicity of bands from stool samples. However, combination with Southern blotting. Taq polymerase amplification detected virus in 13 of a panel of 30 clinical samples known to contain these viruses and also detected astroviruses in a mixed infection. Amplification using Tet-z DNA polymerase was less efficient (6/30) and detected predominantly viruses typed as UK type 2 by solid phase immune electron microscopy.     

Detection of Chlamydia psittaci using DNA probes and the polymerase chain reaction

Fewer than 10(5) elementary bodies of Chlamydia psittaci could be detected by using DNA hybridisation with a plasmid probe specific for avian chlamydial strains. PCR amplification of chlamydial DNA using primers specific for conserved regions of the major outer membrane protein gene enabled the detection of fewer than 10 elementary bodies. DNA could be amplified from 22 of the 24 chlamydial strains tested including avian, feline, ovine, caprine, koala and lymphogranuloma venereum strains.     

Fluorescence-based temperature control for polymerase chain reaction

The ability to accurately monitor solution temperature is important for the polymerase chain reaction (PCR). Robust amplification during PCR is contingent on the solution reaching denaturation and annealing temperatures. By correlating temperature to the fluorescence of a passive dye, noninvasive monitoring of solution temperatures is possible. The temperature sensitivity of 22 fluorescent dyes was assessed. Emission spectra were monitored and the change in fluorescence between 45 and 95 °C was quantified. Seven dyes decreased in intensity as the temperature increased, and 15 were variable depending on the excitation wavelength. Sulforhodamine B (monosodium salt) exhibited a fold change in fluorescence of 2.85. Faster PCR minimizes cycling times and improves turnaround time, throughput, and specificity. If temperature measurements are accurate, no holding period is required even at rapid speeds. A custom instrument using fluorescence-based temperature monitoring with dynamic feedback control for temperature cycling amplified a fragment surrounding rs917118 from genomic DNA in 3 min and 45 s using 35 cycles, allowing subsequent genotyping by high-resolution melting analysis. Gold-standard thermocouple readings and fluorescence-based temperature differences were 0.29 ± 0.17 and 0.96 ± 0.26 °C at annealing and denaturation, respectively. This new method for temperature cycling may allow faster speeds for PCR than currently considered possible.     

Detection by multiplex polymerase chain reaction and typing of Chlamydia trachomatis isolates

Abstract The multiplex polymerase chain reaction (PCR) was applied for the detection of the Chlamydia trachomatis chromosome and plasmid. The multiplex PCR demonstrated a sensitivity of 0.8 fg of chlamydial DNA, corresponding to the detection of about 5 copies of the plasmid. Analysis of 195 genital specimens collected randomly from a female population, showed that the multiplex PCR is more sensitive and rapid than culturing for detecting Chlamydia trachomatis . Moreover, sequencing of the II variable domain of the ompl gene, directly from DNA of the clinical specimens, appears to be a simple and rapid method for determining serovar isolates.     

Identification of the anginosus group within the genus Streptococcus using polymerase chain reaction

The aim of this study was to establish an identification method for the anginosus group within the genus Streptococcus by polymerase chain reaction (PCR). Using a primer pair based on the group-specific sequences of penicillin-binding protein 2B (pbp2b) gene, a 275-bp fragment was amplified from each species in the group but no size-matched products were obtained in other streptococci. Further identification in the species or subspecies level was possible by a multiplex PCR with primers for the 16S ribosomal RNA gene of Streptococcus anginosus, the hyaluronate lyase genes both of Streptococcus intermedius and Streptococcus constellatus subsp. constellatus, and the intermedilysin (ily) gene of S. intermedius. In the case ofStreptococcus constellatus subsp. pharyngis, the amplified fragment from the S. intermedius-type hyaluronate lyase gene was obtained, while that from the ily gene was not. These results also indicate that two different hyaluronate lyase genes are distributed among the anginosus group.     

Development of a polymerase chain reaction assay for Mycoplasma salivarium by using the nucleotide sequence within aminopeptidase My gene

A polymerase chain reaction assay for a 278-nucleotide DNA fragment within aminopeptidase My gene of Mycoplasma salivarium was developed. The assay amplified M. salivarium DNA, but did not amplify DNAs of other mollicutes, bacteria and mammalian cells. The detection limit of the assay was 10 fg of DNA, approximately equivalent to 10 organisms.     

Direct quantification of polymerase chain reaction fragments using field-amplified sample injection in capillary zone electrophoresis for gene dosage estimation

To assess gene dosages for clinical application, especially for prognostication of cancer, we developed a direct quantification method for polymerase chain reaction products. We report on an application of field amplified sample injection (FASI) to capillary zone electrophoresis which allows the quantification of PCR products without sample preparation. Using an external standard and UV detection for the quantification of DNA, a low coefficient of variation has been obtained. Overall, the described method provides a fast and easy tool for PCR product quantification in clinical laboratories.     

Computing by polymerase chain reaction

A mathematical notation is introduced to represent, at a symbolic level, different mechanisms of DNA recombination, and a 'PCR lemma' is proven by analytically describing the combinatorial properties of the polymerase chain reaction process. This approach led to the discovery of novel techniques, based on a form of PCR which we called cross pairing PCR (briefly XPCR). They were mathematically analyzed and already experimentally proven in different contexts, such as DNA extraction and recombination. Thus, a mathematical analysis of standard methodologies may highlight novel mechanisms of DNA recombination and this can provide new technologies for DNA manipulation.     

Combined influence of magnesium concentration and polymerase chain reaction specificity enhancers

Abstract The importance of an optimal magnesium concentration (6.5 mM), as well as the inclusion of a specific polymerase chain reaction enhancer, to obtain the correct fragment after amplification was examined during this study. In contrast to tetramethylammonium chloride, dimethyl sulphoxide did not enhance the specificity of amplification at different magnesium concentrations, but rather altered the specificity, resulting in the amplification of a larger fragment.     

Recombinant DNA probes and polymerase chain reaction for detection of Mycoplasma gallisepticum strains

Abstract Two recombinant DNA clones, pMG286.2 and pMG301.1, were isolated from the partial genomic library of Mycoplasma gallisepticum strain S6. Recombinant M. gallisepticum specific fragments were used as probes in Southern hybridisation with 10 M. gallisepticum strains whose DNA was digested by Eco RI, Hin dIII, Bgl II, Rsa I and Bam HI. The 1.5 kb fragment pMG301.1 did not show polymorphism in hybridisation patterns with M. gallisepticum strains, while the 3.5 kb fragment pMG286.2 enabled differentiation of M. gallisepticum strains into clusters. The DNA sequence of pMG301.1 was used to design a pair of 27-mer oligonucleotides flanking a 1.3 kb genomic region. These two primers directed specific in vitro amplification of all M. gallisepticum strains assayed giving an expected 1.3 kb product. Digestion of polymerase chain reaction products by Dde I enabled simple differentiation between clusters of M. gallisepticum strains and may be useful for improved epizootiological studies of M. gallisepticum infections in poultry.     

Detection of Mycobacterium leprae in nasal mucosa biopsies by the polymerase chain reaction

Several discoveries about leprosy indicate that Mycobacterium leprae transmission mainly occurs by inhalation, and the nose is major port of entry and exit. The present study evaluated the clinical application of PCR for detection of M. leprae DNA in nasal mucosa biopsies in untreated leprosy patients (52) and their contacts (99) from the State Reference Center in Sanitary Dermatology and Leprosy, Uberlandia, MG, Brazil. PCR detection of a 372-base pair DNA fragment from M. leprae was accomplished in 36 (69.2%) patients, from which 34 (91.9%) of them were multibacillaries. Furthermore, PCR was positive in 3 (16.7%) of 18 slit-skin smear negative, 4 (25.0%) of 16 skin lesion BI negative, 8 (33.3%) of 24 nasal mucosa BI negative patients, and 10 of 99 contacts (10.1%). The presence of bacilli in 10.1% of the contacts may potentially reflect an occult leprosy, and these patients must be accompanied, followed by a chemoprophylaxy treatment. Considering all PCR results against clinical and BI classification of patients and controls, we have found a sensitivity of 69.2%, a specificity of 89.9%, and an accuracy of 82.8%. It has been demonstrated here through PCR of nasal biopsies that the bacillus invades the mucosa, passing through the nasal inferior turbinate to reach peripheral blood. Therefore, the molecular investigation of invasive nasal biopsies by PCR tests has proven to be useful in defining patients of higher risk of transmission and risk-group contacts, which is an important step to reach the World Health Organization objective towards the elimination of leprosy as a public health problem.     

Detection of urovirulence factors in Escherichia coli by multiplex polymerase chain reaction

Abstract Primers to amplify the genes encoding the virulence factors of uropathogenic Escherichia coli , such as pilus associated with pyelonephritis ( pap ), haemolysin ( hly ), aerobactin ( aer ) and cytotoxic necrotizing factor 1 ( cnf 1) genes, were designed. The above primers along with previously reported primers for S fimbriae ( sfa ) and afimbrial adhesin I ( afaI ) genes were combined to develop a multiplex polymerase chain reaction (PCR) for detection of the respective virulence factors and for the identification of uropathogenic E. coli . The multiplex PCR to detect pap, sfa, afa I, hly, aer and cnf 1 genes was highly specific and the sensitivity was found to be about 5 × 10³ colony forming units of E. coli per ml. A total of 194 E. coli strains isolated from patients with simple acute cystitis were examined by the multiplex PCR and the results were in complete agreement with that obtained by DNA colony hybridization test. The multiplex PCR developed was, therefore, concluded to be a useful, sensitive and rapid assay system to identify uropathogenic E. coli .     

Differentiation of Naegleria fowleri and other naegleriae by polymerase chain reaction and hybridization methods

Abstract In order to detect and identify Naegleria fowleri strains an assay based on the Polymerase Chain Reaction (PCR) was evaluated. The amplified DNA fragments were detected by gel electrophoresis and ethidium bromide staining, followed by Southern blot hybridization with an internal digoxigenin-labeled probe. A set of primers (B1B2) which flank a 678-bp region within a virulence-associated gene, allowed for the highly specific identification of N. fowleri , since Naegleriae ( N. lovaniensis, N. australiensis, N. gruberi, N. andersoni and N. jadini ) and other Protozoa did not react. These primers did not detect amplification products from various organisms: Gram-positive bacteria, algae, y, yeasts and human DNA. Whereas a second set of primers (A1A2), which flank a different sequence, detected various Naegleriae and Acanthamoebae strains. After 40 amplification cycles, the limit of detection was a single cell (cyst or trophozoite). Thus, the PCR appears to be a rapid and powerful tool for identification and detection of N. fowleri .     

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.     

Evaluation of the Applied Biosystems automated Taqman polymerase chain reaction system for the detection of meningococcal DNA

In a period where the proportion of culture confirmed cases in the UK has been steadily declining, diagnosis by PCR has been used to increase the number of confirmed cases and provide additional epidemiological data. This report presents a comparative evaluation of the fluorogenic probe-based 5' exonuclease assay (Taqman) using the Perkin-Elmer Applied Biosystems automated sequence detection system 7700 with previously reported polymerase chain reaction enzyme-linked immunosorbent (PCR ELISA) assays for the detection of meningococcal DNA in CSF, plasma and serum samples. Taqman assays developed were based on the detection of a meningococcal capsular transfer gene (ctrA), the insertion sequence IS1106 and the sialytransferase gene (siaD) for serogroup B and C determination and compared with similar assays in a PCR ELISA format. The Taqman ctrA assay was specific for Neisseria meningitidis, however the IS1106 assay gave false positive reactions with a number of non-meningococcal isolates. Sensitivity of the Taqman ctrA, IS1106 and siaD assays testing samples from culture-confirmed cases were 64, 69 and 50%, respectively, compared with 26, 67 and 43% for the corresponding PCR ELISA assays. Improvements to the DNA extraction procedure has increased the sensitivity to 93 and 91% for the TaqMan ctrA and siaD assays, respectively, compared to culture confirmed cases. Since the introduction of Taqman PCR a 56% increase in laboratory confirmed cases of meningococcal disease has been observed compared to culture only confirmed cases. The developed Taqman assays for the diagnosis of meningococcal disease enables a high throughput, rapid turnaround of samples with considerable reduced risk of contamination.     

The polymerase chain reaction and hepatitis C virus diagnosis

Abstract: In the absence of tissue culture, electron microscopy or assays for viral antigen, the direct detection of hepatitis C virus (HCV) is by necessity dependent upon nucleic acid hybridisation methods. Of the available methods, amplification of HCV cDNA by polymerase chain reaction (PCR) commends itself by virtue of its extreme sensitivity and its consequent ability to detect the very low levels of HCV-RNA that are present in many clinical samples. In this review the development and evolution of PCR techniques for HCV detection are described and a number of clinical applications are considered in detail. The application include diagnosis of acute infection during the seronegative window period prior to the appearance of HCV antibodies, and diagnosis of HCV infection in the immunosuppressed. PCR also enables identification of chronic viraemic carrier state and it permits accurate monitoring of the antiviral effects of drugs such as interferon. Confirmation of the specificity HCV antibody assays and detection of HCV contamination of blood donations and blood products are other important areas in which PCR techniques have proved invaluable. In addition, PCR-based techniques underlie an increasing number of molecular epidemiological and genotyping studies and they are providing insights into the details of HCV cellular tropism and replication. A number of logistic problems and operational difficulties are also discussed. Despite these limitations it is concluded that PCR will continue to make significant contributions to both clinical practice and to our understanding of the basic biology of HCV infection.     

Detection of Nitrosomonas spp. by polymerase chain reaction

Abstract A unique genomic DNA fragment was isolated from Nitrosomonas europaea ATCC 19718. Based on the sequence of this fragment, oligonucleotide primers for polymerase chain reaction amplification were prepared which amplify sequences of 775 and 658 bp. The predicted DNA fragments were both amplified from the genome of N. europaea and a Nitrosomonas spp. isolated from a local oxidation pond. The primers failed to amplify DNA from the genomes of the ammonia oxidiser Nitrosolobous multiformis , the nitrite oxidiser Nitrococcus mobilis as well as from the genomes of other unrelated heterotrophic bacteria. These DNA sequences could be amplified from 0.01 ng of N. europaea genomic DNA or from 100 intact cells, and it was possible to detect Nitrosomonas DNA in a DNA mixture extracted from water samples drawn from a local oxidation pond.     

Rapid identification of Aerococcus viridans using the polymerase chain reaction and an oligonucleotide probe

A polymerase chain reaction/oligonucleotide probe method was developed for the specific identification of the Gram-positive bacterium Aerococcus viridans. Primers for the enzymatic amplification reaction were designed from specific sequences within the 16S rRNA. The method was also highly sensitive and 10 cfu of A. viridans could be detected in 5 h although the reliability of detection was poor in mixed cultures with Escherichia coli.