Comparison of 14 PCR systems for the detection and subtyping of stx genes in Shiga-toxin-producing Escherichia coli

The specificity of 14 polymerase chain reaction (PCR) systems designed for the detection and subtyping of stx genes was tested on a set of Escherichia coli strains with known sequences of stx genes. Systems designed for the detection of genes of the stx1 type did not detect any variant genes of the stx2 type and conversely, no stx2 type-specific systems detected stx1 variant genes. Among five stx2 type-specific systems, none detected the stx2ev gene, and two detected the stx2e gene. Among systems designed for screening genes of the both stx1 and stx2 types with a single primer pair, only one system (the Lin system) was able to detect stx genes in all studied strains. Shiga-toxin-producing E. coli frequently carry more than one stx variant gene. Coamplification of stx genes present in the same strain was demonstrated by restriction of PCR products with endonucleases generating fragments of variant-specific size. The amplification product obtained by the Lin system restricted by Hincll yielded fragments of different size for stx1, stx2, stx2c, stx2e and stx2ev. Thus it was possible to identify different genes carried in a single strain with a simple two-step PCR/endonuclease restriction protocol.     

Detection of Salmonella enteritidis in eggs by the polymerase chain reaction

A polymerase chain reaction (PCR) for the specific detection of the gene sequence, sefA, encoded by all isolates of Salmonella enteritidis, was developed. The PCR could detect as few as four S enteritidis washed bacterial cells but egg contents inhibited the PCR. Eggs spiked with 50 S enteritidis bacterial cells were homogenised, inoculated into buffered peptone water and grown at 37 degrees C for 16 hours, when the PCR was successful. A positive internal control was developed to differentiate between true and false negative PCR results for the detection of S enteritidis. In a limited trial of the egg handling procedures and the PCR, one of 250 chickens' eggs from retail outlets was found to be contaminated with S enteritidis.     

Meta-analysis of the risk of metachronous hernia in infants and children

In French Polynesia, Aedes polynesiensis (Marks) is the vector of the human filarial parasite Wuchereria bancrofti (Cobbold) and dog heartworm, Dirofilaria immitis (Leidy). A multiplex polymerase chain reaction (PCR) assay was designed to screen pools of field-collected Ae. polynesiensis for the presence of both parasites simultaneously using primers specific for each parasite. The sensitivity of detection on purified DNA was 1 and 10 pg, equivalent to 0.1 and 1 L3 larva per pool for W. bancrofti and D. immitis, respectively. Codetection was performed at an hybridization temperature of 58 degrees C to avoid competition between heterologous DNA and primers that was observed at 55 degrees C. In addition, D. immitis was detected by PCR in the blood of infected dogs.     

Efficacy of venlafaxine and placebo during long-term treatment of depression: a pooled analysis of relapse rates

An assay was developed for the specific detection of Salmonella enterica serotype Enteritidis, using a novel application of the polymerase chain reaction (PCR). This PCR assay is based on the mismatch amplification mutation assay, an allele-specific reaction, and can discriminate Enteritidis from all other salmonella. PCR primers were selected to amplify a 351-base pair (bp) DNA fragment from the salmonella plasmid virulence A (spv A) gene of Enteritidis. A single base difference at position 272 is present between the nucleotide sequence of the spvA gene of Enteritidis and other salmonellae. The downstream PCR primer, that encompasses position 272 of the Enteritidis spvA gene, was designed to contain a single base mismatch at the penultimate position, resulting in a 1-base mismatch with Enteritidis and a 2-base mismatch with other salmonellae that harbour the virulence plasmid. The upstream primer was completely homologous with the region immediately 5' to the spvA gene. When these primers were used and the annealing and extension reactions were performed at the same temperature, the PCR assay was specific for Enteritidis; no PCR product was detected for 40 other serotypes and 28 different genera examined. In pure culture, 120 colony forming units (c.f.u.) could be detected; a PCR product was observed from template derived from a 5 h enrichment broth culture of chicken seeded with 1 c.f.u. per gram of Enteritidis. This PCR assay is specific, reproducible, and less time consuming than the standard bacteriological methods used to detect Enteritidis.     

High-dose chemotherapy supported by peripheral blood stem cells to treat intermediate--and high-grade non-Hodgkin''s lymphomas

The presence or absence of the mecA gene, the determinant of resistance to all beta-lactam antibiotics, was examined in clinical isolates of Staphylococcus aureus by multiplex polymerase chain reaction (MPCR). Two pairs of primers were used, which yielded two specific products; a 280-bp nuc- based PCR fragment (amplification product of the nuc gene encoding specific Staphylococcus aureus nuclease) and a 533-bp mecA-based PCR fragment (amplification product of the mecA gene). The MPCR system was designed to be incorporated into the work flow in clinical diagnostic laboratories as a routine analysis.     

A randomized study of the efficacy of the PROPATH Program for patients with Parkinson disease

By polymerase chain reaction (PCR) using a pair of primers specific for Salmonella phoE gene a 365-bp specific gene fragment could be amplified from yolk of infertile eggs and dead-in-shell chicken embryos, and from environmental samples. Out of 45 dead-in-shell embryo samples, 20 (44.4%) were found positive for Salmonella DNA by PCR compared to 11 (24.4%) by bacteria isolation. Salmonella DNA could also be detected from infertile eggs, chicken faeces, floor litter and chick fluff, which incidence was higher than that by bacteria isolation.     

Semi-automated detection of the factor V mutation by allele specific amplification and capillary electrophoresis

Recently a point mutation (G1691A) in the coagulation factor V gene was shown to cause resistance for cleavage by activated protein C. The mutation is associated with an increased thrombotic risk and thus-far the most common genetic cause of thrombophilia. Current techniques to investigate the single base pair mutation at the DNA level use an assay based upon the polymerase chain reaction followed by restriction enzyme digestion or Southern blotting and allele specific probing. The method we describe here consists of a single PCR in which two specially designed allele specific primers and two consensus primers were used in one reaction to distinguish between homozygous normal, heterozygous and homozygous mutant individuals. Amplification products were analysed using Capillary Electrophoresis and on line UV monitoring. The Allele Specific Amplification Protocol and subsequent CE analysis (ASAP-CE) is a convenient, fast, automated and highly reproducible method that can be used in a routine laboratory setting.     

Excess hepatobiliary cancer mortality among munitions workers exposed to dinitrotoluene

This study was conducted to determine the sex of buffalo embryos produced in vitro by amplifying male specific DNA sequences using the polymerase chain reaction (PCR). This method uses three different pairs of bovine Y-chromosome specific primers and a pair of bovine satellite specific primers. Buffalo in vitro fertilized embryos at the 4-cell to blastocyst stage were collected at days 3, 4, 6, and 8 postinsemination, and the sex of each embryo was determined using all three different Y-chromosome specific primers. The bovine satellite sequence specific primers recognize similar sequences in buffalo and are amplified both in males and in females. Similarly, Y-chromosome specific primers amplify the similar Y-chromosome specific sequences in male embryos of buffalo. Upon examining genomic DNA from lymphocytes of adult males and females, and embryos, the results demonstrate the feasibility of embryo sexing in buffaloes. Furthermore, sex determination by PCR was found to be a rapid and accurate method.     

Quantitative analysis of polymerase chain reaction using anisotropy ratio and relative hydrodynamic volume of fluorescence polarization method

The method based on the combination of polymerase chain reaction (PCR) and fluorescence polarization is presented. A targeted DNA was amplified with a 5'-fluorescein labeled primer, using a 256 bp DNA fragment of stx2 gene in Escherichia coli O157:H7 (188-443 bp) as a template. The fluorescence anisotropy of the 5'-fluorescein labeled primer increased upon the polymerization through Taq polymerase. The conversion of primer to PCR product was quantitatively monitored by anisotropy ratio and relative hydrodynamic volume. This system was also applied to the determination of E.coli O157:H7.     

The effect of chemical anti-inhibitors on fibrinolytic enzymes and inhibitors

Based on published gene sequences of bovine viral diarrhoea virus (BVDV) type I and classical swine fever virus (CSFV), genus- and species-specific primers were designed to detect and identify pestivirus cDNA sequences in a nested polymerase chain reaction (PCR). The PCR primers were validated using cDNA synthesized from 146 pestivirus isolates, comprising representatives of all four so far described genotypes (BVDV type I, BVDV type II, CSFV and border disease virus), as well as others of uncertain classification. PCR products of the predicted size were amplified from all viruses with the genus-specific primers. All 53 cattle isolates, including 5 typed antigenically as BVDV type II were amplified by the internal BVDV-specific primers, but not the CSFV-specific primers. The same result was found for other BVDV type I and II viruses isolated from sheep and pigs. Seventy-seven CSF viruses were amplified by their respective internal primers. Available information strongly indicate that 4 CSF viruses also amplified by the BVDV-specific primers had been contaminated with BVDV in cell cultures. Border disease viruses were mostly not detected by the BVDV-specific primers, but were detected weakly by the CSFV-specific primer pair. Using carrier RNA for extraction of viral RNA, the sensitivity of detection of the single and nested PCR was, respectively, 5 and 50 times higher than obtained with a cell culture assay. The RT-PCR also detected BVDV in all of 15 commercial batches of fetal calf serum examined, and verified three earlier diagnoses of CSFV by detecting specific gene sequences in 30 year old frozen archival organ samples.     

Detection of Mycobacterium bovis in milk by polymerase chain reaction

A simple method was developed for DNA extraction of Mycobacterium bovis in milk and further detection of the bacterium by polymerase chain reaction (PCR). Milk previously seeded with M. bovis was used as the starting material. The procedure involved overnight digestion of a milk sample with proteinase K at 56 degrees C and phenol extraction, followed by ethanol precipitation and PCR. The amplification pattern obtained was analyzed with primers BW8-BW9 which amplify a 248 bp in strains of M. bovis. By using the BW8-BW9 primers, 10(3) CFU were detected on silver-stained PAGE gels. The procedure was validated by PCR analysis of milk in tuberculin-positive animals. It is anticipated that this method can be used for routine diagnosis of M. bovis in milk samples.     

Inhibition of PCR amplification by a point mutation downstream of a primer

A T-->C point mutation is shown to specifically inhibit PCR amplification when compared to wild-type controls in exon H of the factor IX gene. Multiple primers of different lengths and locations were designed to examine this phenomenon. The experiments suggest that poor annealing and/or extension from the downstream primer are responsible for the observed inhibition and that the mutation can exert an inhibitory effect upon PCR amplification at a distance of at least 84 bp. The inhibition was not alleviated when amplification conditions such as annealing temperature, time of extension, type of DNA polymerase or concentration of DNA template, primer or DNA polymerase were varied. The inhibitory factor(s) are likely to be contained within the amplified segment itself because neither the use of a previously amplified PCR product as template for nested PCRs nor the restriction enzyme digestion of that previously amplified product relieved the inhibition of PCR amplification in the mutant sample. Computer analyses with the FOLDRNA and FOLDDNA programs did not reveal the mechanism of inhibition. Although dramatic inhibition, as shown here, may be uncommon, more subtle inhibition may be frequent. Documentation of differential amplification caused by a single-base substitution in template sequence has implications for certain commonly used PCR-based methods such as quantitative PCR, differential display and DNA fingerprinting. In addition, heterozygous single-base pair mutations down-stream of a primer may be missed if the PCR is inhibited; alternatively; the mutation may appear to be homozygous if amplification of the mutated allele is selectively enhanced.     

Central GABAA and GABAB receptor modulation of basal and stress-induced plasma interleukin-6 levels in mice

The development of control strategies for loiasis is of crucial importance in endemic areas and depends heavily on the accurate identification of occult-infected individuals. A polymerase chain reaction (PCR) and nested polymerase chain reaction (nested PCR) were developed and based on sequences of the repeat 3 region (15r3) of the gene encoding a Loa loa 15-kD protein. The assays was performed on 20 blood samples from occult-infected subjects and 30 from field-collected amicrofilaremic individuals. The size of the initial PCR product was 396 basepairs (bp). When this initial amplification using primers 15r3(1) and 15r3(2) was carried out for 30 cycles, the PCR products from three of the 20 occult-infected and five of the 30 amicrofilaremic individuals were visualized after electrophoresis by staining the gel with ethidium bromide. Subsequent Southern blotting and hybridization with the specific probe revealed hybridization in 19 of 20 occult-infected and 23 of 30 amicrofilaremic samples but only after two days of exposure of the blot to the x-ray film. When the nested PCR was carried out (product size = 366 bp, primers 15r3(3) and 15r3(4)), 19 of 20 occult-infected and 23 of 30 amicrofilaremic samples that were positive by Southern hybridization of the initial PCR products were strongly positive by staining with ethidium bromide. Qualitative Southern blotting of the nested PCR products using the same probe previously described confirmed the ethidium bromide staining results after a very short exposure time of 4 hr. These results demonstrate that the nested PCR amplification product is specific and that its sensitivity in detecting occult loiasis is 95%. This approach has significant promise for the screening of large human populations for active loiasis without the requirement for blotting and hybridization of the PCR products.     

Direct sequencing of double-stranded PCR products incorporating a chemiluminescent detection procedure

A simple and reliable method is described for direct sequencing of material generated by the polymerase chain reaction (PCR). Sequencing reactions can be performed directly on PCR products without the need for purification of the template by removal of residual deoxyribonucleoside triphosphates or primers. The coupling of a chemiluminescent detection system with the use of the same primers in the initial and sequencing PCR's allows for sequencing of a number of PCR products on the one gel.     

Construction of recombination-deficient strains of Streptococcus gordonii by disruption of the recA gene

Degenerate oligonucleotide primers were used in a polymerase chain reaction (PCR) to amplify a region of the recA sequence of Streptococcus gordonii Challis. The resulting PCR fragment was cloned into the suicide vector pAM6199 and introduced into strain Challis, giving rise to recombination-deficient strains in which the recA gene was specifically inactivated.     

Simultaneous analysis of various mutations on the 21-hydroxylase gene by multi-allele specific amplification and capillary gel electrophoresis

A detailed study is presented on the detection of various known point mutations using polymerase chain reaction (PCR) based multi-allele specific amplification (MASA) in conjunction with capillary gel electrophoresis (CGE) separation. The resulting PCR products, corresponding to the individual mutations, are labeled with ethidium bromide during CGE separation, and detected by laser-induced fluorescence. MASA proved to be a novel, fast and cost-effective method for simultaneous analysis of multiple known mutation sites, employing more than one allele specific primers in a single PCR reaction. It results in coexisting amplification of numerous DNA fragments differing in size, which are subsequently separated by CGE. In the present study, several point mutations were analyzed simultaneously by MASA-CGE on the 21-hydroxylase gene of a patient with congenital adrenal hyperplasia.     

Application of the polymerase chain reaction for the detection of Ehrlichia canis in tissues of dogs

Doxycycline treatment resulted in a carrier status in 3 dogs and complete clearance of Ehrlichia canis in 2 dogs (Iqbal and Rikihisa, 1994). Using specimens obtained during that study applicability of polymerase chain reactions (PCRs) in detecting E. canis DNA in tissue specimens and correlation of PCR results with our previous cell culture isolation results were evaluated. PCRs using a pair of primers specific to E. canis 16SrRNA gene sequence were used to detect DNA of E. canis in tissues of 5 experimentally-infected dogs 2 months after doxycycline treatment. An approximately 600 bp product defined by the specific primers was amplified in blood, kidney, lymph nodes, liver, and/or spleen of 3 dogs from which E. canis was reisolated in cell culture. In contrast, E. canis DNA was not detected in tissue or blood specimens of the 2 dogs from which E. canis was not reisolated after doxycycline treatment or in 2 control uninfected dogs. The findings indicate PCR is effective in detecting E. canis in tissues.