Comparative Evaluation of Quantitative Polymerase Chain Reaction Methods for Routine Enumeration of Specific Bacterial DNA in Aquatic Samples

Summary Three quantitative polymerase chain reaction (PCR) methods, the internal standard method (IS-PCR), competitive PCR (cPCR) and most probable number-PCR (MPN-PCR), were compared in terms of their ability to quantify specific bacterial DNA in environmental samples. Serially diluted Pseudomonas putida BH, the target bacterium, was inoculated into sterilized potassium phosphate buffer (PPB), river water and activated sludge, total DNA was extracted, and the number of pheB genes carried by P. putida BH in each sample was enumerated. IS-PCR and cPCR could not quantify the pheB gene at low concentrations (1.0 × 10³ copies ml^-1 in all samples and 1.0 × 10⁴ copies ml^--1 in some samples) and tended to give overestimations because of differences in amplification efficiencies between pheB gene and the internal standard/competitor in a reaction tube. Although reproducibility of MPN-PCR was slightly lower than that of the other two methods, MPN-PCR was the most sensitive, enabling us to quantify the pheB gene at 1.0 × 10³ copies ml^--1, and it had a good correlation with the inoculum size of P. putida BH. These results suggest that MPN-PCR is the best suited for routine microbial monitoring in natural environmental samples because of the simple handling, the ease of modification as occasion demands and the wide detection range, especially at low cell densities of the target microbe.     

Identification of specific gene sequences in preimplantation embryos by genomic amplification: detection of a transgene

Endogenous and foreign DNA sequences can be detected in an extremely small number of cells via sequence amplification in vitro. The polymerase chain reaction (PCR) technique applied in multiple cycles allows the amplification of specific short regions of the genome to levels that can be detected by DNA blotting techniques. Cow and mouse blastocysts were analyzed by PCR for the presence of an endogenous singlecopy gene or an integrated foreign gene. The endogenous single-copy gene encoding the beta chain of bovine luteinizing hormone was detectable in cow blastocysts and in purified bovine genomic DNA representing as few as 25 cells. To determine whether exogenous genes (transgenes) can be detected in preimplantation embryos, transgenic male mice hemizygous for the prokaryotic gene encoding neomycin resistance were bred to nontransgenic females, and the resulting blastocysts were analyzed. The neo gene was detected in approximately half of the embryos. The capability to identify specific gene sequences in a limited number of embryonic cells affords investigators the opportunity to study genetics in early development.     

Benzoxazinoid biosynthesis in dicot plants

Benzoxazinoids are common defence compounds of the grasses and are sporadically found in single species of two unrelated orders of the dicots. In the three dicotyledonous species Aphelandra squarrosa, Consolida orientalis and Lamium galeobdolon the main benzoxazinoid aglucon is 2,4-dihydroxy-2H-1,4-benzoxazin-3(4H)-one (DIBOA). While benzoxazinoids in Aphelandra squarrosa are restricted to the root, in Consolida orientalis and Lamium galeobdolon DIBOA is found in all above ground organs of the adult plant in concentrations as high as in the seedling of maize. The initial biosynthetic steps in dicots and monocots seem to be identical. Indole is most probably the first specific intermediate that is oxygenated to indolin-2-one by a cytochrome P450 enzyme. C. orientalis has an active indole-3-glycerolphosphate lyase for indole formation that evolved independently from its orthologous function in maize. The properties and evolution of plant indole-3-glycerolphosphate lyases are discussed.     

Rapid detection of Yersinia pestis with multiplex real-time PCR assays using fluorescent hybridisation probes

The objective of the present study was to establish a system of real-time polymerase chain reactions (PCRs) for the specific detection of Yersinia pestis using the LightCycler (LC) instrument. Twenty-five strains of Y. pestis, 94 strains of other Yersinia species and 33 clinically relevant bacteria were investigated. Assays for the 16S rRNA gene target and the plasminogen activator gene (resides on the 9.5-kb plasmid) and for the Y. pestis murine toxin gene and the fraction 1 antigen gene (both on the 100-kb plasmid) were combined for the use in two multiplex assays including an internal amplification control detecting bacteriophage lambda-DNA. Applying these multiplex assays, Y. pestis was selectively identified; other bacteria yielded no amplification products. The lower limit of detection was approximately 0.1 genome equivalent. Rat or flea DNA had no inhibitory effects on the detection of Y. pestis. The results obtained using the multiplex real-time assays showed 100% accuracy when compared with combinations of conventional PCR assays. We developed and evaluated a highly specific real-time PCR strategy for the detection of Y. pestis, obtaining results within 3 h including DNA preparation.     

Detection of the low-germination-rate resting oospores of Pythium myriotylum from soil by PCR

AIMS: To establish a sensitive and specific polymerase chain reaction (PCR)-based method for detecting Pythium myriotylum in soils. METHODS AND RESULTS: Oospores of P. myriotylum were separated from large soil particles by flotation in sucrose solution. The thick-walled oospores were disrupted by vortex with sea sand and its DNA was extracted by the Cetyl trimethyl Ammonium Bromide (CTAB) method. The recovered DNA was verified by PCR amplification of a 150-bp target sequence of P. myriotylum. Samples of 10 g of soil were assayed; thus, the detection limit by PCR-based method was 10 oospores per gram soil. The method was successfully applied for the detection of P. myriotylum in soils collected in March, prior to planting of ginger crops. CONCLUSIONS: A PCR-based method for detecting P. myriotylum from soil was achieved. SIGNIFICANCE AND IMPACT OF THE STUDY: The PCR method has allowed us to monitor the presence of P. myriotylum in soil prior planting season as a way of reducing or eliminating disease.     

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.     

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.     

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.     

多聚酶链反应技术鉴别肾综合征出血热病毒血清型的初步研究

肾综合征出血热(HFRS)为一组抗原性密切相关的布尼亚科汉坦病毒引起的急性传染病。在我国存在至少两种临床表现、动物宿主及流行特征截然不同的血清型别,即血清Ⅰ型(汉坦型)和血清Ⅱ型(汉城型)。这两型病毒间的血清学定型已有报道。近年来,除啮齿类动物外,从临床病人以及非啮齿类动物体内也分离到了HFRS病毒。同时出现两类型别毒株共存,以及从家鼠体内分离到野鼠型毒株或从野鼠体内分离到家鼠型毒株的复杂情形。为此,准确检定并鉴别不同来源毒株型别,将为深入了解其病原学、流行病学以及制定疫苗生产策略提供重要信息。     

Biotype and macromolecular profiles of cytotoxin-producing strains of Helicobacter pylori from antral gastric mucosa

Biotype, genome, protein and plasmid profile diversity amongst 40 epidemiologically unrelated strains of Helicobacter pylori was studied. Strains were API Zym biotypes II, III and IV but most (87%) were biotype II. Four subsets of strains were defined on a combination of motility (56% positive) and cytotoxin production (44% positive). A close association (P = 0.45) between these two features was observed for 69% of strains. Each strain of H. pylori had a unique DNA type defined by either HaeIII or HindIII total digest patterns and by ribopatterns, except for DNA of the rare strains not cut by these endonucleases. Strain diversity was confirmed by one-dimensional SDS-PAGE electrophoretic protein patterns. No consistent associations between cytotoxin activity and overall ribopattern or band subsets within a ribopattern were detected. Some strains (39%) contained a plasmid but the presence of plasmids was not consistently associated with either cytotoxin activity, biotype, motility or ribopattern. We conclude that the cytotoxin-producing strains of H. pylori were genomically as diverse as the non-cytotoxin producing strains.     

Polymerase chain reaction optimization for amplification of Guanine-Cytosine rich templates using buccal cell DNA

CONTEXT:

Amplification of Guanine-Cytosine (GC) -rich sequences becomes important in screening and diagnosis of certain genetic diseases such as diseases arising due to expansion of GC-rich trinucleotide repeat regions. However, GC-rich sequences in the genome are refractory to standard polymerase chain reaction (PCR) amplification and require a special reaction conditions and/or modified PCR cycle parameters.

AIM:

Optimize a cost effective PCR assay to amplify the GC-rich DNA templates.

SETTINGS AND DESIGN:

Fragile X mental retardation gene (FMR 1) is an ideal candidate for PCR optimization as its GC content is more than 80%. Primers designed to amplify the GC rich 5’ untranslated region of the FMR 1 gene, was selected for the optimization of amplification using DNA extracted from buccal mucosal cells.

MATERIALS AND METHODS:

A simple and rapid protocol was used to extract DNA from buccal cells. PCR optimization was carried out using three methods, (a) substituting a substrate analog 7-deaza-dGTP to dGTP (b) in the presence of a single PCR additive and (c) using a combination of PCR additives. All PCR amplifications were carried out using a low-cost thermostable polymerase.

RESULTS:

Optimum PCR conditions were achieved when a combination of 1M betaine and 5% dimethyl sulfoxide (DMSO) was used.

CONCLUSIONS:

It was possible to amplify the GC rich region of FMR 1 gene with reproducibility in the presence of betaine and DMSO as additives without the use of commercially available kits for DNA extraction and the expensive thermostable polymerases.     

Cloning and partial sequence of transferrin-binding protein 2 of Neisseria meningitidis using a novel method: Twin N-terminal PCR

Abstract The genes encoding transferrin-binding proteins (TBPs) 1 and 2 of Neisseria meningitidis and N. gonorrhoeae were used as model loci in a novel method of cloning (twin N-terminal polymerase chain reaction; TNT-PCR) involving amplification between the 5' ends of two genes. Primers were based on N-terminal amino-acid sequences. A 2.1-kb product amplified from N. meningitidis strain SD (B15 P1.16) was cloned into a plasmid vector and partially sequenced. Translated sequence immediately downstream of the primer at both ends of this product correlated to the additional known N-terminal amino acids of TBP-1 and 2. The protein encoded by the cloned sequence reacted with TBP-2-specific antiserum. The size of products generated in TNT-PCR correlated exactly with the different sized TBP-2 produced by 10 strains of the Neisseria spp. examined, indicating successful cloning of the gene for TBP-2 and showing it to be adjacent to and preceding TBP-1 on the chromosome for both N. meningitidis and N. gonorrhoeae .