Peter Rådström

Pre-PCR processing - Strategies to generate PCR-compatible samples

Polymerase chain reaction (PCR) is recognized as a rapid, sensitive, and specific molecular diagnostic tool for the analysis of nucleic acids. However, the sensitivity and kinetics of diagnostic PCR may be dramatically reduced when applied directly to biological samples, such as blood and feces, owing to PCR-inhibitory components. As a result, pre-PCR processing procedures have been developed to remove or reduce the effects of PCR inhibitors. Pre-PCR processing comprises all steps prior to the detection of PCR products, that is, sampling, sample preparation, and deoxyribonucleic acid (DNA) amplification. The aim of pre-PCR processing is to convert a complex biological sample with its target nucleic acids/cells into PCR-amplifiable samples by combining sample preparation and amplification conditions. Several different pre-PCR processing strategies are used: (1) optimization of the DNA amplification conditions by the use of alternative DNA polymerases and/or amplification facilitators, (2) optimization of the sample preparation method, (3) optimization of the sampling method, and (4) combinations of the different strategies. This review describes different pre-PCR processing strategies to circumvent PCR inhibition to allow accurate and precise DNA amplification.

Standardization of diagnostic PCR for the detection of foodborne pathogens.

In vitro amplification of nucleic acids using the polymerase chain reaction (PCR) has become, since its discovery in the 1980s, a powerful diagnostic tool for the analysis of microbial infections as well as for the analysis of microorganisms in food samples. However, despite its potential, PCR has neither gained wide acceptance in routine diagnostics nor been widely incorporated in standardized methods. Lack of validation and standard protocols, as well as variable quality of reagents and equipment, influence the efficient dissemination of PCR methodology from expert research laboratories to end-user laboratories. Moreover, the food industry understandably requires and expects officially approved standards. Recognizing this, in 1999, the European Commission approved the research project, FOOD-PCR (http://www.PCR.dk), which aims to validate and standardize the use of diagnostic PCR for the detection of pathogenic bacteria in foods. The present review focuses on the harmonization procedure and standardization criteria for detection of foodborne pathogens by PCR. The progress of standardization so far and future perspectives of diagnostic PCR are discussed.

Site-specific recombination promotes linkage between trimethoprim- and sulfonamide resistance genes. Sequence characterization of dhfrV and sulI and a recombination active locus of Tn21

SummaryA new gene for trimethoprim resistance, dhfrV, found in several plasmid isolates with different characteristics, was sequenced and found to correspond to a peptide of 157 amino acids showing 75% similarity with the previously characterized, drug resistant dihydrofolate reductase of type I. The sequenced surroundings of dhfrV in plasmid pLMO20, were found to be almost identical with genetic areas surrounding resistance genes in transposon Tn21 and in R plasmid R388. The trimethoprim resistance genes of pLMO20 and R388 and the spectinomycin resistance gene of Tn21 could be regarded as having been inserted, by recombination, into an evolutionary older structure containg the sulfonamide resistance gene, sulI. The latter gene was sequenced and found to correspond to a peptide of 279 amino acids and with a molecular weight of 30126 daltons. The inserted genes were found to be governed by a promoter situated in the highly conserved structure and also controlling expression of sulI. The insertion points of the different resistance genes were precisely defined, and at the 3′ ends of the inserted genes inverted repeats allowing the formation of stem and loop structures were found. Similar structures were found at the 3′ ends of the antibiotic resistance genes in Tn7, which could indicate similar recombination mechanisms to be effective in the evolutionary construction of all these different resistance elements.

Removal of PCR inhibitors from human faecal samples through the use of an aqueous two-phase system for sample preparation prior to PCR

Abstract A sample preparation method based on a two-phase aqueous polymer system, composed of 8% (w/w) polyethylene glycol 4000 and 11% (w/w) dextran 40, was employed to remove PCR-inhibitory substances from human faeces prior to PCR. The majority of the PCR-inhibitory substances, including bile salts, were shown to be distributed in the polyethylene glycol-rich top phase, whereas target bacteria were detected by PCR in the dextran-rich bottom phase. The efficiency of the aqueous two-phase system in removing PCR-inhibitory substances from faeces was evaluated with a standardised PCR assay containing different concentrations of pure Helicobacter pylori DNA. The detection level for a pure water solution of DNA was in the range 10 −12 to 10 −13 g DNA per reaction tube. Untreated faecal homogenate totally inhibited PCR even after 1000 times dilution in water. A positive PCR result was obtained when 10 −6 g H. pylori DNA was present in a 50 μl reaction mixture containing 10 μl homogenised faeces, which had been briefly centrifuged, boiled and diluted 100 times in water. After extraction of an undiluted heat-treated homogenate in the aqueous two-phase system, the detection level was lowered to the range 10 −10 to 10 −11 g DNA. When the aqueous two-phase system was evaluated on five faecal samples the detection level was decreased by three to five orders of magnitude. Finally, the aqueous two-phase system was applied to faecal samples inoculated with H. pylori cells. The sensitivity of the PCR assay after extraction was found to be 40 times above the detection level of H. pylori in a pure water solution. The target bacteria were detected directly by PCR in the dextran-rich bottom phase. Furthermore, most of the H. pylori cells were shown to be present in the dextran-rich bottom phase.

Sample preparation methods in PCR-based detection of food pathogens

Abstract Since its introduction in the mid-1980s, polymerase chain reaction (PCR) technology has been recognized as a promising method for the examination of microbes in food. The PCR represents a rapid method with both high sensitivity and specificity for the immediate detection of pathogenic microorganisms. Although this technique can be extremely effective with pure microbial cultures, the sensitivity may be reduced dramatically when it is applied directly to food samples. One important reason for this is that the complex composition of food matrices can inhibit the PCR. This review describes sample preparation methods that are used to facilitate PCR detection either by separating the microorganisms from the PCR inhibitors and/or by concentrating the microorganisms to detectable concentrations.

Biotechnical use of polymerase chain reaction for microbiological analysis of biological samples.

Since its introduction in the mid-80s, polymerase chain reaction (PCR) technology has been recognised as a rapid, sensitive and specific molecular diagnostic tool for the analysis of micro-organisms in clinical, environmental and food samples. Although this technique can be extremely effective with pure solutions of nucleic acids, its sensitivity may be reduced dramatically when applied directly to biological samples. This review describes PCR technology as a microbial detection method, PCR inhibitors in biological samples and various sample preparation techniques that can be used to facilitate PCR detection, by either separating the micro-organisms from PCR inhibitors and/or by concentrating the micro-organisms to detectable concentrations. Parts of this review are updated and based on a doctoral thesis by Lantz [1] and on a review discussing methods to overcome PCR inhibition in foods [2].

Detection of pathogenic Yersinia enterocolitica in enrichment media and pork by a multiplex PCR: a study of sample preparation and PCR-inhibitory components

A multiplex PCR assay including sample preparation was developed to detect viable pathogenic strains of Yersinia enterocolitica in PCR-inhibitory samples, such as pork and enrichment media. The method developed was used to simultaneously detect the plasmid-borne virulence gene yadA and a Yersinia-specific region of the 16S rRNA gene. According to an auto-agglutination test for virulence-plasmid-bearing strains of Y. enterocolitica, all potential pathogenic strains tested were detected by the assay. A DNA extraction procedure, an aqueous two-phase system composed of polyethylene glycol 4000 and dextran 40 and a buoyant density centrifugation method, based on Percoll, were compared with regard to their efficiency in separating Yersinia enterocolitica from PCR inhibitors originating from enrichment media and pork. Using the density gradient centrifugation method resulted in a detection level of 4.0 x 10(2) CFU Y. enterocolitica per ml enrichment media. To ensure detection of viable bacteria a short enrichment step was included in the sample preparation together with the density gradient centrifugation. When this sample treatment method was evaluated with a selective enrichment medium together with a background flora inoculated with approximately 1.0 x 10(1) CFU per ml of Y. enterocolitica and incubated at 25 degrees C, a positive PCR result was obtained after 6 to 8 h. Our results indicate that selective enrichment followed by buoyant density gradient centrifugation provides a convenient and user-friendly sample preparation method prior to PCR.

Enhanced Exopolysaccharide Production by Metabolic Engineering of Streptococcus thermophilus

ABSTRACT It is possible that the low levels of production of exopolysaccharides (EPSs) by lactic acid bacteria could be improved by altering the levels of enzymes in the central metabolism that influence the production of precursor nucleotide sugars. To test this hypothesis, we identified and cloned the galU gene, which codes for UDP glucose pyrophosphorylase (GalU) in Streptococcus thermophilus LY03. Homologous overexpression of the gene led to a 10-fold increase in GalU activity but did not have any effect on the EPS yield when lactose was the carbon source. However, when galU was overexpressed in combination with pgmA, which encodes phosphoglucomutase (PGM), the EPS yield increased from 0.17 to 0.31 g/mol of carbon from lactose. A galactose-fermenting LY03 mutant (Gal+) with increased activities of the Leloir enzymes was also found to have a higher EPS yield (0.24 g/mol of carbon) than the parent strain. The EPS yield was further improved to 0.27 g/mol of carbon by overexpressing galU in this strain. However, the highest EPS yield, 0.36 g/mol of carbon, was obtained when pgmA was knocked out in the Gal+ strain. Measurements of the levels of intracellular metabolites in the cultures revealed that the Gal+ strains had considerably higher glucose 1-phosphate levels than the other strains, and the strain lacking PGM activity had threefold-higher levels of glucose 1-phosphate than the other Gal+ strains. These results show that it is possible to increase EPS production by altering the levels of enzymes in the central carbohydrate metabolism.

Rapid and specific detection of Salmonella spp. in animal feed samples by PCR after culture enrichment.

ABSTRACT A PCR procedure has been developed for routine analysis of viable Salmonella spp. in feed samples. The objective was to develop a simple PCR-compatible enrichment procedure to enable DNA amplification without any sample pretreatment such as DNA extraction or cell lysis. PCR inhibition by 14 different feed samples and natural background flora was circumvented by the use of the DNA polymerase Tth. This DNA polymerase was found to exhibit a high level of resistance to PCR inhibitors present in these feed samples compared to DyNAzyme II, FastStart Taq, Platinum Taq, Pwo, rTth, Taq, and Tfl. The specificity of the Tth assay was confirmed by testing 101 Salmonella and 43 non-Salmonella strains isolated from feed and food samples. A sample preparation method based on culture enrichment in buffered peptone water and DNA amplification with Tth DNA polymerase was developed. The probability of detecting small numbers of salmonellae in feed, in the presence of natural background flora, was accurately determined and found to follow a logistic regression model. From this model, the probability of detecting 1 CFU per 25 g of feed in artificially contaminated soy samples was calculated and found to be 0.81. The PCR protocol was evaluated on 155 naturally contaminated feed samples and compared to an established culture-based method, NMKL-71. Eight percent of the samples were positive by PCR, compared with 3% with the conventional method. The reasons for the differences in sensitivity are discussed. Use of this method in the routine analysis of animal feed samples would improve safety in the food chain.

Rapid diagnosis of meningococcal meningitis by polymerase chain reaction

Rapid diagnosis of meningococcal disease followed by early treatment is essential. However, culture of blood or cerebrospinal fluid (CSF) may be unsuccessful because antibiotic treatment is often started before adequate specimens are collected, and because bacteria may die during transportation to the laboratory. We have used the polymerase chain reaction (PCR) to detect meningococcal DNA in a culture-negative CSF of a 15-year-old girl with meningococcal disease. Two oligonucleotides flanking the dihydropteroate synthase gene (dhps) of Neisseria meningitidis were used as primers. The PCR reaction is a rapid technique for the early detection of meningococcal meningitis, and also when culture is negative.