Rickard Knutsson

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.

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.

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 Quantification of Yersinia enterocolitica in Pork Samples by a Novel Sample Preparation Method, Flotation, Prior to Real-Time PCR

ABSTRACT The development of real-time PCR thermal cycles in the late 1990s has opened up the possibility of accurate quantification of microorganisms in clinical, environmental, and food samples. However, a lack of suitable sample preparation methods that allow rapid quantification of the nucleic acids, remove PCR inhibitors, and prevent false-positive results due to DNA originating from dead cells has limited the use of quantitative PCR. We have used for the first time a new variant of density gradient centrifugation, called flotation, as a user-friendly sample preparation method prior to PCR. This paper describes the use of this sample preparation method, without DNA purification, for direct detection and quantification of Yersinia enterocolitica in PCR-inhibitory meat juice from pork. Flotation combined with qPCR could overcome PCR interference in juice from pork, as was shown by amplification efficiencies of 1.006 ± 0.021 and 1.007 ± 0.025, which are comparable to the amplification efficiency obtained for purified DNA samples (1.005 ± 0.059). Applying flotation to meat juice samples containing natural background flora and spiked with different levels of Y. enterocolitica showed that direct quantification of Y. enterocolitica was possible down to a level of at least 4.2 × 103 CFU per ml of meat juice, even in the presence of 106 CFU of background flora per ml. Finally, the results showed that samples containing large amounts of Y. enterocolitica DNA did not result in a positive PCR signal. This indicates that the risk of false-positive results due to detection of DNA originating from dead cells can be greatly reduced by using flotation prior to PCR.

Modeling of 5′ Nuclease Real-Time Responses for Optimization of a High-Throughput Enrichment PCR Procedure for Salmonella enterica

ABSTRACT The performance of a 5′ nuclease real-time PCR assay was studied to optimize an automated method of detection of preenriched Salmonella enterica cells in buffered peptone water (BPW). The concentrations and interactions of the PCR reagents were evaluated on the basis of two detection responses, the threshold cycle (CT) and the fluorescence intensity by a normalized reporter value (ΔRn). The CT response was identified as the most suitable for detection modeling to describe the PCR performances of different samples. DNA extracted from S. enterica serovar Enteritidis was studied in double-distilled H2O (ddH2O) and in two different enrichment media (brain heart infusion and BPW) with two PCR mixtures based on AmpliTaq Gold or rTth. A descriptive model was proposed and fitted to the available experimental data. Equivalent PCR performances for the two PCR mixtures were obtained when DNA was diluted in ddH2O. However, the level of detection of DNA was affected when BPW was present during amplification. Use of the rTth mixture generated a 1-log-unit wider linear range of amplification, and the DNA detection levels were 2 × 10−13 g/microwell for the rTth mixture and 2 × 10−12 g/microwell for the AmpliTaq Gold mixture. To verify the improved amplification capacity of the rTth mixture, BPW was inoculated with 1 CFU of S. enterica serovar Enteritidis per ml and the mixture was incubated at 30°C. Samples for PCR were withdrawn every 4 h during a 36-h enrichment. Use of the rTth mixture resulted in an earlier PCR detection during enrichment than use of the AmpliTaq Gold mixture. For accurate detection (CT ≤ 30) of S. enterica serovar Enteritidis inoculated in BPW, the rTth mixture required 8.4 h of enrichment, while the AmpliTaq Gold mixture needed 11.6 h. In conclusion, the principle applied can improve the methodology of 5′ nuclease real-time PCR for numerical optimization of sample pretreatment strategies to provide automated diagnostic PCR procedures.

Strategies for overcoming PCR inhibition.

INTRODUCTIONThe use of conventional and real-time PCR is to some extent restricted by the presence of PCR inhibitors. This is particularly so when the techniques are applied directly to complex biological samples such as clinical, environmental, or food samples for the detection of microorganisms. PCR inhibitors can originate from the sample itself, or as a result of the method used to collect or otherwise prepare the sample. Either way, inhibitors can dramatically reduce the sensitivity and amplification efficiency of PCR. This article discusses methods of reducing inhibition and designing reliable and sensitive conventional and real-time PCR experiments.

Evaluation of selective enrichment PCR procedures for Yersinia enterocolitica.

Four enrichment PCR protocols for detecting unlysed cells of pathogenic Yersinia enterocolitica were studied. First, the probability of detecting Y. enterocolitica cells of known concentrations by a multiplex PCR assay was determined, and it was found to follow a logistic regression model. From this model, the probability of detecting Y enterocolitica at a specific concentration could be estimated; for example, the detection probability of 10(4) CFU/ml was estimated to be 85.4%. The protocols were evaluated on enrichment cultures inoculated with 10(2) CFU/ml Y. enterocolitica and 10(2)-10(6) CFU/ml of a defined background flora. For each protocol, the time for sample withdrawal and the presence of background flora were studied with respect to PCR detection. The optimal point in time of sample withdrawal was found to be different for each protocol employed. Early detection was favoured by concentrating the target cells, and the most rapid PCR detection of Y. enterocolitica was achieved with enrichment in Yersinia-PCR-compatible-enrichment (YPCE) medium for 3 h at 25 degrees C, followed by a centrifugation prior to PCR analysis. For detection of Y. enterocolitica in the presence of high concentrations (10(6) CFU/ml) of background flora, a long incubation time followed by density centrifugation and a dilution step was most successful. The protocol that gave the most reliable PCR detection in the presence of 10(6) CFU/ml background flora included 24 h incubation in Yersinia-selective-enrichment (YSE) broth at 25 degrees C, followed by Percoll density centrifugation, and a 100 times dilution prior to PCR analysis.

A multiplex real-time PCR for identifying and differentiating B. anthracis virulent types.

Bacillus anthracis is closely related to the endospore forming bacteria Bacillus cereus and Bacillus thuringiensis. For accurate detection of the life threatening pathogen B. anthracis, it is essential to distinguish between these three species. Here we present a novel multiplex real-time PCR for simultaneous specific identification of B. anthracis and discrimination of different B. anthracis virulence types. Specific B. anthracis markers were selected by whole genome comparison and different sets of primers and probes with optimal characteristic for multiplex detection of the B. anthracis chromosome, the B. anthracis pXO1 and pXO2 plasmids and an internal control (IC) were designed. The primer sets were evaluated using a panel of B. anthracis strains and exclusivity was tested using genetically closely related B. cereus strains. The robustness of final primer design was evaluated by laboratories in three different countries using five different real-time PCR thermocyclers. Testing of a panel of more than 20 anthrax strains originating from different locations around the globe, including the recent Swedish anthrax outbreak strain, showed that all strains were detected correctly.

Real-time PCR for Clostridium botulinum type C neurotoxin (BoNTC) gene, also covering a chimeric C/D sequence—Application on outbreaks of botulism in poultry

In recent years, botulism type C has become a serious problem in poultry flocks in Sweden. A real-time PCR assay for Clostridium botulinum (C. botulinum) type C neurotoxin (BoNTC) gene was developed as an alternative to the mouse bioassay for detection and identification of C. botulinum type C. The complete method consists of an optimized enrichment protocol followed by automated DNA extraction prior to real-time PCR. The sensitivity of the PCR assay was determined with purified DNA to approximately 50 copies per PCR reaction. The specificity of the PCR assay was evaluated on a panel of about thirty relevant bacteria and on samples of caecum from birds collected in connection with botulism outbreaks on Swedish poultry farms. The PCR assay also covers a previously reported chimeric C/D sequence of the gene. Caecum samples from the outbreaks were positive by real-time PCR. Some of these samples were also examined with a set of conventional PCR methods, to distinguish the gene for the chimeric form from the conserved type C gene. Interestingly, the caecum samples were found to be positive for the chimeric C/D sequence. This is the first study in Europe demonstrating the chimeric C/D sequence. When the toxin gene in two of the samples was sequenced, it was closely identical (99-100%) with several previously reported C/D chimeric sequences. DNA extraction and the real-time PCR assay were both performed in a 96-well format, facilitating for future large-scale detection in outbreak situations and prevalence studies.