pre-PCR Treatment Research Articles

Toward an international standard for PCR-based detection of foodborne Escherichia coli O157: validation of the PCR-based method in a multicenter interlaboratory trial.

The performance of a polymerase chain reaction (PCR) method for detection of Escherichia coli O157, previously validated on DNA extracted from pure cultures, was evaluated on spiked cattle swabs through an interlaboratory trial, including 12 participating laboratories from 11 European countries. Twelve cattle swab samples, spiked at 4 levels (0, 1-10, 10-100, and 100-1000 colony-forming units, in triplicate) with E. coli O157 were prepared centrally in the originating laboratory; the receiving laboratories performed pre-PCR treatment followed by PCR. The results were reported as positive when the correct amplicons were present after gel electrophoresis. The statistical analysis, performed on 10 sets of reported results, determined the diagnostic sensitivity to be 92.2%. The diagnostic specificity was 100%. The accordance (repeatability) was 90.0%, calculated from all positive inoculation levels. The concordance (reproducibility) was 85.0%, calculated from all positive inoculation levels. The concordance odds ratio (degree of interlaboratory variation calculated from all positive inoculation levels) was 1.58, indicating the robustness of the PCR method. Thus, the interlaboratory variation due to personnel, reagents, minor temperature or pH fluctuations and, not least, thermal cyclers, did not affect the performance of the method, which is currently being considered as part of an intenational PCR standard.

A simple genotyping procedure without DNA extraction to identify rare blood donors.

Transfusion-induced alloimmunization has severe clinical consequences including haemolytic transfusion reactions, impaired transfused RBCs longevity and greater difficulty in finding compatible blood. Molecular analysis of genomic DNA now permits prediction of blood group phenotypes based on identification of single nucleotide polymorphisms. Implementation of molecular technologies in donor centres would be helpful in finding RBC units for special patient populations, but DNA extraction remains an obstacle to donor genotyping. We propose a simple method compatible with high throughput that allows blood group genotyping using a multiplex commercial kit without the need for DNA extraction. The principle relies on pre-PCR treatment of whole blood using heating/cooling procedure in association with a recombinant hotstart polymerase. In a prospective analysis, we yielded 5628 alleles identification and designated 63 donors with rare blood, that is either negative for a high-frequency antigen or with a rare combination of common antigens. The procedure was optimized for simplicity of use in genotyping platform and would allow not only to supply antigen-matched products to recipients but also to find rare phenotypes. This methodology could also be useful for establishing a donor repository for human platelet antigens (HPA)-matched platelets since the same issues are involved for patients with neonatal alloimmune thrombocytopenia or post-transfusion purpura.

Pre-PCR treatments as a key factor on the probability of detection of Listeria monocytogenes and Salmonella in ready-to-eat meat products by real-time PCR

The aim of this study was to compare the performance of different pre-PCR treatments for the detection of Listeria monocytogenes and Salmonella spp. by real-time PCR in spiked ready-to-eat meat samples (cooked ham, dry-cured ham and fermented sausage). Three different pre-PCR treatments were assayed: (i) PrepMan® Ultra Sample Preparation Reagent, (ii) DNA purification using DNeasy Tissue Kit and QIAcube automated sample preparation system, and (iii) BAX® system. The analysis range was 2 to 2 × 106 CFU/ml. PCR was done in quintuplicate and the experiment was repeated five times. The ‘probability of detection’ was used to compare the efficiency of the pre-PCR treatments and to calculate the theoretical limit of detection of each treatment.The best pre-PCR treatment was DNA purification using DNeasy Tissue kit and QIAcube; it showed the highest detection probability and the lowest limit of detection for each food type and pathogen assayed, followed by PrepMan and BAX treatments. Probability of detection showed differences among food matrices and between L. monocytogenes and Salmonella detection probability. Despite being an expensive method, DNeasy Tissue kit would be the recommended treatment to reduce the risk of false negative results and to improve detection of foodborne pathogens in RTE-meat products by real-time PCR.

Cryptosporidium propidium monoazide-PCR, a molecular biology-based technique for genotyping of viable Cryptosporidium oocysts.

Cryptosporidium is an important waterborne protozoan parasite that can cause severe diarrhea and death in the immunocompromised. The current methods used to monitor for Cryptosporidium oocysts in water are the microscopy-based USEPA methods 1622 and 1623. These methods assess total levels of oocysts in source waters, but do not determine oocyst viability or genotype. Recently, propidium monoazide (PMA) has been used in conjunction with molecular diagnostic tools to identify species and assess the viability of bacteria. The goal of this study was the development of a Cryptosporidium PMA-PCR (CryptoPMA-PCR) assay that includes PMA treatment prior to PCR analysis in order to prevent the amplification of DNA from dead oocysts. The results demonstrated that PMA penetrates only dead oocysts and blocks amplification of their DNA. The CryptoPMA-PCR assay can also specifically detect live oocysts within a mixed population of live and dead oocysts. More importantly, live oocysts, not dead oocysts, were detected in raw waste or surface water samples spiked with Cryptosporidium oocysts. This proof-of-concept study is the first to demonstrate the use of PMA for pre-PCR treatment of Cryptosporidium oocysts. The CryptoPMA-PCR assay is an attractive approach to specifically detect and genotype viable Cryptosporidium oocysts in the water, which is critical for human health risk assessment.

Application of conventional and real-time fluorescent ITS1 rDNA PCR for detection of Besnoitia besnoiti infections in bovine skin biopsies

Besnoitia besnoiti, an apicomplexan protozoan parasite, is the causative agent of bovine besnoitiosis. This infection may dramatically affect body condition, and, in males, lead to irreversible infertility. While identification of clinical cases and their histopathological confirmation is relatively simple to carry out, finding subclinical forms of infection is more difficult, thus a more sensitive test for the identification of the etiological agent may be an appropriate diagnostic tool. We have developed the ITS1 rDNA-sequence-based conventional and real-time PCR which are highly sensitive and specific for the detection of B. besnoiti infection in cattle. A recombinant internal positive control was introduced to assess possible sample-related inhibitory effects during the amplification reaction and, in order to prevent false-positive results, a pre-PCR treatment of potentially contaminating dU-containing PCR product with uracyl-DNA-glycosylase (UDG) was followed.

Characterization of the PCR inhibitory effect of bile to optimize real-time PCR detection of Helicobacter species

The inhibitory effect of human and porcine bile samples to detect Helicobacter DNA was studied by adding different concentrations of bile samples to PCR mixtures of six thermostable DNA polymerases containing cagA specific primers and Helicobacter pylori DNA. PCR products were amplified by using the Rotorgene system and SYBR Green I. Among the six DNA polymerases tested, r Tth had the lowest sensitivity to bile inhibitors, whereas Taq and Tfl had the highest sensitivity. Bile proteins did not inhibit Ampli Taq DNA polymerase, whereas the fraction containing mainly bile acids and their salts inhibited the amplification capacity of Ampli Taq. Heating human bile at 98 °C and adding casein and formamide to the reaction mixture reduced the PCR inhibitory effect of bile. Therefore, a pre-PCR treatment based on dilution and heating of bile, adding casein and formamide to the reaction mixture of r Tth DNA polymerase was found efficient to amplify DNA directly in bile.

Toward an International Standard for PCR-Based Detection of Foodborne Escherichia coli O157: Validation of the PCR-Based Method in a Multicenter Interlaboratory Trial

Abstract The performance of a polymerase chain reaction (PCR) method for detection of Escherichia coli O157, previously validated on DNA extracted from pure cultures, was evaluated on spiked cattle swabs through an interlaboratory trial, including 12 participating laboratories from 11 European countries. Twelve cattle swab samples, spiked at 4 levels (0, 1–10, 10–100, and 100–1000 colony-forming units, in triplicate) with E. coli O157 were prepared centrally in the originating laboratory; the receiving laboratories performed pre-PCR treatment followed by PCR. The results were reported as positive when the correct amplicons were present after gel electrophoresis. The statistical analysis, performed on 10 sets of reported results, determined the diagnostic sensitivity to be 92.2%. The diagnostic specificity was 100%. The accordance (repeatability) was 90.0%, calculated from all positive inoculation levels. The concordance (reproducibility) was 85.0%, calculated from all positive inoculation levels. The concordance odds ratio (degree of interlaboratory variation calculated from all positive inoculation levels) was 1.58, indicating the robustness of the PCR method. Thus, the interlaboratory variation due to personnel, reagents, minor temperature or pH fluctuations and, not least, thermal cyclers, did not affect the performance of the method, which is currently being considered as part of an international PCR standard.

Towards an international standard for PCR-based detection of foodborne thermotolerant campylobacters: interaction of enrichment media and pre-PCR treatment on carcass rinse samples

As part of a large EU project for standardisation of polymerase chain reaction (PCR), a systematic evaluation of the interaction of enrichment media, type of DNA polymerase and pre-PCR sample treatment for a PCR detecting thermotolerant campylobacters was carried out. The growth-supporting capacity and PCR compatibility of enrichment in Preston, Mueller–Hinton and Bolton broth (blood-containing and blood-free) were evaluated. The effect of resin-based DNA extraction and DNA extraction by boiling on the final PCR assay was investigated. The time-course studies indicated that a 20-h sample enrichment in blood-containing Bolton broth, followed by a simple resin-based extraction of DNA and a PCR amplification using T th polymerase, resulted in strong and clear PCR amplicons for target (287 bp) and internal amplification control (IAC, 124 bp). The enrichment PCR-based method, tested on 68 presumably naturally contaminated poultry-rinse samples, showed a diagnostic sensitivity of 97.5% (39 PCR-positive/40 total positive samples) and a diagnostic specificity of 100% (28 PCR-negative/28 total negative samples; P=0.32) when compared to a standard bacteriological method (ISO 10272).

Immunomagnetic separation and PCR for detection of Helicobacter pylori in water and stool specimens.

The detection of Helicobacter pylori in clinical and environmental samples by PCR sometimes requires removal of polymerase inhibitors. We have used a magnetic immunoseparation technique as pre-PCR treatment to facilitate direct detection of H. pylori in stool and water specimens. Rabbit hyperimmune antiserum was produced and magnetic beads were coated with purified immunoglobulin G, which reacted with and bound to both coccoid and rod-shaped forms of H. pylori. When PCR was applied for the detection of H. pylori from cultured samples, the number of organisms that was required for positive scores varied significantly. For a 3-day culture of H. pylori, samples containing 10(2) bacteria per ml are needed for a positive score; for a 6-day culture, samples containing 10(4) bacteria per ml are needed; and for a 10-day culture, samples containing 10(6) bacteria per ml are needed. These results indicate that the coccoid forms of H. pylori may have a different antigenicity and DNA content and are therefore more difficult to detect by immunomagnetic separation and PCR than the rod-shaped forms. Spiked samples with the addition of feces, spiked water samples, and a patient stool specimen were all scored positive with this technique.

Preventing false positives: quantitative evaluation of three protocols for inactivation of polymerase chain reaction amplification products.

False-positive results because of carryover contamination by previously amplified nucleic acids are currently the greatest impediment to routine implementation of nucleic acid amplification protocols. We evaluated three methods for inactivation of a 156-bp Borrelia burgdorferi polymerase chain reaction (PCR) product: (i) post-PCR cross-linking with isopsoralen (IP), (ii) pre-PCR treatment of a dU-containing PCR product with uracil N-glycosylase (UNG), and (iii) post-PCR alkaline hydrolysis (primer hydrolysis) of PCR products synthesized by using primers containing 3' ribose residues. The sensitivities of the PCR performed under the conditions of each protocol were comparable. Inactivation of amplified DNA was highly efficient for all three protocols; the IP and UNG protocols eliminated at least to 3 x 10(9) copies of the product. The primer hydrolysis protocol varied in efficiency depending on the number and position of the 3' ribose residues, but inactivation ranged from 10(4) to 10(9) copies. We conclude that with some modifications, all three systems are effective for eliminating amplified DNA products. Routine implementation of at least one method should help to avoid false-positive results because of carryover contamination.