Nuclease Real-time PCR Research Articles

Simple and Robust Detection of CYP2D6 Gene Deletions and Duplications Using CYP2D8P as Reference.

Genotyping of the CYP2D6 gene is the most commonly applied pharmacogenetic test globally. Significant economic interests have led to the development of a plurality of assays, available for almost any genotyping platform or DNA detection chemistry. Of all the genetic variants, copy number variations are particular difficult to detect by polymerase chain reaction. Here, we present two simple novel approaches for the identification of samples carrying either deletions or duplications of the CYP2D6 gene; by relative quantification using a singleplex 5′nuclease real-time PCR assay, and by high-resolution melting of PCR products. These methods make use of universal primers, targeting both the CYP2D6 and the reference gene CYP2D8P, which is necessary for the analysis. The assays were validated against a reference method using a large set of samples. The singleplex nature of the 5′nuclease real-time PCR ensures that the primers anneal with equal affinity to both the sequence of the CYP2D6 and the reference gene. This facilitates robust identification of gene deletions and duplications based on the cycle threshold value. In contrast, the high-resolution melting assay is an end-point PCR, where the identification relies on variations between the amount of product generated from each of the two genes.

Abstract 951: Evolution of antibody titers, growth factor levels and in vitro activity in the sera of patients enrolled in the EPICAL trial of afatinib combined with anti-EGF vaccination

Abstract Background: We have recently demonstrated that antibodies generated by vaccination (anti-EGFVacAbs) potentiate the effects of EGFR TKIs in epidermal growth factor receptor mutant (EGFR-mut) non-small cell lung cancer (NSCLC) cells growing in vitro and prevent the emergence of resistance to EGFRTKIs (1). Based on these preclinical results we have started the Epical Phase Ib clinical trial(NCT03623750), testing anti-EGF vaccination in combination with EGFR TKI in advanced EGFR-mut NSCLC patients. Methods: The EPICAL trial is a Multicenter, Open-Label, Exploratory Phase Ib Clinical Study in which 23 patients have been enrolled. Anti-EGF VacAbs is a vaccine for intramuscular administration that is designed to be used in conjunction with standard of care treatment which, in this study, is afatinib. Blood samples have been collected at baseline and every three months and sent to a central laboratory. The presence of EGFR mutations has been determined on purified DNA by 5′-nuclease real-time PCR (Taqman®) assay in presence of PNA. All the serum samples have been analyzed by ELISA to determine the levels of EGF, TGFα, HGF and GAS6, together with anti-EGF antibody titers. Sera of patients were added to EGFR-mutant cell lines growing in vitro and Western blot was used to analyze EGFR, AKT andERK activation. Results: Median EGF levels in the sera of enrolled patients was 179.5 pg/mL at baseline and no anti-EGFantibodies were detected. Three months after first immunization, median serum EGF levels decreased to30.8 pg/mL (p=0.0008) while the anti-EGF vaccination titers increased to 1:6000 (p=0.0012). Anti-EGF titers remained high and EGF levels low in the patient's sera for the duration of the trial. Regarding serum TGFα and HGF levels baseline were 19.4 pg/mL and 861.2 pg/mL, respectively, and after three months dropped to 7.9 pg/mL (p=0.0037) and 341.3 pg/mL (p=0.0259). In EGFR-mutant cell lines in culture, the activation of the EGFR, AKT or ERK was abrogated by the sera of immunized patients. Conclusions: Anti-EGF vaccination induces high anti-EGF antibody titers and effectively reducing the levels of EGF and other growth factors in the blood of advanced NSCLC patients. 1J Thorac Oncol. 2018 Sep;13(9):1324-1337. Citation Format: Silvia Garcia-Roman, Jordi Codony, Miguel Angel Molina-Vila, Nuria Jordana-Ariza, Beatriz Garcia, Monica Garzon, Clara Mayo de las Casas, Maria Jose Catalan, Andres Aguilar, Santiago Viteri, Andres F. Cardona, Delvys Rodríguez, Manuel Cobo, Noemi Reguart, Erik d'Hondt, Rafael Rosell. Evolution of antibody titers, growth factor levels and in vitro activity in the sera of patients enrolled in the EPICAL trial of afatinib combined with anti-EGF vaccination [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 951.

P2.03-16 Agreement Between Different Methodologies for Non-Invasive p.T790M and EGFR Sensitizing Mutation Testing

Tyrosine kinase inhibitors (TKIs) are the current standard of care for patients with advanced EGFR-mutant non-small cell lung cancer (NSCLC). However, most patients progressed within 1 to 2 years. The EGFR p.T790M mutation is the most common resistance mechanism to first and second generation EGFR TKIs. The identification of p.T790M mutation is of considerable clinical relevance as osimertinib has demonstrated clinical efficacy in this setting. Guidelines recommend testing for the p.T790M mutation in blood at relapse to TKIs, and re-biopsy only in case of a negative result. Several blood based methodologies for detection of EGFR mutations have been developed in the recent years. However, the number of comparison studies between platforms is very limited. This is a multicenter, cross-sectional study (ClinicalTrials.gov Identifier: NCT03363139) performed by the Spanish Lung Cancer Group. Samples from 75 consecutive EGFR mutant NSCLC patients were collected at disease progression to first line TKI treatment. The presence of EGFR mutations in the cfDNA was evaluated in 39 samples by 7 methodologies, namely: Cobas® EGFR Mutation Test v2 (Roche Diagnostics), Therascreen EGFR Plasma RGQ PCR Kit (Qiagen), QuantStudio® 3D Digital PCR System (Thermofisher), a 5′-nuclease real-time PCR (TaqMan®) assay in presence of PNA, OncoBEAM EGFR (Sysmex Inostics), NGS with two different gene panels: Oncomine® (Thermofisher) and Lung Cancer Panel (Qiagen). The agreement between methodologies was assessed using the kappa coefficient (K) and its corresponding 95% confidence intervals (95% CI). For quantitative variables the concordance correlation coefficient (ccc) was used. Complete results are available for 39 patients. Overall, the agreement between all methodologies for the detection of p.T790M mutation as well as the original EGFR sensitizing mutation was good (K=0.669; 95CI: 0.504-0.835 and K=0.750 95CI: 0.599-0.899 respectively). Remarkably, the agreement between FDA-approved methodologies for p.T790M detection was almost perfect (K=0.926; 95CI: 0.712-1) and good for the EGFR sensitizing mutations (K=0.657; 95CI: 0.417-0.902). Similarly, the agreement between NGS-based methodologies for the detection of p.T790M and the EGFR activating mutations was very high (K=0.843; 95CI: 0.567-1 and K=0.872 95CI: 0.595-1 respectively). Moreover, concordance between both technologies for p.T790M and EGFR sensitizing mutation mutant allele frequency was excellent (ccc=0.956; 95CI: 0.906-1 and ccc=0.980 95CI: 0.950-1 respectively). The proportion of samples that were positive for p.T790M detection varied from 28% (PCR based technologies) to 37% depending on the methodology. NGS and PCR-based methodologies show a good to excellent agreement for the detection of EGFR mutations, including the p.T790M. Our results support the use of liquid biopsies for non-invasive testing of clinically relevant mutations (Data from the whole cohort will be presented at the meeting).

Assessment of a quantitative 5′ nuclease real-time polymerase chain reaction using groEL gene for Ehrlichia and Anaplasma species in rodents in Brazil

New genotypes of Anaplasmataceae agents have been detected in wild carnivores, birds and deer in Brazil. The present work aimed to investigate the presence of Ehrlichia and Anaplasma species in rodents sampled in Brazil. Additionally, a newly designed quantitative 5′ nuclease real-time multiplex PCR for Ehrlichia and Anaplasma spp. detection based on groEL gene amplification was designed, showing high specificity and sensitivity (10 groEL fragment copy/μL). Between 2000 and 2011, different rodent species [n=60] were trapped in 5 Brazilian biomes. Among 458 rodent spleen samples, 0.4% (2/458) and 2.4% (11/458) were positive for Ehrlichia and Anaplasma spp., respectively. Of 458 samples, 2.0% (9/458) and 1.1% (5/458) were positive for Anaplasma sp. and Ehrlichia sp., respectively, using conventional 16S rRNA PCR assays. Maximum Likelihood phylogenetic analyse based on a small region of 16S rRNA genes positioned the Anaplasma genotypes in rodents near Anaplasma phagocytophilum or Anaplasma marginale and Anaplasma odocoilei isolates. Ehrlichia genotypes were closely related to E. canis. There was a low occurrence of Anaplasma and Ehrlichia in wild and synanthropic rodents in Brazil, suggesting the circulation of new genotypes of these agents in rodents in the studied areas.

Relative Accuracy, Specificity and Sensitivity of a 5′ Nuclease Real-Time PCR Assay for Listeria monocytogenes Detection in Naturally Contaminated Pork Cuts

In the present study, the relative sensitivity, specificity and accuracy of a real-time PCR assay for Listeria monocytogenes detection in naturally contaminated pork cuts were evaluated in comparison to the ISO 11290-1:2004 reference culture method. One hundred and sixty meat samples were collected from ten different lots over a year. The PCR method included a 24-h primary enrichment step, a DNA extraction step, and a final 5′ nuclease real-time PCR assay, including an internal amplification control (IAC) and targeting the hlyA gene. Based on the analysis of 480 sub-units (three sub-units for each sample), the relative sensitivity, specificity and accuracy of the real-time PCR assay were 77.3 %, 67.0 % and 73.1 %, respectively, corresponding to a Cohen’s kappa value of 0.69 (good agreement). Considering that real samples from the worse storage scenarios were included, these results suggest the PCR method as a rapid and accurate alternative method for the quick check of L. monocytogenes in meat lots before distribution.

Relative accuracy, specificity and sensitivity of a 5′ nuclease Real-Time PCR assay for Salmonella detection in naturally contaminated pork cuts

In the present study the relative sensitivity, specificity and accuracy of a Real-Time PCR assay for Salmonella detection in naturally contaminated pork cuts were evaluated in comparison with the ISO 6579:2004 reference culture method. Meat samples were collected from packaging up to the end of shelf life from 10 different lots over a year. The PCR method included an 18 h pre-enrichment step in buffered peptone water, a DNA extraction step, and a final 5′ nuclease Real-Time PCR assay, including an Internal Amplification Control (IAC) and targeting the ttrRSBCA locus. Based on the analysis of 480 sub-units (three sub-units for each sample), the relative sensitivity, specificity and accuracy of the Real-Time PCR assay were 90, 78.7, and 82.9% respectively, corresponding to a Cohen's kappa value of 0.81 (very good agreement). These results suggest the PCR method as a rapid and accurate method for the quick check of meat lots before distribution. The ISO reference method might be applied only on positive Real-Time PCR samples for confirmatory and isolation purposes, mandatory in epidemiological investigations.

Mitochondrial Genome Microsatellite Instability and Copy Number Alteration in Lung Carcinomas

Mitochondrial DNA (mtDNA) is considered a hotspot of mutations in various tumors. However, the relationship between microsatellite instability (MSI) and mtDNA copy number alterations in lung cancer has yet to be fully clarifieds. In the current study, we investigated the copy number and MSI of mitochondrial genome in lung carcinomas, as well as their significance for cancer development. The copy number and MSI of mtDNA in 37 matched lung carcinoma/adjacent histological normal lung tissue samples were examined by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) assays for sequence variation, followed by sequence analysis and fluorogenic 5'-nuclease real-time PCR. Student's t test and linear regression analyses were employed to analyze the association between mtDNA copy number alterations and mitochondrial MSI (mtMSI). The mean copy number of mtDNA in lung carcinoma tissue samples was significantly lower than that of the adjacent histologically normal lung tissue samples (p < 0.001). mtMSI was detected in 32.4% (12/37) of lung carcinoma samples. The average copy number of mtDNA in lung carcinoma samples containing mtMSI was significantly lower than that in the other lung carcinoma samples (P < 0.05). Results suggest that mtMSI may be an early and important event in the progression of lung carcinogenesis, particularly in association with variation in mtDNA copy number.

Development and Evaluation of a Genus-Specific, Probe-Based, Internal-Process-Controlled Real-Time PCR Assay for Sensitive and Specific Detection of Blastocystis spp

Blastocystis is a common intestinal parasite of unsettled clinical significance, which is not easily detected by standard parasitological methods. The genus comprises at least 13 subtypes (STs) (which likely represent separate species), 9 of which have been found in humans. Recent data indicate that at least one of the subtypes is associated with intestinal disease. A quantitative TaqMan 5' nuclease real-time PCR (TaqMan PCR) including an internal process control (IPC) was developed for the detection of Blastocystis and shown to be applicable to genomic DNAs extracted directly from feces. The assay enabled successful amplification of DNAs from all relevant subtypes within the genus (ST1 to ST9). For assay evaluation, 153 samples previously tested by xenic in vitro culture (XIVC) were screened by the TaqMan assay. A total of 49/51 samples positive by XIVC and 13/102 samples negative by XIVC were positive by the TaqMan assay; samples positive by the TaqMan assay and negative by XIVC were subsequently tested by conventional PCR, and amplicons could be identified to the subtype level by sequencing in 69% of the cases. Compared to the TaqMan assay, XIVC had a sensitivity of 79%. This is the first time that a genus-specific, probe-based, internal-process-controlled real-time PCR assay for the detection Blastocystis has been introduced.

The transcobalamin (TCN2) 776C&gt;G polymorphism affects homocysteine concentrations among subjects with low vitamin B12 status

Methionine synthase catalyzes the conversion of 5-methyltetrahydrofolate to tetrahydrofolate and homocysteine (Hcy) to methionine using vitamin B(12) as a cofactor. Transcobalamin is the main transporter of vitamin B(12) from blood into cells. This study was undertaken to assess the relationship between the transcobalamin P259R (TCN2 776C>G) polymorphism and both serum vitamin B(12) and total Hcy (tHcy) levels. The population comprised 613 men from Northern Ireland, aged 30-49 years, for whom tHcy, serum vitamin B(12) and serum folate concentrations were available. TCN2 776C>G genotypes were determined using a TaqMan 5' nuclease Real-Time PCR assay. Standard statistical tests of association were applied to assess the relationships between the polymorphism and phenotypic variables. The TCN2 776CC homozygous genotype was associated with lower serum vitamin B(12) concentrations compared with the 776CG (P(unadjusted)=0.01; P(adjusted)=0.03) and 776GG genotypes (P(unadjusted)=0.015; P(adjusted)=0.045). Among individuals with vitamin B(12) concentrations in the lower half of the distribution, tHcy concentrations were higher in TCN2 776GG homozygotes than in individuals with the other genotypes (P(unadjusted)=0.015; P(adjusted)=0.06). These data suggest that, relative to transcobalamin with arginine at position 259 (776G), transcobalamin with proline at this position (776C) is either more efficient at vitamin B(12) transport from blood to tissues or has higher affinity for vitamin B(12). Furthermore, vitamin B(12) status influences the relationship between TCN2 776C>G genotype and tHcy concentrations. Thus, the TCN2 776C>G polymorphism may contribute to the risk of pathologies associated with a low B(12), and high tHcy phenotype.

Evaluation of a new 5'-nuclease real-time PCR assay targeting the Toxoplasma gondii AF146527 genomic repeat

AbstractToxoplasma gondii can be responsible for congenital toxoplasmosis leading to mild or severe sequelae, and for life-threatening infections in immunocompromised hosts. A new 5'-nuclease real-time PCR assay that targets the 300-fold repeated AF146527 DNA sequence (TaqMan-AF-PCR) has been developed and its performance for diagnosis of toxoplasmosis and treatment follow-up has been assessed. A retrospective analysis was first performed with 144 clinical specimens previously analysed for the presence of T. gondii DNA by a PCR–ELISA assay that targets the B1 gene of T. gondii (B1-PCR–ELISA). Fifteen samples, all from patients with clinically proven toxoplasmosis, were negative according to B1-PCR–ELISA and positive according to TaqMan-AF-PCR. A prospective analysis was then performed with 203 consecutive clinical specimens received at the laboratory of Parasitology of Saint-Louis Hospital during a 4-month period. The diagnosis of toxoplasmosis in two patients was made according to the TaqMan-AF-PCR whereas the B1-PCR–ELISA failed to make diagnosis. Additionally, iterative samples from a patient with cerebral and disseminated toxoplasmosis, already tested using a B1 real-time PCR assay, were tested using the TaqMan-AF-PCR and a Light Cycler real-time PCR assay targeting the same repetitive AF146527 sequence (LC-AF-PCR). Detection was achieved with the TaqMan-AF-PCR, with a mean gain of 7.1 and 3.3 amplification cycles when compared with the B1 real-time PCR and the LC-AF-PCR, respectively. This study demonstrates the higher sensitivity of the 5'-nuclease real-time PCR assay developed for the AF146527 DNA sequence and confirms the interest of using this highly repeated target to improve the diagnosis of toxoplasmosis.

A real-time PCR method for the detection of Salmonella enterica from food using a target sequence identified by comparative genomic analysis

A 5′-nuclease real-time PCR assay using a minor groove binding probe was developed for the detection of Salmonella enterica from artificially contaminated foods. S. enterica-specific sequences were identified by a comparative genomic approach. Several species-specific target sequences were evaluated for specificity. A real-time PCR assay was developed targeting a nucleotide sequence within the putative type III secretion ATP synthase gene ( ssaN). An internal amplification control (IAC) probe was designed by randomly shuffling the target probe sequence and a single-stranded oligonucleotide was synthesized to serve as an IAC. The assay demonstrated 100% inclusivity for the 40 Salmonella strains tested and 100% exclusivity for 24 non- Salmonella strains. The detection limit of the real-time PCR assay was 41.2 fg/PCR with Salmonella Typhimurium genomic DNA and 18.6 fg/PCR using Salmonella Enteritidis genomic DNA; 8 and 4 genome equivalents, respectively. In the presence of a natural background flora derived from chicken meat enrichment cultures, the sample preparation and PCR method were capable of detecting as few as 130 Salmonella cfu/mL. Using the developed real-time PCR method to detect Salmonella in artificially contaminated chicken, liquid egg and peanut butter samples, as few as 1 cfu/10 g of sample was detectable after a brief (6 h) non-selective culture enrichment.

Assessment of a quantitative multiplex 5’ nuclease real-time PCR for spotted fever and typhus group rickettsioses and Orientia tsutsugamushi

Department of Pathology,The Johns Hopkins University School of Medicine, Baltimore, MD, USAINTRODUCTIONScrub typhus, murine typhus and spotted fevergroup rickettsioses are re-emerging causes ofacute febrile illness in south and southeast Asia[1,2], whereas spotted fever group and typhusgroup rickettsioses are globally distributed. Datafrom the United States suggest both increasingincidence and serious under-reporting (fewerthan a quarter of cases) [3]. Lack of accurate,rapid diagnostic tools has limited assessment ofthe global burden, distribution and clinical fea-tures of rickettsioses, including the pathogenicityof different species [4]. Paired sera, the currentgold standard, at best informs epidemiology,because diagnoses can be made in retrospect onlyif clinical suspicion, follow-up and laboratoryinfrastructure enable a convalescent blood sampleto be obtained and tested [5]. Serology also doesnot reliably distinguish among species. Becauserickettsial illnesses clinically mimic other causesof undifferentiated fever that require differenttreatment, better diagnostic tools are needed toguide management and avert needless morbidityand mortality [1,2]. To address this need, wesought to develop a quantitative, multiplex5¢-nuclease real-time PCR assay to diagnosespotted fever (SFGR) and typhus group (TGR)rickettsioses, as well as Orientia tsutsugamushi(OT), the cause of scrub typhus.METHODS

Rapid quantification of Salmonella Typhimurium inoculated to meat products by real-time PCR

The objective of this study was to use a 5'-nuclease (TaqMan) real-time PCR method with primers and probe specific to the spaQ gene as a rapid approach to quantitatively determine Salmonella Typhimurium. The result showed that the correlation coefficient between real-time PCR estimates and bovine serum albumin (BSA) plate counts of S. Typhimurium was 0.99, independently of 10(5)-fold numbers of bystander Escherichia coli O157:H7 or total viable counts. The sensitivity of the real-time quantitative PCR assay was 10 CFU/mL for pure S. Typhimurium culture without enrichment. A known number of S. Typhimurium target cells were inoculated to dumpling fillings and chicken nuggets and DNA was extracted for real-time PCR analysis. The sensitivity was 60 CFU/g for S. Typhimurium inoculated to the food samples without any preceding procedure of enrichment. The duration of the entire experiment from DNA isolation and purification to PCR amplification was less than 12 h. This study demonstrated that real-time PCR is a rapid and reliable technique for quantifying S. Typhimurium possessing the spaQ gene in pure culture and in meat products.

Rapid Real-Time PCR Detection of HPdel Directly from Diluted Blood Samples

Anhaptoglobinemic patients have been reported to experience severe anaphylactic reactions to transfusions due to the production of antihaptoglobin (anti-HP) antibodies(1)(2). Anhaptoglobinemia in patients homozygous for HP del , which is a deletion of an approximately 28-kb segment of chromosome 16 extending from the promoter region of the HP (haptoglobin) gene to exon 5 of HPR (haptoglobin-related protein), has been adequately characterized only recently(1). Use of a simple duplex PCR method has detected the HP del allele in East and Southeast Asian populations at frequencies of 1%–3% but this allele has not been detected in African, West and South Asian, and European populations(1)(3)(4). Thus, diagnosing HP del homozygosity prior to blood transfusion or the infusion of blood components into individuals from East and Southeast Asian populations is effective for preventing anaphylactoid shock due to anti-HP antibodies. We have developed a simple method that uses a 5′ nuclease real-time PCR assay (TaqMan; Applied Biosystems) to detect the HP del allele without having to isolate genomic DNA. The ethics committee of Kurume University School of Medicine approved this study protocol. To distinguish alleles, we performed real-time PCR assays that detect the 2 regions that encompass the HP del breakpoint and the 5′ …

Detection of Salmonella enterica in food using two-step enrichment and real-time polymerase chain reaction

A new real-time polymerase chain reaction-based method was developed for the detection of Salmonella enterica in food. The method consisted of a novel two-step enrichment involving overnight incubation in buffered peptone water and a 5-h subculture in Rappaport-Vassiliadis medium, lysis of bacterial cells and a Salmonella-specific 5'-nuclease real-time PCR with an exogenous internal amplification control. Because a two-step enrichment was used, the detection limit for dead S. enterica cells in artificially contaminated ice cream and salami samples was high at 10(7 )CFU (25 g)(-1), eliminating potential false-positive results. When the method was evaluated with a range of 100 naturally contaminated food samples, three positive samples were detected by both the real-time PCR-based method and by the standard microbiological method, according to EN ISO 6579. When the real-time PCR-based method was evaluated alongside the standard microbiological method according to EN ISO 6579 with 36 food samples artificially contaminated at a level of 10(0 )CFU (25 g)(-1), identical results were obtained from both methods. The real-time PCR-based method involving a two-step enrichment produced equivalent results to EN ISO 6579 on the day after sample receipt. The developed method is suitable for rapid detection of S. enterica in food.

Genetic susceptibility to progressive massive fibrosis in coal miners

Progressive massive fibrosis (PMF) is a chronic interstitial lung disease with a complex aetiology that can occur after cumulative dust exposure. A case-control study was conducted to test the hypothesis that single nucleotide polymorphisms (SNPs) within genes involved in inflammatory and fibrotic processes modulate the risk of PMF development. The study population consisted of 648 underground coal miners participating in the National Coal Workers Autopsy Study, of which 304 were diagnosed with PMF. SNPs that influence the regulation of interleukin (IL)-1, IL-6, tumour necrosis factor-alpha, transforming growth factor-beta1, vascular endothelial growth factor (VEGF), epidermal growth factor intercellular cell adhesion molecule (ICAM)-1 and matrix metalloproteinase-2 genes were determined using a 5'-nuclease real-time PCR assay. There were no significant differences in the distribution of any individual SNP or haplotype between the PMF and control groups. However, the polygenotype of VEGF +405/ICAM-1 +241/IL-6 -174 (C-A-G) conferred an increased risk for PMF (odds ratio 3.4, 95% confidence interval 1.3-8.8). The present study suggests that the examined genetic variations that help regulate inflammatory and fibrotic processes are unlikely to strongly influence susceptibility to this interstitial lung disease, although the role of vascular endothelial growth factor, intercellular cell adhesion molecule-1 and interleukin-6 polymorphisms in the development of progressive massive fibrosis may require further investigation.

Estimation of Viable Salmonella Cell Numbers in Meat and Meat Product Using Real-Time PCR

Recently we developed a novel method for estimating viable Salmonella cell numbers by means of a 5'-nuclease real-time PCR [Fujikawa et al., J. Food Hyg. Japan, 47, 151-156 (2006)]. The method was based on the increase kinetics of the target DNA region (inv A) of the microorganism growing in a culture medium during incubation. The index for the PCR was the threshold cycle. In this study, we validated the method for application in food. Namely, Salmonella cells spiked into ground chicken, pork, and beef and raw hamburger patty at various cell concentrations were cleaned up using buoyant density centrifugation and the Salmonella cell numbers were estimated with our method. Linear decreases in the threshold cycle value were observed during incubation of the samples. The standard curves for the cell number in all food samples were almost identical. With a standard curve using the mean parameter values, we successfully estimated viable Salmonella cell concentrations of the foods. The results indicate that our method is applicable for viable cell number estimation of the target microorganism in foods. Further, we used this method to study Salmonella growth in ground chicken stored at a constant temperature.

A real-time PCR for the detection of Salmonella Enteritidis in poultry meat and consumption eggs

A robust duplex 5′ nuclease (TaqMan) real-time PCR was developed and in-house validated for the specific detection of Salmonella enterica subspecies enterica serovar Enteritidis in whole chicken carcass rinses and consumption eggs. The assay uses specifically designed primers and a TaqMan probe to target the Prot6e gene located on the S. Enteritidis specific 60-kb virulence plasmid. As an internal amplification control to monitor Salmonella DNA in the sample, a second primer/TaqMan probe set detects simultaneously the Salmonella specific invA gene. The assay identified correctly 95% of the 79 Salmonella Enteritidis strains tested comprising 19 different phage types. None of the 119 non-Enteritidis strains comprising 54 serovars was positive for the Prot6e gene. The assay detection probability was for 10 2 or more genome equivalents 100% and for 10 equivalents 83%. A pre-PCR sample preparation protocol including a pre-enrichment step in buffered peptone water, followed by DNA extraction was applied on low levels of artificially contaminated whole chicken carcass rinses and eggs from hens as well as 25 potentially naturally contaminated chickens. The detection limit was less than three CFU per 50 ml carcass rinse or 10 ml egg. The sensitivity and specificity compared to the traditional culture-based detection method and serotyping were both 100%. Twenty-five potentially naturally contaminated chickens were compared by the real-time PCR and the traditional cultural isolation method resulting in four Salmonella positive samples of which two were positive for the Prot6e gene and serotyped as S. Enteritidis. We show also that Salmonella isolates which have a rough lipopolysaccharide structure could be assigned to the serovar Enteritidis by the real-time PCR. This methodology can contribute to meet the need of fast identification and detection methods for use in monitoring and control measures programmes.

Diagnostic real-time PCR assays for the detection of emetic Bacillus cereus strains in foods and recent food-borne outbreaks.

Cereulide-producing Bacillus cereus can cause an emetic type of food-borne disease that mimics the symptoms provoked by Staphylococcus aureus. Based on the recently discovered genetic background for cereulide formation, a novel 5' nuclease (TaqMan) real-time PCR assay was developed to provide a rapid and sensitive method for the specific detection of emetic B. cereus in food. The TaqMan assay includes an internal amplification control and primers and a probe designed to target a highly specific part of the cereulide synthetase genes. Additionally, a specific SYBR green I assay was developed and extended to create a duplex SYBR green I assay for the one-step identification and discrimination of the two emesis-causing food pathogens B. cereus and S. aureus. The inclusivity and exclusivity of the assay were assessed using a panel of 100 strains, including 23 emetic B. cereus and 14 S. aureus strains. Different methods for DNA isolation from artificially contaminated foods were evaluated, and established real-time assays were used to analyze two recent emetic food poisonings in southern Germany. One of the food-borne outbreaks included 17 children visiting a day care center who vomited after consuming a reheated rice dish, collapsed, and were hospitalized; the other case concerned a single food-poisoning incident occurring after consumption of cauliflower. Within 2 h, the etiological agent of these food poisonings was identified as emetic B. cereus by using the real-time PCR assay.

A multiplex real-time PCR assay for detection of Xanthomonas campestris from brassicas

To develop a sensitive real-time PCR-based protocol for the detection of Xanthomonas campestris pathovars from Brassica seed. A 5' nuclease real-time PCR assay was developed to screen Brassica spp. seed for the presence of X. campestris pathovars that cause black rot. The assay amplifies a 78-bp segment of the X. campestris hrpF gene and a 100-bp segment of the Brassica spp. 18S-25S internal transcribed spacer region. The Brassica spp. target provides an internal control for the amplification process to prevent false negatives that may arise from inhibitors that are often present in extracts from plant material. Whilst the primers were compatible with SYBR Green I assays, the use of fluorescently labelled probes in a 5' nuclease assay afforded greatest sensitivity and specificity. Seed batches carrying one artificially infected seed among 10,000 were readily detected using the assay. The multiplex real-time PCR assay permitted the rapid detection of pathogenic strains of X. campestris from bacterial colonies, Brassica seed and plants. Strains of X. campestris pathogenic to brassicas were readily detected from seed via a multiplex 5' nuclease real-time PCR assay. The real-time assay offers an improvement in sensitivity and a reduced turn-around time over the conventional multiplex PCR. Real-time PCR can be used to rapidly screen Brassica spp. seed batches for the presence of X. campestris pathovars. This assay provides a means for growers and the seed industry to be aware of the black rot status of their planting material, so that they may more effectively employ disease control measures or seed disinfection.