Pg Of Genomic DNA Research Articles

Development of a highly resolved loop-mediated isothermal amplification method to detect the N526K ftsI mutation of β-lactamase-negative ampicillin-resistant Haemophilus influenzae

Rapid and easy detection of sequence polymorphisms, including nucleotide point mutations of bacterial pathogens responsible for amino acid substitutions linked to drug resistance, is essential for the proper use of antimicrobial agents. Here, a detection method using loop-mediated amplification (LAMP) combined with amplification refractory mutation system (ARMS) to accurately distinguish a different single nucleotide in the target sequence was established, named ARMS-SNP LAMP. This procedure is capable of species-specific detection of a nucleotide (1578T) in the ftsI gene on Haemophilus influenzae without amplifying the sequence carrying the point mutations (T1578G/A) in β-lactamase-negative ampicillin resistant (BLNAR) strains. Reactions were performed at 61°C for 45min. Successful target gene amplifications were detected by measuring real-time turbidity using a turbidimeter and visual detection. The assay had a detection limit of 10.0pg of genomic DNA per reaction and showed specificity against 52 types of pathogens, whereas amplifications were completely blocked in even 100.0ng/μL of genomic DNA with point mutations at T1578G and T1578A. The expected ARMS-SNP LAMP products were confirmed through identical melting curves in real-time LAMP procedures. This novel procedure was also used to analyze 57 clinical isolates of H. influenzae. All 25 clinical isolates with the naïve sequence of 1578T gave positive results. In addition, concordant negative results were obtained for 31 of the BLNAR strains with the T1578G mutation and one strain with the T1578A mutation. The ARMS-SNP LAMP method is a simple and rapid method for SNP-genotyping of a clinical isolate as point-of-care testing (POCT) technology. It is suitable for use in both resource-limited situations and well-equipped clinical settings because of its simplicity and convenience.

Development of a nested-PCR assay for the rapid detection of Pilidiella granati in pomegranate fruit

Pilidiella granati, a causal agent of twig blight and crown rot of pomegranate, is an emerging threat that may cause severe risk to the pomegranate industry in the future. Development of a rapid assay for the timely and accurate detection of P. granati will be helpful in the active surveillance and management of the disease caused by this pathogen. In this study, a nested PCR method was established for the detection of P. granati. Comparative analysis of genetic diversity within 5.8S rDNA internal transcribed spacer (ITS) sequences of P. granati and 21 other selected fungal species was performed to design species-specific primers (S1 and S2). This primer pair successfully amplified a 450 bp product exclusively from the genomic DNA of P. granati. The developed method can detect 10 pg genomic DNA of the pathogen in about 6 h. This technique was successfully applied to detect the natural infection of P. granati in the pomegranate fruit. The designed protocol is rapid and precise with a high degree of sensitivity.

Rapid and Specific Polymerase Chain Reaction-Enzyme Linked Immunosorbent Assay for Detection of Escherichia coli LT Toxin From Clinical Isolates

Background: Enterotoxigenic Escherichia coli (ETEC) is the most common agent, which causes diarrhea. ETEC is colonized along the cells and produces enterotoxins leading to diarrhea. Different detection methods have been utilized for detection of ETEC heat Labile Toxin (LT) toxins or respective genes. These methods have disadvantages such as high costs and labor time and limitations in handling many samples simultaneously. Objectives: The aim of this study was detection of LT toxin genes in E. coli clinical strains by polymerase chain reaction-enzyme linked immunosorbent assay (PCR-ELISA). Methods: This experimental study was conducted on Iranian children communities from May to November 2014. Forty stool samples were obtained from laboratories and investigated for heat-labile toxin (LT). Specific primers were designed and the DIG -labeled PCR products were bounded to streptavidin-coated wells of a microtiter plate and detected by anti-DIG-peroxidase conjugate. An internal biotin-labeled probe was designed for LT gene and detected with streptavidin. Sensitivity and specificity of the PCR-ELISA method were determined using Enterobacteria strains. Results: Overall, 7.5% of clinically isolated strains were detected as LT positive. The specificity of PCR-ELISA method was 100%. The detection limit of PCR-ELISA was 1.9 pg of genomic DNA. Conclusions: Results showed that this method is fast and sensitive for diagnosing bacteria. Polymerase chain reaction-ELISA is a suitable substitute for all the above factors because it is a quite sensitive, specific and rapid way for detection of LT toxin gene from ETEC strains.

A facile cascade signal amplification strategy using DNAzyme loop-mediated isothermal amplification for the ultrasensitive colorimetric detection of Salmonella

To make the direct readout of loop-mediated isothermal amplification (LAMP) simpler and more reliable and to enhance the visual sensitivy of LAMP detection, we developed a facile cascade signal-on colorimetric DNAzyme LAMP (dLAMP) sensor that integrates the amplification power of LAMP and the inherent catalytic activity of the DNAzyme for the simple and ultrasensitive analysis of targets. First, a signal inner primer with a 17-nt DNAzyme complementary sequence (a signal precursor) in its middle insertable region was asymmetrically designed for the next amplification and colorimetric reaction. In the presence of target DNA, the target specifically initiated LAMP, and the signal precursor was amplified to a larger number of DNAzyme sequences during the target DNA amplification. With the addition of hemin, free DNAzyme fragments, which were released by ultrasonication and pPNA, formed G-quadruplex-hemin conjugates that act as a signal readouts for the direct colorimetric assay. Even less 0.5pg genomic DNA were detected using the established one-step cascade signal amplification strategy without the need for gels or other apparatus. Moreover, the novel colorimetric strategy showed high fidelity in DNA detection and visual sensitivity of LAMP. This method may pave the way for the point-of-care detection and other PCR-based colorimetric analyses.

A primer and probe set for detecting multiple types of EGFR exon 19 deletions

EGFR exon 19 deletion is an important indicator for tyrosine kinase inhibitor treatment in non-small cell lung cancer. However, detection of exon 19 deletions faces a challenge: there are more than 30 types of mutations reported at the hotspot. Moreover, considering the application in body fluid samples, assays with high sensitivity and specificity are necessary for the detection of rare mutant alleles. Here, we describe a single tube reaction which could detect at least 29 types of exon 19 deletions with only an unlabeled peptide nucleic acid (PNA) clamp and a pair of DNA probes. The PNA clamp was used to inhibit amplification of wild-type templates; and the DNA probes were used to generate melting peaks for multiple types of mutations. Under optimal condition, the assay was able to detect as low as 0.01% mutant DNA in wild-type background, and had a limit of detection of 10 pg genomic DNA. Feasibility of the assay was tested in body fluid samples from lung cancer patients. The assay detected 100% and 60% of deletions in pleural effusions and plasma, respectively. We believe the present assay can be used in the clinical laboratories and has potential to be adapted for a microfluidic device.

Desarrollo de un ensayo de PCR para detectar los genes codificadores de la toxina del cólera (ctxAB) en preparaciones del candidato vacunal vivo atenuado CV638

The live oral vaccine candidate against cholera CV638 is produced under good manufacturer practices. The active ingredient of this vaccine candidate is the genetically modified strain Vibrio cholerae (V. cholerae) 638, developed by researchers at the National Center of Scientific Research, from the strain V. cholerae serogroup O1 biotype El Tor C7258, by removing the cholera toxin genes (ctxAB). Since the strain 638 lacks these genes in its genome, the presence of ctxAB in vaccine preparations, would be given by a contamination with a toxigenic V. cholerae strain. The present study was aimed at designing a specific PCR to detect ctxAB genes in DNA isolated from preparations of the vaccine candidate CV638, artificially contaminated with toxigenic V. cholerae strain. The sensitivity of the optimized PCR assay was 1 pg of genomic DNA from toxigenic strain of V. cholerae C7258, corresponding to approximately 200 genomic copies. The sensitivity of the PCR method for detecting toxigenic strains in CV638 vaccine preparations contaminated with a toxigenic strain was ∼7×103 colony forming units of the toxigenic strain per dose of CV638. Once validated, this method could be used in the quality control of the production of the live vaccine candidate CV638.

Ultrasensitive determination of 5-methylcytosine and 5-hydroxymethylcytosine in genomic DNA by sheathless interfaced capillary electrophoresis-mass spectrometry.

A newly developed sheathless interface for capillary electrophoresis-mass spectrometry, using a porous tip sprayer, was first applied for highly sensitive determination of cytosine modifications. The system performed well in identification and quantification of both 5-methylcytosine and 5-hydroxymethylcytosine using only 125 pg (∼20 cells) genomic DNA samples.

Simultaneous Whole Mitochondrial Genome Sequencing with Short Overlapping Amplicons Suitable for Degraded DNA Using the Ion Torrent Personal Genome Machine.

ABSTRACTWhole mitochondrial (mt) genome analysis enables a considerable increase in analysis throughput, and improves the discriminatory power to the maximum possible phylogenetic resolution. Most established protocols on the different massively parallel sequencing (MPS) platforms, however, invariably involve the PCR amplification of large fragments, typically several kilobases in size, which may fail due to mtDNA fragmentation in the available degraded materials. We introduce a MPS tiling approach for simultaneous whole human mt genome sequencing using 161 short overlapping amplicons (average 200 bp) with the Ion Torrent Personal Genome Machine. We illustrate the performance of this new method by sequencing 20 DNA samples belonging to different worldwide mtDNA haplogroups. Additional quality control, particularly regarding the potential detection of nuclear insertions of mtDNA (NUMTs), was performed by comparative MPS analysis using the conventional long‐range amplification method. Preliminary sensitivity testing revealed that detailed haplogroup inference was feasible with 100 pg genomic input DNA. Complete mt genome coverage was achieved from DNA samples experimentally degraded down to genomic fragment sizes of about 220 bp, and up to 90% coverage from naturally degraded samples. Overall, we introduce a new approach for whole mt genome MPS analysis from degraded and nondegraded materials relevant to resolve and infer maternal genetic ancestry at complete resolution in anthropological, evolutionary, medical, and forensic applications.

Development of a DNA Macroarray for the Detection and Identification of Fungal Pathogens Causing Decline of Young Grapevines.

Young vine decline (YVD) is a complex disease caused by at least 51 different fungi and responsible for important economic losses to the grapevine industry worldwide. YVD fungi are known to occur in planting material. Hence, detection prior to planting is critical to assure longevity of newly established vineyards. A DNA macroarray based on reverse dot-blot hybridization containing 102 oligonucleotides complementary to portions of the β-tubulin region was developed for detection of YVD fungi. Specificity of the array was first evaluated against 138 pure fungal cultures representing 72 different taxa from nine genera, including 37 YVD species. In total, 61 species, including 34 YVD pathogens, were detected and identified by the array. The detection limit of the array was below 0.1 pg of genomic DNA. The array was validated against artificially inoculated canes and soil and commercial planting material, with the latter showing a high incidence of YVD fungi in nursery plants otherwise not detected by traditional plating and culturing. This DNA array proved to be a rapid and specific tool to simultaneously detect and identify most YVD fungi in a single test, which has the potential to be used in commercial diagnostics or by the grapevine nursery industry to determine the health status of the planting material.

Promising Nucleic Acid Lateral Flow Assay Plus PCR for Shiga Toxin-ProducingEscherichia coli

Shiga toxin (Stx)-producing Escherichia coli (STEC) is a frequent cause of foodborne infections, and methods for rapid and reliable detection of STEC are needed. A nucleic acid lateral flow assay (NALFA) plus PCR was evaluated for detecting STEC after enrichment. When cell suspensions of 45 STEC strains, 14 non-STEC strains, and 13 non-E. coli strains were tested with the NALFA plus PCR, all of the STEC strains yielded positive results, and all of the non-STEC and non-E. coli strains yielded negative results. The lower detection limit for the STEC strains ranged from 0.1 to 1 pg of genomic DNA (about 20 to 200 CFU) per test, and the NALFA plus PCR was able to detect Stx1- and Stx2-producing E. coli strains with similar sensitivities. The ability of the NALFA plus PCR to detect STEC in enrichment cultures of radish sprouts, tomato, raw ground beef, and beef liver inoculated with 10-fold serially diluted STEC cultures was comparable to that of a real-time PCR assay (at a level of 100 to 100,000 CFU/ml in enrichment culture). The bacterial inoculation test in raw ground beef revealed that the lower detection limit of the NALFA plus PCR was also comparable to that obtained with a real-time PCR assay that followed the U.S. Department of Agriculture guidelines. Although further evaluation is required, these results suggest that the NALFA plus PCR is a specific and sensitive method for detecting STEC in a food manufacturing plant.

Microarray on digital versatile disc for identification and genotyping of Salmonella and Campylobacter in meat products.

Highly portable, cost-effective, and rapid-response devices are required for the subtyping of the most frequent food-borne bacteria; thereby the sample rejection strategies and hygienization techniques along the food chain can be tailor-designed. Here, a novel biosensor is presented for the generic detection of Salmonella and Campylobacter and the discrimination between their most prevalent serovars (Salmonella Enteritidis, Salmonella Typhimurium) and species (Campylobacter jejuni, Campylobacter coli), respectively. The method is based on DNA microarray developed on a standard digital versatile disc (DVD) as support for a hybridization assay and a DVD driver as scanner. This approach was found to be highly sensitive (detection limit down to 0.2pg of genomic DNA), reproducible (relative standard deviation 4-19%), and high working capacity (20 samples per disc). The inclusivity and exclusivity assays indicated that designed oligonucleotides (primers and probes) were able to discriminate targeted pathogens from other Salmonella serovars, Campylobacter species, or common food-borne pathogens potentially present in the indigenous microflora. One hundred isolates from meat samples, collected in a poultry factory, were analyzed by the DVD microarraying and fluorescent real-time PCR. An excellent correlation was observed for both generic and specific detection (relative sensitivity 93-99% and relative specificity 93-100%). Therefore, the developed assay has been shown to be a reliable toolto be used in routine food safety analysis, especially in settings with limited infrastructure due to the excellent efficiency-cost ratio of compact disc technology. Graphical Abstract DNA microarray performed by DVD technology for pathogen genotyping.

Real-time fluorescence ligase chain reaction for sensitive detection of single nucleotide polymorphism based on fluorescence resonance energy transfer.

Most of practical methods for detection of single nucleotide polymorphism (SNP) need at least two steps: amplification (usually by PCR) and detection of SNP by using the amplification products. Ligase chain reaction (LCR) can integrate the amplification and allele discrimination in one step. However, the detection of LCR products still remains a great challenge for highly sensitive and quantitative SNP detection. Herein, a simple but robust strategy for real-time fluorescence LCR has been developed for highly sensitive and quantitative SNP detection. A pair of LCR probes are firstly labeled with a fluorophore and a quencher, respectively. When the pair of LCR probes are ligated in LCR, the fluorophore will be brought close to the quencher, and thus, the fluorescence will be specifically quenched by fluorescence resonance energy transfer (FRET). The decrease of fluorescence intensity resulted from FRET can be real-time monitored in the LCR process. With the proposed real-time fluorescence LCR assay, 10 aM DNA targets or 100 pg genomic DNA can be accurately determined and as low as 0.1% mutant DNA can be detected in the presence of a large excess of wild-type DNA, indicating the high sensitivity and specificity. The real-time measuring does not require the detection step after LCR and gives a wide dynamic range for detection of DNA targets (from 10 aM to 1 pM). As LCR has been widely used for detection of SNP, DNA methylation, mRNA and microRNA, the real-time fluorescence LCR assay shows great potential for various genetic analysis.

Species-specific detection of Vibrio alginolyticus in shellfish and shrimp by real-time PCR using the groEL gene

Vibrio alginolyticus is an important opportunistic pathogen in humans and marine animals. Culture-based methods and the traditional polymerase chain reaction (PCR) cannot quantify the pathogen with sufficient sensitivity. Thus, reliable, rapid, and accurate detection and quantification methods are essential to prevent and control V. alginolyticus. We developed a real-time PCR assay using SYBR Green I targeting the groEL gene to detect and quantify V. alginolyticus. A species-specific primer was designed based on the groEL gene. Primer specificity was confirmed using 3 V. alginolyticus strains and 32 other Vibrio and non-Vibrio strains. Only the V. alginolyticus strain showed a positive result in the specificity test. A melting curve analysis showed a specific peak with a melting temperature of 85.80 ± 0.15 °C. A standard curve was produced to permit quantification of the target organism. Detection sensitivity was 0.14 pg of genomic DNA (equivalent to 10 cells per ml) for a pure culture of V. alginolyticus. V. alginolyticus was also quantified in artificially inoculated shellfish and living shrimp. The results indicated that SYBR Green I-based quantitative real-time polymerase chain reaction targeting the groEL gene enabled accurate, sensitive, and rapid quantitative detection of V. alginolyticus in seawater, shellfish, and shrimp.

A duplex qPCR for the simultaneous detection of Escherichia coli O157:H7 and Listeria monocytogenes using LNA probes.

In this study, a duplex qPCR assay was developed for the needs of the Irish fish industry to screen for the two major food-borne pathogens of fish, Listeria monocytogenes and Escherichia coli O157:H7. The assay can claim positive or negative results for two pathogens in one go in only 20 h including 16 h universal pre-enrichment and compared to traditional ISO approved plate culture methods the labour and the cost involved in testing of one sample is reduced to minimum. The highly specific genomic areas targeted for PCR amplification in the assay are the hly gene for listeriolysin O (LLO) of L. monocytogenes and the stx gene for Shiga-like toxin expressed by E. coli O157:H7. The detection limit of the assay is consistent with the consumer protection limits of 1 pg genomic DNA or 1 CFU 25 g(-1) fish meat (with enrichment) allowing the test to be considered as a substitute to standard plate culture methods. The study highlights a novel duplex qPCR for Listeria monocytogenes and Escherichia coli O157:H7 that could be used as an alternative to plate-based ISO or singleplex PCR methods while minimizing the costs. The assay uses rapid DNA extraction methods and locked nucleic acid probes. Sensitivity and specificity are 100 and 98·95% respectively. The potential for quantitative rage of the assay is 10(8) -10(1) CFU ml(-1) .

Multiplex Real-Time PCR Method for Simultaneous Identification and Toxigenic Type Characterization of Clostridium difficile From Stool Samples.

BackgroundThe aim of this study was to develop and validate a multiplex real-time PCR assay for simultaneous identification and toxigenic type characterization of Clostridium difficile.MethodsThe multiplex real-time PCR assay targeted and simultaneously detected triose phosphate isomerase (tpi) and binary toxin (cdtA) genes, and toxin A (tcdA) and B (tcdB) genes in the first and sec tubes, respectively. The results of multiplex real-time PCR were compared to those of the BD GeneOhm Cdiff assay, targeting the tcdB gene alone. The toxigenic culture was used as the reference, where toxin genes were detected by multiplex real-time PCR.ResultsA total of 351 stool samples from consecutive patients were included in the study. Fifty-five stool samples (15.6%) were determined to be positive for the presence of C. difficile by using multiplex real-time PCR. Of these, 48 (87.2%) were toxigenic (46 tcdA and tcdB-positive, two positive for only tcdB) and 11 (22.9%) were cdtA-positive. The sensitivity, specificity, negative predictive value (NPV), and positive predictive value (PPV) of the multiplex real-time PCR compared with the toxigenic culture were 95.6%, 98.6%, 91.6%, and 99.3%, respectively. The analytical sensitivity of the multiplex real-time PCR assay was determined to be 103colonyforming unit (CFU)/g spiked stool sample and 0.0625 pg genomic DNA from culture. Analytical specificity determined by using 15 enteric and non-clostridial reference strains was 100%.ConclusionsThe multiplex real-time PCR assay accurately detected C. difficile isolates from diarrheal stool samples and characterized its toxin genes in a single PCR run.

Rapid and sensitive identification of the herbal tea ingredient Taraxacum formosanum using loop-mediated isothermal amplification.

Taraxacum formosanum (TF) is a medicinal plant used as an important component of health drinks in Taiwan. In this study, a rapid, sensitive and specific loop-mediated isothermal amplification (LAMP) assay for authenticating TF was established. A set of four specific LAMP primers was designed based on the nucleotide sequence of the internal transcribed spacer 2 (ITS2) nuclear ribosomal DNA (nrDNA) of TF. LAMP amplicons were successfully amplified and detected when purified genomic DNA of TF was added in the LAMP reaction under isothermal condition (65 °C) within 45 min. These specific LAMP primers have high specificity and can accurately discriminate Taraxacum formosanum from other adulterant plants; 1 pg of genomic DNA was determined to be the detection limit of the LAMP assay. In conclusion, using this novel approach, TF and its misused plant samples obtained from herbal tea markets were easily identified and discriminated by LAMP assay for quality control.

Development of a Quantitative Polymerase Chain Reaction Detection Protocol for Cercospora kikuchii in Soybean Leaves and Its Use for Documenting Latent Infection as Affected by Fungicide Applications.

Cercospora leaf blight (CLB) of soybean, caused by Cercospora kikuchii, is a serious disease in the southern United States. A sensitive TaqMan probe-based real-time quantitative polymerase chain reaction (qPCR) assay was developed to specifically detect and quantify C. kikuchii in naturally infected soybean plants. The sensitivity was 1 pg of genomic DNA, which was equivalent to about 34 copies of genome of C. kikuchii. Using this qPCR assay, we documented a very long latent infection period for C. kikuchii in soybean leaves beginning at the V3 growth stage (as early as 22 days after planting). The levels of biomass of C. kikuchii remained low until R1, and a rapid increase was detected from the R2/R3 to R4/R5 growth stages shortly before the appearance of symptoms at R6. The efficacy of various fungicide regimens under field conditions also was evaluated over a 3-year period using this qPCR method. Our results showed that multiple fungicide applications beginning at R1 until late reproductive stages suppressed the development of C. kikuchii in leaves and delayed symptom expression. Different fungicide chemistries also had differential effects on the amount of latent infection and symptom expression during late reproductive growth stages.

Multiplex real-time PCR assay for detection of pathogenic Vibrio parahaemolyticus strains

Foodborne disease caused by pathogenic Vibrio parahaemolyticus has become a serious public health problem in many countries. Rapid diagnosis and the identification of pathogenic V. parahaemolyticus are very important in the context of public health. In this study, an EvaGreen-based multiplex real-time PCR assay was established for the detection of pathogenic V. parahaemolyticus. This assay targeted three genetic markers of V. parahaemolyticus (species-specific gene toxR and virulence genes tdh and trh). The assay could unambiguously identify pathogenic V. parahaemolyticus with a minimum detection limit of 1.4 pg genomic DNA per reaction (concentration giving a positive multiplex real-time PCR result in 95% of samples). The specificity of the assay was evaluated using 72 strains of V. parahaemolyticus and other bacteria. A validation of the assay with clinical samples confirmed its sensitivity and specificity. Our data suggest the newly established multiplex real-time PCR assay is practical, cost-effective, specific, sensitive and capable of high-throughput detection of pathogenic V. parahaemolyticus.

A new method to detect Lonsdalea quercina in infected plant tissues by real‐time PCR

SummaryPresymptomatic and accurate diagnoses of pathogens are essential for disease prediction and the timely application of bactericide. The bacterium Lonsdalea quercina (=Brenneria quercina) has been reported as the causal agent of drippy nut and bark canker disease on oak in California (US) and Europe. In recent years, it is also found on Populus × euramericana trees in Henan province of China. This bacterium causes longitudinal cankers of a few centimetres in size on the bark surface of the upper trunk. In this study, we developed two species‐specific PCR assays using primer pairs LqfF/LqfR and LqgF/LqgR for the rapid and accurate detection of the pathogenic bacteria in diseased plant tissues. The results show that the LqfF/LqfR primers amplified only a single PCR band of approximately 382 bp and the LqgF/LqgR primers yielded a PCR product of approximately 286 bp. The two primers were successfully adapted to real‐time PCR based on SYBR Green I used with the ABI 7500 system. The detection limit of the reaction was 0.1 pg genomic DNA per 20 μl PCR reaction volumes. The pathogen was mainly detected in the phloem of cankers as well as in the exudates of diseased trees, but was not found in the xylem or leaves. The size of pathogen in distribution was larger than the lesion. The results demonstrate that real‐time PCR assays can be used to detect the pathogen by extracting DNA directly from infected plant tissues. This method is a rapid, reliable method for the presymptomatic and accurate detection of L. quercina, providing a useful insight into epidemiological studies.

Development of 23 individual TaqMan® real-time PCR assays for identifying common foodborne pathogens using a single set of amplification conditions

Most of the acute intestinal diseases are caused by foodborne pathogens with infants and elderly people being at major risk. The aim of this study was to develop a procedure to simultaneously detect 20 foodborne pathogens in complex alimentary matrices such as milk, cheese and meat. The list of targets include, among the others, Listeria spp., Salmonella spp., Shigella spp., Escherichia coli spp., Campylobacter spp., Clostridium spp. and Staphylococcus aureus. The accuracy of detection was determined by using ATCC strains as positive and negative controls. The achieved sensitivity of each of assays was 1 pg of genomic DNA, which was equivalent to ∼1 cfu. The working ranges of the TaqMan® Real-time PCR assays, when used quantitatively on cheese and meat samples inoculated with serial dilution of Listeria spp., Listeria monocytogenes, S. aureus, Salmonella enterica, Shigella boydii, E. coli O157:H7, Bacillus cereus, Campylobacter coli, Yersinia enterocolitica, Enterobacter sakazakii and Pseudomonas aeruginosa was 108 cfu/g to 104 cfu/g. No matrix interferences were observed.