Multiplex Real-time PCR Method Research Articles

Comparison of Multiplex Real-Time PCR and PCR-Reverse Blot Hybridization Assays for the Direct and Rapid Detection of Porcine Circovirus Type 2 Genotypes

Porcine circovirus type 2 (PCV2), the causative agent of porcine circovirus-associated diseases (PCVAD), poses a serious economic threat for the swine industry. Currently, PCV2 is classified into five major genotypes: PCV2a, PCV2b, PCV2c, PCV2d, and PCV2e. The aim of this study is to evaluate the performance of two commercially available methods, multiplex real-time PCR assay and PCR-reverse blot hybridization assay (REBA), for the rapid detection of PCV2 and direct identification of PCV2 genotypes from clinical samples as well as to compare the results with that of sequence analysis. Molecular diagnostic methods were used to evaluate a total of 180 samples, including tissues and blood samples from pigs that were suspected of PCVAD infection. The results of this study showed that the detection rate for positive PCV2 was 48.3% (n = 87) in both multiplex real-time PCR and PCR-REBA methods. Using sequence analysis, which is the gold standard, and multiplex real time PCR assay, the sensitivity, specificity, positive predictive value, and negative predictive value of PCV2 genotyping were found to be 97.1% (n = 67, 95% CI 0.894–0.998, p < 0.001), 100% (n = 93, 95% CI 0.966–1.000, p < 0.001), 100% (95% CI 0.953–1.000, p < 0.001), 97.9% (95% CI 0.921–0.998, p < 0.001), respectively. The results of PCR-REBA were found to be consistent with those of sequence analysis for all the samples and showed good agreement (κ = 1). The most prevalent genotypes detected in this study were PCV2d (n = 53, 60.9%), followed by PCV2a (n = 17, 19.5%), PCV2b (n = 14, 16.1%), and PCV2a/b co-infection (n = 3, 3.5%). Both the methods required ~3 h for completion. Therefore, we conclude that two molecular methods are rapid and reliable for the characterization of the causative pathogen with PCV2 genotypes.

Evaluation of multiplex SYBR green real-time PCR assay for detection of pathogenic Escherichia coli

Pathogenic Escherichia coli (E. coli) has been implicated in a wide range of disease causing infections. It is essential to generate a method for detecting and differentiating each pathotype of E. coli which is more quickly and efficiently by using less reagent. This study aimed to evaluate a SYBR Green multiplex real-time PCR method for detecting four types of pathogenic E. coli. Two of multiplex real-time PCR system, 6-plex and 3-plex, were set to detect six different virulence factors from ETEC, EPEC, EHEC, and EIEC and evaluate the melting curves and specificity compared to simplex method. The results showed that 3-plex rt-PCR method gave more reliable melting curves than 6-plex. The 3-plex rt-PCR also provided similar melting value (Tm) to simplex system. The results of this specificity assay supported the selection of 3-plex rt-PCR conditions for detection of pathogenic E. coli.

Multiplex real-time PCR for the detection of Clavibacter michiganensis subsp. michiganensis, Pseudomonas syringae pv. tomato and pathogenic Xanthomonas species on tomato plants.

A multiplex real-time PCR method based on fluorescent TaqMan® probes was developed for the simultaneous detection of the tomato pathogenic bacteria Clavibacter michiganensis subsp. michiganensis, Pseudomonas syringae pv. tomato and bacterial spot-causing xanthomonads. The specificity of the multiplex assay was validated on 44 bacterial strains, including 32 target pathogen strains as well as closely related species and nontarget tomato pathogenic bacteria. The designed multiplex real-time PCR showed high sensitivity when positive amplification was observed for one pg of bacterial DNA in the cases of Clavibacter michiganensis subsp. michiganensis and Pseudomonas syringae pv. tomato bacteria and 100 pg for bacterial spot-causing xanthomonads. The reliability of the developed multiplex real-time PCR assay for in planta detection was verified by recognition of the target pathogens in 18 tomato plants artificially inoculated by each of the target bacteria and tomato samples from production greenhouses.

Rapid Multiplex Real-Time PCR Method for the Detection and Quantification of Selected Cariogenic and Periodontal Bacteria.

Dental caries and periodontal diseases are associated with a shift from symbiotic microbiota to dysbiosis. The aim of our study was to develop a rapid, sensitive, and economical method for the identification and quantification of selected cariogenic and periodontal oral bacteria. Original protocols were designed for three real-time multiplex PCR assays to detect and quantify the ratio of 10 bacterial species associated with dental caries (“cariogenic” complex) or periodontal diseases (red complex, orange complex, and Aggregatibacter actinomycetemcomitans). A total number of 60 samples from 30 children aged 2–6 years with severe early childhood caries and gingivitis were tested. In multiplex assays, the quantification of total bacterial (TB) content for cariogenic bacteria and red complex to eliminate differences in quantities caused by specimen collection was included. The mean counts for the TB load and that of ten evaluated specimens corresponded to previously published results. We found a significant difference between the microbial compositions obtained from the area of control and the affected teeth (p < 0.05). Based on this comprehensive microbiological examination, the risk of dental caries or periodontal inflammation may be determined. The test could also be used as a tool for behavioral intervention and thus prevention of the above-mentioned diseases.

Simultaneous detection of classical PRRSV, highly pathogenic PRRSV and NADC30-like PRRSV by TaqMan probe real-time PCR

Porcine Reproductive and Respiratory Syndrome (PRRS), an acute infectious disease caused by the porcine reproductive and respiratory syndrome virus (PRRSV), is one of the most devastating diseases affecting the global swine industry. In order to establish a multiplex real-time PCR method for the simultaneous detection of the classical PRRSV (C-PRRSV) strain, the highly pathogenic PRRSV (HP-PRRSV) strain and NADC30-like PRRSV (NL-PRRSV) strain, we designed specific primers and TaqMan fluorescent probes based on the Nsp2 target gene sequence of these three different PRRSV strains, and designed American-type PRRSV (PRRSV-U) special primers and probes based on the relatively conserved target gene sequence of ORF7. The method established in this study can quickly and accurately detect and differentiate three types of strains of clinical tissue samples, respectively. This method plays a key role in the rapid diagnosis and determination of PRRSV.

1777. Metagenomic Approach for the Detection of Viruses in Stool Samples from Infants and Children with Acute Gastroenteritis in Kuwait

BackgroundMetagenomics techniques are target-independent tools that enable the identification of uncommon disease etiologies and genomic characterization of all microorganisms present in a sample in less time and at a lower cost than previous sequencing techniques. In this study, we developed a metagenomic approach using next-generation sequencing technology to identify known and unknown viruses in stool samples from infants and children with acute gastroenteritis in Kuwait.MethodsWe have investigated 84 stool samples from infants and children aged one month to 10 years old with signs and symptoms of gastroenteritis who attended Mubarak Al-Kabeer and Al-Amiri hospitals in Kuwait from January to December 2017 using both multiplex real-time PCR and metagenomics sequencing (Illumina Miseq instrument) methods.ResultsThe metagenomics analysis of viral sequences verified that human adenovirus was the leading cause of gastroenteritis among infants and children in Kuwait, and was detected in 23% of the samples, rotavirus A was detected in 16% of the samples, and the combined infection of human adenovirus and rotavirus was detected in 7% of the samples. Also, newly discovered viruses known to cause gastroenteritis were identified; astrovirus MLB2 and primate bocaparvovirus-1 were detected in 5% of the samples. Also, each of the following new viruses was detected in 2% of the samples; aichivirus A, cardiovirus, parechovirus A, astrovirus VA4, cosavirus F, and bufavirus-3. On the other hand, multiplex real-time PCR showed that the combined infection of human adenovirus and rotavirus was the leading cause of gastroenteritis among infants and children in Kuwait, which was detected in 27% of the samples. However, the rotavirus was the second most common cause of diarrhea, which was detected in 20% of the samples. And the human adenovirus alone was detected in 18% of the samples. Our results showed a 69% agreement between both methods. By applying the Cohen’s Kappa statistics for a measure of agreement, the result gave fair agreement between the two methods (k = 0.388, P = 0.0).ConclusionOur findings revealed the capability of a metagenomic approach to detect many viruses causing gastroenteritis in stool samples from infants and children in Kuwait.Disclosures All authors: No reported disclosures.

A multiplex real-time PCR method applied to detect eight pollen species in food for the prevention of allergies

Pollen-induced anaphylaxis is recognized as the most common allergic disease. Methods for pollen species identification are thus regarded as important and urgent for the protection of sensitive consumers. In our study, a multiplex real-time PCR assay was established targeting eight pollens that have been reported recently as potential allergens or were uncommon species that have been newly exploited for marketing purposes (pine pollen, canola bee pollens, kiwi bee pollens, willow bee pollens, corn poppy bee pollens, rose bee pollens, lotus bee pollens, and camellia bee pollens). This method proved to be highly specific and efficient, with four pollen species being identified simultaneously from a single reaction. The absolute sensitivity was 1 pg/μL to 1 ng/μL for DNA from the eight pollen species, and as low as 0.5% (w/w) mixed bee pollen powder could be discriminated. A microscopy method using SEM was compared with our multiplex real-time PCR method. Results demonstrated that SEM was much less sensitive than multiplex real-time PCR, with the sensitivity of the former being approximately 10–50% (w/w).

Development of a rapid and reliable single-tube multiplex real-time PCR method for HLA-A*24:02 genotyping.

Aim:HLA-A*24:02 is significantly associated with cutaneous adverse drug reactions caused by aromatic antiepileptic drugs. Here, we aimed to establish a fast and reliable detection method for HLA-A*24:02 genotyping. Methods: A single-tube multiplex quantitative real-time polymerase chain reaction(qPCR) assay for HLA-A*24:02 genotyping was established by combining allele-specific primers with TaqMan probes. Results: A 100% concordance was observed between qPCR and SBT result in 106 Han subjects. The detection limit of the new method was 0.05ng DNA. The positive rate of HLA-A*24:02 in Tibetans (55.6%, n=81) was significantly higher than those in Han (34%, n=106), Uighur (27.5%, n=102), Bouyei (25.9%, n=116) and Miao populations (26.5%, n=113). Conclusion: The newly established qPCR assay was reliable for HLA-A*24:02 screening in clinical applications.

Rapid Multiplex Genotyping of 20 HLA-A*02:01 Restricted Minor Histocompatibility Antigens.

A subset of MHC-associated self-peptides presented by the recipient's cells and immunologically foreign to the donor can induce an allogeneic immune response after hematopoietic stem cell transplantation (HSCT). These immunogenic peptides originate from the genomic polymorphisms and are known as minor histocompatibility antigens (MiHA). MiHA mismatches trigger the post-transplant immune response, which could manifest in both the deleterious “graft-vs.-host” disease and the beneficial “graft-vs.-leukemia” effect. Importantly, some MiHAs are considered to be promising targets for posttransplant T-cell immunotherapy of hematopoietic malignancies. This creates a demand for a robust and fast approach to genotyping MiHA-encoding polymorphisms. We report a multiplex real-time PCR method for the genotyping of 20 polymorphisms that are encoding HLA-A*02:01-restricted MiHAs. This method uses allele-specific primers and gene-specific hydrolysis probes. In 1 h it allows for the detection of MiHA mismatches in a donor-recipient pair without the need for electrophoresis, sequencing, or other time-consuming techniques. We validated the method with Sanger and NGS sequencing and demonstrated good performance over a wide range of DNA concentrations. We propose our protocol as a fast and accurate method of identifying mismatched MiHAs. The information on the MiHA mismatches is useful for studying the allogeneic immune response following HSCT and for selecting the targets for post-transplant T-cell therapy.

Simultaneous detection of five pig viruses associated with enteric disease in pigs using EvaGreen real-time PCR combined with melting curve analysis

In recent years, a series of porcine diarrhea viruses such as porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), rotaviruses of group A (RVA), rotaviruses of group C (RVC), and porcine circovirus 2 (PCV2) caused enormous economic losses all over the world. While any of these viruses is capable to cause disease alone, there is often concurrent infection with more than one virus on pig farms. In this study, a multiplex real-time PCR method based on EvaGreen fluorescent dye and melting curve analysis was established to simultaneously detect these five viruses in a single closed tube. Five distinct melt peaks were obtained with different melting temperature (Tm) value corresponding to each of the five viruses. This method was highly sensitive to detect and distinguish TGEV, RVA, RVC, PEDV and PCV2 with the limits of detection ranging from 5 to 50 copies/μL. The intra-assay and inter-assay reproducibility were good with coefficient of variation of Tm and cycle threshold values less than 0.32% and 2.86%, respectively. Testing of 90 field samples by the single and multiplex real-time PCR assays demonstrated a concordance of 91.1%. Thus, the EvaGreen multiplex real-time PCR is a rapid, sensitive and low-cost diagnostic tool for differential detection and routine surveillance of TGEV, RVA, RVC, PEDV and PCV2 in pigs.

A multiplex real-time PCR method for differentiation of beef, buffalo meat and pork

The objective of this study was to optimize a multiplex real-time PCR protocol for detection of DNA of beef, buffalo meat and pork, serving for food authenticity. The optimized concentrations were 200 nM primer and 100 nM specific probe for beef/buffalo meat, and 300 nM primer and 150 nM probe for pork. The amplification was performed using initial denaturation at 50oC for 2 min, 95oC for 2 min, followed by 45 cycles of denaturation at 95oC for 15 sec, and annealing and extension at 60oC for 40 sec. This protocol had high sensitivity and specificity. The detection limit of this method was found to be 0.1% in raw and heat-treated meat mix (80 – 121oC/15 min) or 0.005 ng DNA/reaction. The protocol of testing was applied for the commercial products both fresh and processed meats. The results demonstrated that 50% of raw beef sample (6/12) weren't found beef DNA. Eight of twelve beef sausage samples (66.67%) contained buffalo DNA. Beef DNA were found in all 12 samples of beef meatballs, but eight out of the 12 meatball samples were confirmed to have buffalo DNA (66.67%) and two out of the 12 meatball samples (16.67%) also contained porcine DNA.

Multiplex real-time PCR for the detection of insect DNA and determination of contents of Tenebrio molitor, Locusta migratoria and Achaeta domestica in food

In Europe, edible insects are not a part of a common diet. But according to the Food and Agriculture Organization of the united nations (FAO), animal protein from insects could be an ecological, economic and healthy addition to human diets. In the EU there are currently no regulations on insects as food for human consumption. The production and sales of products containing insects are not prohibited. Several companies are producing and marketing food products containing insects already. In Switzerland, the recently revised food law allows and regulates insect production and products for human consumption. Like other ingredients, such food additives, insects have to be labeled correctly. To enforce such labeling, food control laboratories need analytical tools to detect insect ingredients and determine their species and quantity. We, therefore, developed a multiplex real-time PCR method detecting insect DNA generally and the three approved species specifically. The performance of this multiplex real-time PCR method was assessed during validation including data from other laboratories. These results indicate that the method is fit for purpose.

Fast Multiplex real time PCR method for sex-identification of medaka (Oryzias latipes) by non-invasive sampling

Medaka fish (Oryzias latipes) has been widely used in fish screening and multi-generation tests to provide relevant data to assess impacts of endocrine disrupting chemicals (EDCs) in fish populations.The genotypic differentiation of Medaka sex allows diagnosing the sex reversal, and is required in current test guidelines (e.g. OECD TG 240, 2015). DNA isolation for genetic sex-identification requires sample collection, which has been normally conducted using invasive (fish sacrifice) or semi-invasive (fin-clip) procedures, which conflicts with the need for a fast, simple, and stress-free method. Swabbing skin mucus to collect DNA has been adopted in ecological studies of larger fish, however for smaller fish, it has to be established.To handle larger number of samples, real-time PCR represents a faster and sensitive method compared to conventional PCR. In this study, we aimed to develop a multiplex real-time PCR method for Medaka genetic sex-identification, using DNA sampled by swabbing as less invasive technique. In this approach, the male-determining gene DMY was used in combination with the cytochrome b housekeeping gene.•The method developed is a robust, rapid and a sensitive multiplex real-time PCR for Medaka genetic sex-identification.•This method allows the use of DNA isolated from fish by swabbing, as non-invasive sampling method.

Development and validation of multiplex real-time PCR assays for rapid detection of cytomegalovirus, Epstein-Barr virus, and polyomavirus BK in whole blood from transplant candidates.

Transplant recipients are more susceptible to bacterial and viral infections. Cytomegalovirus (CMV), Epstein-Barr virus (EBV), and polyomavirus BK (BK) are risk factors for graft dysfunction. All three of them are latent viruses that can cause serious disease in immunocompromised patients. Mainly qualitative PCR tests are required for diagnosis and quantitative monitoring, which are used to follow the response to transplantation. We developed a multiplex real-time PCR (qPCR) method to detect these viruses during blood screenings of transplant recipients. We also validated analytical and clinical performance tests using the developed multiplex qPCR. The limit of detection (LOD) was 100, 125, and 183 copies/ml for CMV, EBV, and BK, respectively. These results had high linearity (R2 = 0.997) and reproducibility (CV range, 0.95-2.38%, 0.52-3.32%, and 0.31-2.45%, respectively). Among 183 samples, we detected 8 samples that were positive for CMV, while only 6 were positive for EBV, and 3 were positive for BK. Therefore, the viral infection prevalence in transplant candidates was 4.40% for CMV, 3.29% for EBV, and 1.64% for BK. This multiplex qPCR method should be used widely for diagnosing and monitoring latent viral infections in transplant recipients.

Development of a multiplex real-time PCR method for early diagnosis of three bacterial diseases in fish: A real-case study in trout aquaculture

Lactococcus garvieae, Yersinia ruckeri and Flavobacterium psychrophilum are three of the most important pathogens in worldwide rainbow trout aquaculture (Oncorrhynchus mykiss Walbaum). In this work, a multiplex quantitative PCR (qPCR) method for the simultaneous detection of the three pathogens was developed and tested in a commercial trout farm. Fifty-four spleen and brain samples from a trout farm were analyzed using both multiplex qPCR and classic microbiological methods. When comparing qPCR results and classic plate diagnostic techniques no negative deviations were observed, which resulted in a 100% relative sensitivity of the multiplex qPCR method developed. In addition, efficiency of the qPCR method ranged between 97.5% and 108.8%. The specificity of the combination of primers and probes was successfully tested in 57 target and non-target bacterial strains Hence, the multiplex qPCR method developed in the present study could be used as a reliable diagnostic tool for the detection of L. garvieae, Y. ruckeri and F. psychrophilum in rainbow trout farms, enabling faster diagnostics (only a few hours) and contributing to a quick response and management of bacterial disease outbreaks.

New rapid method to detect BCR-ABL fusion genes with multiplex RT-qPCR in one-tube at a time

Fast identification of BCR-ABL fusion genes is critical for precise diagnosis, risk stratification and therapy scheme selection in leukemia. More convenient methods are needed for quickly detection of the BCR-ABL fusion genes. Multiplex fluorescent reverse transcription quantitative real-time PCR (Multiplex RT-qPCR) methods are developed for detection of the at least 14 subtypes of BCR-ABL fusion genes in one tube at a time by using patients’ bone marrow samples. The new Multiplex RT-qPCR method could quickly detect BCR-ABL fusion genes with sensitivity up to 10–106 copies. It can detect the fusion genes in patients’ bone marrow samples containing any subtypes of the major bcr (M-bcr) e13a2, e14a2, e13a3 and e14a3, the minor bcr (m-bcr) e1a2 and e1a3, the micro bcr (μ-bcr) e19a2 and e19a3, and the nano bcr (n-bcr) e6a2 and e6a3. The specificity is comparable to the FISH methods. The cutoff for clinical diagnosis of BCR-ABL(+) is also determined by testing in clinical chronic myeloid leukemia samples. This is a new fast method with high sensitivity and specificity for clinical detection of BCR-ABL fusion genes. It will benefit the precise diagnosis, targeted therapy and minimal residual disease (MRD) monitoring in leukemia.

Clinical utility of an optimised multiplex real-time PCR assay for the identification of pathogens causing sepsis in Vietnamese patients

IntroductionFor the identification of bacterial pathogens, blood culture is still the gold standard diagnostic method. However, several disadvantages apply to blood cultures, such as time and rather large volumes of blood sample required. We have previously established an optimised multiplex real-time PCR method in order to diagnose bloodstream infections. Material and methodsIn the present study, we evaluated the diagnostic performance of this optimised multiplex RT-PCR in blood samples collected from 110 septicaemia patients enrolled at the 108 Military Central Hospital, Hanoi, Vietnam. ResultsPositive results were obtained by blood culture, the Light Cylcler-based SeptiFast® assay and our multiplex RT-PCR in 35 (32%), 31 (28%), and 31 (28%) samples, respectively. Combined use of the three methods confirmed 50 (45.5%) positive cases of bloodstream infection, a rate significantly higher compared to the exclusive use of one of the three methods (P=0.052, 0.012 and 0.012, respectively). The sensitivity, specificity and area under the curve (AUC) of our assay were higher compared to that of the SeptiFast® assay (77.4%, 86.1% and 0.8 vs. 67.7%, 82.3% and 0.73, respectively). Combined use of blood culture and multiplex RT-PCR assay showed a superior diagnostic performance, as the sensitivity, specificity, and AUC reached 83.3%, 100%, and 0.95, respectively. The concordance between blood culture and the multiplex RT-PCR assay was highest for Klebsiella pneumonia (100%), followed by Streptococcus spp. (77.8%), Escherichia coli (66.7%), Staphylococcus spp. (50%) and Salmonella spp. (50%). In addition, the use of the newly established multiplex RT-PCR assay increased the spectrum of identifiable agents (Acintobacter baumannii, 1/32; Proteus mirabilis, 1/32). ConclusionThe combination of culture and the multiplex RT-PCR assay provided an excellent diagnostic accomplishment and significantly supported the identification of causative pathogens in clinical samples obtained from septic patients.

Application of real-time PCR for early diagnosis of diseases caused by Aeromonas salmonicida, Vibrio anguillarum, and Tenacibaculum maritimum in turbot: A field study

ABSTRACTIn the present study a multiplex real-time PCR method was developed for early detection of diseased fish infected by Aeromonas salmonicida, Vibrio anguillarum, and/or Tenacibaculum maritimum. The method consisted of the detection of three species-specific genes after DNA extraction with a commercial kit. Three types of samples were tested, and the results were compared with those of traditional diagnosis. The method obtained a limit of detection of 104 cfu/mL (2 x 102 cfu/tube). Additionally, 27 samples from fish showing signs of disease were correctly diagnosed by the developed methodology, demonstrating its suitability for implementation in aquaculture.

Molecular diagnosis of Shigella, Salmonella and Campylobacter by multiplex Real-time PCR in stool culture samples in Ouagadougou (Burkina Faso)

ABSTRACT:Background: Bacteriological diagnosis of Campylobacter spp, Salmonella spp and Shigella spp could be necessary in the case of infectious gastroenteritis syndrome.The objective of this study was to diagnose concomitantly the three enteropathogenic bacteria by multiplex Real-Time PCR in stool culture samples in Ouagadougou (Burkina Faso).Materials and Methods: The study was conducted from February 5th to March 9th, 2013. Two hundred stool samples were received during the study period. The bacteria were identified by bacterial culture following by multiplex Real-Time PCR.Results: Shigella spp and Campylobacter spp were sought by culture in all 200 samples. Enteropathogenic E. coli was sought only in 37 samples from all children under 2 years old. The bacterial culture was positive in 12 stool samples. Shigella spp and Salmonella spp. were isolated respectively in 5 (2.5%) and 3 samples (1.5%). Enteropathogenic E. coli was isolated in 10.8% (4/37) of the samples tested.The multiplex real-time PCR identified bacteria in 20 patients, including 17 cases of Shigella spp., 1 case of Salmonella spp. and 2 cases of Campylobacter spp.Conclusions: This study has highlighted the low frequency of 3 sought bacterial genera in stool samples. It has also demonstrated a significant difference between the culture and the multiplex Real-Time PCR method in the diagnosis of Shigella.

Detection of Mycobacteria by Culture and DNA-Based Methods in Animal-Derived Food Products Purchased at Spanish Supermarkets.

Mycobacteria include obligate and opportunistic pathogens that cause significant human and animal disease. The burden of tuberculosis has been largely reduced in developed territories but remains a huge problem worldwide. The significance of nontuberculous mycobacteria is growing considerably, especially in developed regions with higher life expectancy and more therapy-related immunosuppressed individuals. Due to their robustness mycobacteria can contaminate animal products by direct transmission from infected individuals or by environmental contamination during processing. The situation at market level is poorly known. Most studies analyzing commercially available foods are limited to a small or local scale and mainly focused on a particular mycobacterial species. There is a need to investigate if animal products that have passed the established controls to be for sale at main supermarkets could represent a route of contact with any mycobacteria. Thus, our goal was to study the prevalence of mycobacteria in these foods to assess if this could represent a source of human exposure. Five stores from the main supermarket chains in Spain were selected. 138 dairy and 119 meat products were purchased. All were processed using culture and multiplex real-time PCR methods. Additional molecular methods were used to specifically identify any positive result. Mycobacterium avium subsp. hominissuis (2), M. avium subsp. avium (1), and M. fortuitum (1) were isolated from powdered infant formula and ground beef, chicken sausage, and mortadella cold cut, respectively. Mycobacterial DNA (M. avium, M. tuberculosis complex and other nontuberculous mycobacteria) was detected in 15% of dairy products and 2% of meat products. These results show that the prevalence of viable mycobacteria in foods of animal origin obtained at the supermarket was not substantial although a considerable proportion of them contained mycobacterial DNA. Contact with mycobacteria through this route could be ensured over time. Further investigation is necessary to determine the real impact of foodborne mycobacterial exposure on human health and identify critical points in the food production system to enable setting up more stringent control measures.