Probe Real-time Polymerase Chain Reaction Research Articles

Genetic Factors of Teeth Impaction: Polymorphic and Haplotype Variants of PAX9, MSX1, AXIN2, and IRF6 Genes.

In recent research, there has been a growing awareness of the role of genetic factors in the positioning and eruption of teeth in the maxilla and mandible. This study aimed to evaluate the potential of specific polymorphic markers of single nucleotide polymorphisms (SNPs) located within the PAX9, MSX1, AXIN2, and IRF6 genes to determine the predisposition to tooth impaction. The study participants were divided into two groups: the first group consisted of individuals with at least one impacted secondary tooth. In contrast, the second group (control group) had no impacted teeth in their jaws. To analyze the genes, real-time PCR (polymerase chain reaction) and TaqMan probes were utilized to detect the selected polymorphisms. The findings suggest that disruptions in the structure and function of the mentioned genetic factors such as polymorphic and haplotype variants of PAX9, MSX1, AXIN2, and IRF6 genes, which play a direct role in tooth and periodontal tissue development, might be significant factors in tooth impaction in individuals with genetic variations. Therefore, it is reasonable to hypothesize that tooth impaction may be influenced, at least in part, by the presence of specific genetic markers, including different allelic variants of the PAX9, AXIN2, and IRF6 genes, and especially MSX1.

Whole-Genome Analysis of Influenza A(H3N2) and B/Victoria Viruses Detected in Myanmar during the COVID-19 Pandemic in 2021.

An influenza circulation was observed in Myanmar between October and November in 2021. Patients with symptoms of influenza-like illness were screened using rapid diagnostic test (RDT) kits, and 147/414 (35.5%) upper respiratory tract specimens presented positive results. All RDT-positive samples were screened by a commercial multiplex real-time polymerase chain reaction (RT-PCR) assay, and 30 samples positive for influenza A(H3N2) or B underwent further typing/subtyping for cycle threshold (Ct) value determination based on cycling probe RT-PCR. The majority of subtyped samples (n = 13) were influenza A(H3N2), while only three were B/Victoria. Clinical samples with low Ct values obtained by RT-PCR were used for whole-genome sequencing via next-generation sequencing technology. All collected viruses were distinct from the Southern Hemisphere vaccine strains of the corresponding season but matched with vaccines of the following season. Influenza A(H3N2) strains from Myanmar belonged to clade 2a.3 and shared the highest genetic proximity with Bahraini strains. B/Victoria viruses belonged to clade V1A.3a.2 and were genetically similar to Bangladeshi strains. This study highlights the importance of performing influenza virus surveillance with genetic characterization of the influenza virus in Myanmar, to contribute to global influenza surveillance during the COVID-19 pandemic.

Rapid Diagnostic Testing for Response to the Monkeypox Outbreak - Laboratory Response Network, United States, May 17-June 30, 2022.

As part of public health preparedness for infectious disease threats, CDC collaborates with other U.S. public health officials to ensure that the Laboratory Response Network (LRN) has diagnostic tools to detect Orthopoxviruses, the genus that includes Variola virus, the causative agent of smallpox. LRN is a network of state and local public health, federal, U.S. Department of Defense (DOD), veterinary, food, and environmental testing laboratories. CDC developed, and the Food and Drug Administration (FDA) granted 510(k) clearance* for the Non-variola Orthopoxvirus Real-time PCR Primer and Probe Set (non-variola Orthopoxvirus [NVO] assay), a polymerase chain reaction (PCR) diagnostic test to detect NVO. On May 17, 2022, CDC was contacted by the Massachusetts Department of Public Health (DPH) regarding a suspected case of monkeypox, a disease caused by the Orthopoxvirus Monkeypox virus. Specimens were collected and tested by the Massachusetts DPH public health laboratory with LRN testing capability using the NVO assay. Nationwide, 68 LRN laboratories had capacity to test approximately 8,000 NVO tests per week during June. During May 17-June 30, LRN laboratories tested 2,009 specimens from suspected monkeypox cases. Among those, 730 (36.3%) specimens from 395 patients were positive for NVO. NVO-positive specimens from 159 persons were confirmed by CDC to be monkeypox; final characterization is pending for 236. Prompt identification of persons with infection allowed rapid response to the outbreak, including isolation and treatment of patients, administration of vaccines, and other public health action. To further facilitate access to testing and increase convenience for providers and patients by using existing provider-laboratory relationships, CDC and LRN are supporting five large commercial laboratories with a national footprint (Aegis Science, LabCorp, Mayo Clinic Laboratories, Quest Diagnostics, and Sonic Healthcare) to establish NVO testing capacity of 10,000 specimens per week per laboratory. On July 6, 2022, the first commercial laboratory began accepting specimens for NVO testing based on clinician orders.

LoopTag FRET Probe System for Multiplex qPCR Detection of Borrelia Species.

Background: Laboratory diagnosis of Lyme borreliosis refers to some methods with known limitations. Molecular diagnostics using specific nucleic acid probes may overcome some of these limitations. Methods: We describe the novel reporter fluorescence real-time polymerase chain reaction (PCR) probe system LoopTag for detection of Borrelia species. Advantages of the LoopTag system include having cheap conventional fluorescence dyes, easy primer design, no restrictions for PCR product lengths, robustness, high sequence specificity, applicability for multiplex real-time PCRs, melting curve analysis (single nucleotide polymorphism analysis) over a large temperature range, high sensitivity, and easy adaptation of conventional PCRs. Results: Using the LoopTag probe system we were able to detect all nine tested European species belonging to the Borrelia burgdorferi (sensu lato) complex and differentiated them from relapsing fever Borrelia species. As few as 10 copies of Borrelia in one PCR reaction were detectable. Conclusion: We established a novel multiplex probe real-time PCR system, designated LoopTag, that is simple, robust, and incorporates melting curve analysis for the detection and in the differentiation of European species belonging to the Borrelia burgdorferi s.l. complex.

Performance Comparison of Multiplexed Fluorescent Resonance Emission Transfer Hybridization Probes Across Roche LightCycler® Real-Time PCR Systems for the Detection of Bartonella species

Abstract Introduction/Objective Molecular assays for Bartonella species are important in diagnosing infection and expediting patient treatment. Real time polymerase chain reaction (RT-PCR) using fluorescent resonance energy transfer (FRET) hybridization probes can be used to detect Bartonella species in blood and fresh/fixed tissue biopsies in RT-PCR instruments. Over time, new technologies and reagents are introduced and existing PCR primers and FRET probes must be re-validated on new platforms. This study aimed to compare the performance of a Bartonella RT-PCR assay using the sunsetting Roche LightCycler® 2.0 (Roche Diagnostics, Indianapolis, IN) and newer LightCycler® 480 RT- PCR instruments. Methods/Case Report DNA was extracted from 132 historically positive, whole organism spiked, and historically negative whole blood and formalin fixed paraffin embedded (FFPE) samples. Samples were run on the LightCycler® 2.0 using instrument specific LightCycler® FastStart DNA Master HybProbe enzyme and compared to results generated using the LightCycler® 480 and its instrument specific LightCycler® 480 Genotyping Master enzyme. During optimization, MgCl2 concentrations and thermocycling profiles were adjusted. Accuracy, specificity, inclusivity, and limit of detection studies were performed. Crossing point (Cp), melting temperature (Tm), fluorescent peak and fluorescent background values were compared between the two instruments. Results (if a Case Study enter NA) The agreement in accuracy between the LightCycler® 2.0 and the LightCycler® 480 was 100% for whole blood samples. For historically positive FFPE samples, LightCycler® 2.0 sensitivity and LightCycler® 480 sensitivity were 86% and 100%, respectively. Specificity and inclusivity of the assay were identical between the two instruments. The limit of detection in whole blood was 5-fold lower on the LightCycler® 480 (50 copies/µL) compared to the LightCycler® 2.0 (250 copies/µL). Mean Cp and fluorescent peak intensity values increased by 5.1% and 65-fold, respectively. Conclusion The study demonstrates similar performance and improved limit of detection for the Bartonella FRET hybridization probe RT-PCR assay on the LightCycler® 480 compared to the LightCycler® 2.0.

Prevalence of H63D and C282Y mutations in hereditary hemochromatosis (HFE) gene in Tripoli region of Libya

Background and Aims: Hereditary hemochromatosis (HH) is an autosomal recessive disorder, characterized by increased intestinal absorption of iron. Excessive amount of iron accumulates in the liver, pancreas, and heart, etc., and eventually leading to organ failure due to iron toxicity and death if untreated. The most common causes of HH are the C282Y and H63D mutations in HFE gene. This study aimed to identify the prevalence of H63D and C282Y alleles among the Libyan population in Tripoli region and to compare the results with other published data. Materials and Methods: This study included 300 randomly selected unrelated Libyan male blood donors, aged between 18 and 50 years. In-house hydrolysis probe real-time polymerase chain reaction and high-resolution melting analysis protocols were developed and employed as screening tools for H63D and C282Y genotyping, respectively, and direct DNA sequencing was used to confirm the results. Results: Seven subjects (2.33%) were detected as homozygous H63D mutation and 72 (24%) were detected as heterozygous, and only one subject was detected as a heterozygous C282Y mutant (0.33%) and no homozygous C282Y mutation was detected. Conclusion: In Libyans residing in Tripoli region, the allele frequency of C282Y was very rare and allele frequency of H63D was common.

Detection of Porcine Adulteration in Cosmetic Cream Formulation via TaqMan Probe Real-Time Polymerase Chain Reaction.

The identification of lard adulterated in cosmetic cream using TaqMan probe real-time Polymerase Chain Reaction was developed in this research. The laboratory prepared cream was formulated with oil in water (o/w) emulsion with the adulteration of 1%, 3% and 5% of lard (w/w). The total DNA from lard-adulterated cosmetic cream was successfully extracted. The detection assay targeting different gene; universal 18S rRNA and porcine-specific multiple porcine repetitive element (MPRE) which employed with the TaqMan probe. This study targeting 187 bp of amplicons for 18S rRNA while 99 bp of amplicons for Porcine-specific probes. A comparison of quantification cycle (Cq) with prior to percentage of adulteration were detected. Lard-adulterated cream was amplified for 18S rRNA while no detection was detected for Porcine-specific probes. The Cq value obtained might be vary as proposed by the hypothesis depending on the total recovery of extracted DNA from the lard-adulterated cream.

Developing molecular diagnostics for detection of red imported fire ants using two genes, Sinv11108 and Sinv11977.

Aggressive red imported fire ants (RIFAs) are expanding their habitat due to active international trade and global warming. To prevent infestation and settlement, RIFAs must be removed during the quarantine process. Because RIFAs are social insects and have different morphological characteristics depending on their castes, non-ant taxonomists have difficulty confirming RIFAs based on their morphological characteristics alone. The disadvantages of previously reported RIFA molecular diagnostics are that they require additional steps, such as restriction enzyme digestion followed by agarose gel electrophoresis separation or DNA sequence verification for polymerase chain reaction (PCR)-amplified products. To overcome these drawbacks, two RIFA-specific genes were selected and used to develop diverse PCR-based RIFA molecular diagnostic techniques. We found that RIFAs could be confirmed by conventional PCR targeting of two RIFA-specific genes followed by agarose electrophoresis separation. In addition, TaqMan probe real-time PCR methods had the advantage of confirming RIFAs immediately after the reactions were completed by observing fluorescence indexes. Finally, multiplex PCRs enhanced RIFA specificity and sensitivity. The new molecular diagnostic methods developed in this study had the advantages of reducing false positive and negative results together with high specificity and sensitivity for RIFAs.

High Expression of FOXP3 mRNA in Blood and Urine as a Predictive Marker in Kidney Transplantation.

Diagnosis of allograft dysfunction by noninvasive biomarker tests is preferable to invasive allograft biopsies and has been extensively considered in recent years. This study aims to evaluate blood and urinary forkhead box P3 (FOXP3) messenger RNA (mRNA) expression in renal transplant recipients in an attempt to determine whether differential diagnosis of graft dysfunction is feasible using mRNA profiles. We analyzed FOXP3 mRNA expression in paired urinary and peripheral blood mononuclear cell (PBMC) samples. A total of 91 kidney transplant recipients enrolled in this study that were classified into 3 groups: biopsy-proven acute rejection (AR; n = 27), chronic allograft nephropathy (n = 19), and well-functioning graft (n = 45). The FOXP3 mRNA expression was quantified by TaqMan probe real-time polymerase chain reaction. Acute rejection patients had a higher expression level of transcription factor FOXP3 compared to the chronic nephropathy and control groups. Analysis of receiver operating characteristic curves showed that rejection could be diagnosed with 100% sensitivity and 96% specificity in urine, and 92% sensitivity and 86% specificity in PBMC samples using the optimal FOXP3 mRNA cutoff value. We subdivided the AR group into progressive and nonprogressive patients, which showed a significant difference in FOXP3 mRNA expression. This result confirmed the role of FOXP3 as a diagnostic marker in predicting transplantation outcomes. Our results suggested that elevated expression of FOXP3 in blood and urine samples from kidney transplant recipients could be a useful noninvasive biomarker to diagnose graft dysfunction.

Development of a real-time polymerase chain reaction assay for the detection of the invasive Mediterranean fanworm, Sabella spallanzanii, in environmental samples.

The Mediterranean fanworm, Sabella spallanzanii Gmelin 1791, was first detected in the Southern Hemisphere in the 1990s and is now abundant in many parts of southern Australia and in several locations around northern New Zealand. Once established, it can proliferate rapidly, reaching high densities with potential ecological and economic impacts. Early detection of new S. spallanzanii incursions is important to prevent its spread, guide eradication or control efforts and to increase knowledge on the species' dispersal pathways. In this study, we developed a TaqMan probe real-time polymerase chain reaction assay targeting a region of the mitochondrial cytochrome oxidase I gene. The assay was validated in silico and in vitro using DNA from New Zealand and Australian Sabellidae with no cross-reactivity detected. The assay has a linear range of detection over seven orders of magnitude with a limit of detection reached at 12.4×10-4ng/μL of DNA. We analysed 145 environmental (water, sediment and biofouling) samples and obtained positive detections only from spiked samples and those collected at a port where S. spallanzanii is known to be established. This assay has the potential to enhance current morphological and molecular-based methods, through its ability to rapidly and accurately identify S. spallanzanii in environmental samples.

Development of hydrolysis probe real-time polymerase chain reaction and high-resolution melting analysis protocols for screening of e280k and c.1055del.g mutations in phenylalanine hydroxylase gene

Background: Phenylketonuria (PKU) is one of the most common inborn errors of amino acids metabolism. It is an autosomal recessive disease that is caused by mutations in phenylalanine hydroxylase (PAH) gene. In the North Africa and Eastern Mediterranean region, E280K missense mutation and c.1055del.G frameshift mutation in PAH gene are one of the most common pathogenic mutations seen in PKU patients. Materials and Methods: In this study, we developed molecular protocols for rapid screening of the PKU patients for these two mutations. These protocols are based on hydrolysis probe real-time polymerase chain reaction technique using allele-specific probes labeled with 6-carboxyfluorescein (FAM) for wild-type (WT) and hexachloro-6-carboxyfluorescein (HEX) for mutant genotypes and Black Hole Quencher 1 as a quencher and high-resolution melting analysis using EvaGreen saturating dye. Results: There was complete accordance between the developed protocols in differentiating genotypes and they proved to be rapid, sensitive, and efficient for the detection and differentiation between WT, mutant, and heterozygous genotypes of the E280K and c.1055del.G mutations. Conclusions: These protocols allow easy molecular screening of the mutations studied among the families of affected people, especially for premarital screening.

Real-time PCR probe optimization using design of experiments approach

Primer and probe sequence designs are among the most critical input factors in real-time polymerase chain reaction (PCR) assay optimization. In this study, we present the use of statistical design of experiments (DOE) approach as a general guideline for probe optimization and more specifically focus on design optimization of label-free hydrolysis probes that are designated as mediator probes (MPs), which are used in reverse transcription MP PCR (RT-MP PCR). The effect of three input factors on assay performance was investigated: distance between primer and mediator probe cleavage site; dimer stability of MP and target sequence (influenza B virus); and dimer stability of the mediator and universal reporter (UR). The results indicated that the latter dimer stability had the greatest influence on assay performance, with RT-MP PCR efficiency increased by up to 10% with changes to this input factor. With an optimal design configuration, a detection limit of 3–14 target copies/10μl reaction could be achieved. This improved detection limit was confirmed for another UR design and for a second target sequence, human metapneumovirus, with 7–11 copies/10μl reaction detected in an optimum case. The DOE approach for improving oligonucleotide designs for real-time PCR not only produces excellent results but may also reduce the number of experiments that need to be performed, thus reducing costs and experimental times.

TaqMan probe real-time polymerase chain reaction assay for the quantification of canine DNA in chicken nugget

ABSTRACTThis paper describes a short-amplicon-based TaqMan probe quantitative real-time PCR (qPCR) assay for the quantitative detection of canine meat in chicken nuggets, which are very popular across the world, including Malaysia. The assay targeted a 100-bp fragment of canine cytb gene using a canine-specific primer and TaqMan probe. Specificity against 10 different animals and plants species demonstrated threshold cycles (Ct) of 16.13 ± 0.12 to 16.25 ± 0.23 for canine DNA and negative results for the others in a 40-cycle reaction. The assay was tested for the quantification of up to 0.01% canine meat in deliberately spiked chicken nuggets with 99.7% PCR efficiency and 0.995 correlation coefficient. The analysis of the actual and qPCR predicted values showed a high recovery rate (from 87% ± 28% to 112% ± 19%) with a linear regression close to unity (R2 = 0.999). Finally, samples of three halal-branded commercial chicken nuggets collected from different Malaysian outlets were screened for canine meat, but no contamination was demonstrated.

Molecular diagnostics: techniques and recommendations for 1p/19q assessment.

Several morphology- and polymerase chain reaction (PCR)-based methods for chromosome 1p 19q deletion status assessment are available. Important prerequisites for all molecular techniques concern tissue quality and selection of regions of interest. The most common methods for diagnostic 1p 19q assessment are fluorescence in situ hybridization and PCR-based microsatellite analysis. While the latter requires the use of autologous blood samples, more advanced techniques such as array comparative genomic hybridization, multiplex ligation-dependent probe amplification or real-time PCR are independent from autologous DNA samples. However, due to high technical demand and experience required their applicability as diagnostic tests remains to be shown. On the other hand, chromogenic in situ hybridization evolves as attractive alternative to FISH. Herein, the available test methods are reviewed and outlined, their advantages and drawbacks being discussed in detail.

Development and validation of a novel hydrolysis probe real-time polymerase chain reaction for agamid adenovirus 1 in the central bearded dragon (Pogona vitticeps).

Agamid adenovirus 1 (AgAdv-1) is a significant cause of disease in bearded dragons (Pogona sp.). Clinical manifestations of AgAdv-1 infection are variable and often nonspecific; the manifestations range from lethargy, weight loss, and inappetence, to severe enteritis, hepatitis, and sudden death. Currently, diagnosis of AgAdv-1 infection is achieved through a single published method: standard nested polymerase chain reaction (nPCR) and sequencing. Standard nPCR with sequencing provides reliable sensitivity, specificity, and validation of PCR products. However, this process is comparatively expensive, laborious, and slow. Probe hybridization, as used in a TaqMan assay, represents the best option for validating PCR products aside from the time-consuming process of sequencing. This study developed a real-time PCR (qPCR) assay using a TaqMan probe-based assay, targeting a highly conserved region of the AgAdv-1 genome. Standard curves were generated, detection results were compared with the gold standard conventional PCR and sequencing assay, and limits of detection were determined. Additionally, the qPCR assay was run on samples known to be positive for AgAdv-1 and samples known to be positive for other adenoviruses. Based on the results of these evaluations, this assay allows for a less expensive, rapid, quantitative detection of AgAdv-1 in bearded dragons.

Rapid and Sensitive Identification of Bacterial Infection and Bacteria Gram Types in Pleural Fluid of Children.

Real-time polymerase chain reaction (RT-PCR) techniques have been increasingly used to detect microbial DNA in clinic for the diagnosis of bacterial infection. This study aims to developing an RT-PCR method to detect bacteria in pleural fluid (PF). We performed a method to simultaneously detect and classify the clinically relevant bacterial pathogens in hydrothorax with Gram probe RT-PCR (GRT-PCR), which targets the conserved region of the 16S rRNA gene. Our results showed this method could specifically and correctly identify 14 clinically important bacterial strains in hydrothorax including 7 gram-positive and 7 gram-negative bacteria. And the sensitivity of this GRT-PCR method in serial dilution can reach 10 CFU/mL. In clinical trial, 180 PF samples from children who were clinically suspected to suffer from bacterial pneumonia and empyema were collected. These samples were detected by GRT-PCR, standard culture, and biochemical routine analysis. The positive rate of the GRT-PCR array was 17.78% (32/180), significantly higher than that of PF culture (11.67%; 21/180; P = .003). When PF culture was used as control, the sensitivity of GRT-PCR was 95.24% (95% confidence interval = 74.13-99.75), and the specificity was 92.45% (95% confidence interval = 86.89-95.86). Our study showed that GRT-PCR is a more effective method for rapid, sensitive, and specific diagnosis of bacterial infection in hydrothorax compared with other traditional methods.

Molecular diagnosis of childhood tuberculosis: history and present

Childhood tuberculosis remains a serious public health threat which draws more and more attention.The unspecific symptoms or signs and the paucibacillary nature of childhood tuberculosis make it hard to diagnose which delays appropriate treatment and increases disease burden and mortality.Molecular methods such as polymerase chain reaction (PCR), real-time PCR, and DNA line probe based on mycobacterium tuberculosis specific nucleotide sequence will make a quick and reliable diagnosis of childhood tuberculosis. Key words: Mycobacterium tuberculosis; Child; Molecular diagnosis

Effects of schizophrenia risk variation in the NRG1 gene on NRG1-IV splicing during fetal and early postnatal human neocortical development.

Neuregulin 1 (NRG1) is a multifunctional neurotrophin that mediates neurodevelopment and schizophrenia risk. The NRG1 gene undergoes extensive alternative splicing, and association of brain NRG1 type IV isoform expression with the schizophrenia-risk polymorphism rs6994992 is a potential mechanism of risk. Novel splice variants of NRG1-IV (NRG1-IVNV), with predicted unique signaling capabilities, have been cloned in fetal brain tissue. The authors investigated the temporal dynamics of transcription of NRG1-IVNV, compared with the major NRG1 isoforms, across human prenatal and postnatal prefrontal cortical development, and they examined the association of rs6994992 with NRG1-IVNV expression. NRG1 type I-IV and NRG1-IVNV isoforms were evaluated with quantitative real-time polymerase chain reaction in human postmortem prefrontal cortex tissue samples at 14 to 39 weeks gestation and postnatal ages 0-83 years. The association of rs6994992 genotype with NRG1-IVNV expression and the subcellular distribution and proteolytic processing of NRG1-IVNV isoforms were also determined. Expression of NRG1 types I, II, and III was temporally regulated during prenatal and postnatal neocortical development. NRG1-IVNV was expressed from 16 weeks gestation until age 3. Homozygosity for the schizophrenia risk allele (T) of rs6994992 conferred lower cortical NRG1-IVNV levels. Assays showed that NRG1-IVNV is a novel nuclear-enriched, truncated NRG1 protein resistant to proteolytic processing. To the authors' knowledge, this study provides the first quantitative map of NRG1 isoform expression during human neocortical development and aging. It identifies a potential mechanism of early developmental risk for schizophrenia at the NRG1 locus, involving a novel class of NRG1 proteins.

Rapid diagnosis and discrimination of bacterial meningitis in children using gram probe real-time polymerase chain reaction.

In this study, we developed a method of simultaneous detection and discrimination of bacteria in cerebrospinal fluid (CSF) with gram probe real-time polymerase chain reaction (PCR). Our results showed 25 clinical strains representing 13 gram-positive and 12 gram-negative bacterial species. They were identified correctly with the corresponding gram probe. The standard curve showed that the amplification efficiency of templates with different concentrations of bacteria was almost the same with a potential detection limit of 10 colony-forming units/mL. A total of 482 children who were clinically suspected of bacterial meningitis were included in this study. A total of 1.0 mL of CSF was collected from every child and was subjected to gram probe-based PCR (GP-PCR), CSF culture, and CSF routine analysis. The positive rate of the GP-PCR array was (32/482, 6.64%) significantly higher than that of CSF culture (23/482, 4.77%). GP-PCR was proved to be an excellent technique for rapid and accurate diagnosis and discrimination of bacterial meningitis, and hence its use as a diagnostic tool in future seems very promising.

Survey of undeclared allergenic pistachio (Pistacia vera) in commercial foods by hydrolysis probe real-time PCR

Tree nut allergies represent an important health problem in industrialized countries. Among these, pistachio (Pistacia vera) kernels which are consumed as snack foods and used as ingredients in confectionery, chocolates, meat products, and ice-cream industries have been reported to cause IgE-mediated allergic reactions. Trace amounts of undeclared pistachio allergens can cause serious health risks for food-allergic consumers. In order to provide an appropriate method for the detection of pistachio in food products, a real-time polymerase chain reaction (PCR) system for the specific and sensitive detection of pistachio was developed. The sensitivity was investigated on spiked wheat flour samples with defined raw and heat-treated pistachio contents (0.1–100,000 mg kg−1). The real-time PCR detected pistachio in these mixtures down to the lowest investigated spike level of 0.1 mg kg−1. In addition, analysis of different retail samples from the market was performed to demonstrate the suitability of the assay in the food industry. The real-time PCR results obtained from the analysis of 229 commercial food products revealed 29 that didn't declare pistachio or traces on the label but were found to contain pistachio. The presented real time PCR method is useful for relatively fast, highly selective, and sensitive detection of pistachio in food samples.