Quantitative detection of species-specific DNA in feedstuffs and fish meals

A sensitive and rapid method for the quantitative detection of bovine-, ovine-, swine-, and chicken-specific mitochondrial DNA sequences based on real-time PCR has been developed. The specificity of the primers and probes for real-time PCR has been tested using DNA samples of other vertebrate species that may also be present in rendered products. The quantitative detection was performed with dual-labeled probes (TaqMan) using absolute quantification with external standards of single species meat-and-bone meals. This method facilitates the detection of 0.01% of the target species-derived material in concentrate feed mixtures and fish meals.     

Towards a quantitative application of real-time PCR technique for fish DNA detection in feedstuffs

A real-time PCR method to detect fish DNA in feedstuffs was developed and optimised. A combination of primers and a Taqman-MGB probe was used to selectively amplify the fish mitochondrial 12S ribosomal RNA gene. Qualitative and also quantitative assessments were performed with different protocols: a relative quantification by a standard curve, and a ΔC_T method, by total plant DNA as endogenous controls. Method specificity was evaluated analysing 40 different tissues (mammalians, avian, fish) and flour samples. Sensitivity was evaluated by LOD (limit of detection) estimation. The designed probe–primers set showed an increased sensitivity compared to previously published PCR end point method, reaching a limit of detection of 0.2 pg of fish DNA, and showing to be a robust assay for fish DNA detection. The quantification results, based on ΔC_T method and the relative standard curve, are well reproducible in our experimental condition but, in lacking of separate pure raw materials of a tested feed, they cannot be applied for reliable and precise quantification on field samples but for now as a semi-quantitative PCR method only.     

Standard and Light-Cycler PCR methods for animal DNA species detection in animal feedstuffs

In this work four species-specific primers and probes were designed and evaluated for the detection and quantification of bovine, ovine, swine and chicken mitochondrial DNA in feeds. PCR primers were optimized using conventional and Real Time PCR, to detect short species-specific sequences amplifiable from heat treated material. Both methods confirmed the high specificity of the primers designed. Real time quantitative PCR assay allowed the detection of as few as 0.01 ng and 0.05 ng of ovine and bovine genomic DNA, respectively. The detection limit for swine and chicken genomic DNA was 0.5 ng. Sensitivity levels observed in DNA extracted from meat samples processed according to EU legislation were different compared to those in genomic DNAs previously described. They resulted in swine 5 fg of MBM DNA, in chicken 25 ng, in ovine and bovine 50 ng. We confirmed the efficiency and specificity of primers in RT-PCR to detect 0.5% of bovine, ovine, swine and chicken MBM in contaminated feedstuffs.Industrial relevanceThe variant Creutzfeldt Jakob disease is a rare and fatal human neurodegenerative condition clearly linked with the bovine spongiform encephalopathies (BSE) of cattle. The ban of using animal derived protein in animal feeds has efficiently controlled the development of the BSE epidemic. The work presented by Frezza and collaborators is an application of the real time polymerase chain reaction (a standard procedure used in molecular biology also known as RT‐PCR) to identify specific DNA of four animal species (bovine, ovine, swine and chicken). This method is applied to the analysis of feeds to detect and eventually estimate the amount of animal derived proteins. The difficult aim to detect DNA derived from heat‐treated material was successfully reached using as target short mitochondrial DNA sequences. The method presented could have important application not only in the control of feed production but also in many fields of the food industry as quality and process control.     

SYBR-Green real-time PCR approach for the detection and quantification of pig DNA in feedstuffs

A real-time polymerase chain reaction assay using primers targeting the porcine-specific mitochondrial 12S rRNA gene and universal eukaryotic primers amplifying a conserved fragment of the nuclear 18S rRNA gene has been developed for the detection and quantification of porcine DNA in food and feedstuffs. The 18S rRNA primers were used as endogenous control for the total content of PCR-amplifiable DNA in the sample. The assay was tested on DNA extracted from raw and heat-treated binary mixtures of porcine tissues in a plant matrix, and on DNA extracted from reference feedstuff samples. Analysis of experimental mixtures demonstrated the suitability of the assay for the detection and quantification of porcine DNA in mixtures containing as little as 0.1%.     

实时荧光定量PCR在食品检测领域中应用

实时荧光定量PCR是在定性PCR技术基础上发展起来的核酸定量技术;该技术不仅实现对DNA模板定量,且具有灵敏度高、特异性强、无污染性、及实时性和准确性等特点。该文主要介绍实时荧光定量PCR原理和在食品检测中应用,及其在食品领域发展前景。     

Modelling the limit of detection in real-time quantitative PCR

The limit of detection (LOD) is a critical performance characteristic of an assay that requires careful evaluation during method validation. However, formal calculations for the LOD do not take into account atypical data sets that are generated from real-time PCR techniques, which can be non-normally distributed, truncated, and heteroscedastic. Experimental data sets for the quantification of genetically modified (GM) material were produced using real-time PCR, in order to model the LOD. A bootstrapping computer simulation calculated the probabilities of detecting PCR positive test results from these data sets, and computer modelling defined a function from the resulting probability plots. The LOD was modelled as a function of sample replication level and cycle threshold values. The broad applicability of this bootstrapping and data modelling approach should be of general interest to laboratories conducting trace-level detection.     

实时荧光定量技术在食品微生物检测和研究中的应用

实时荧光定量PCR技术是通过检测PCR产物中荧光讯号强度来达到定量的目的,不仅实现了对核酸信息量的分析比较,而且与常规PCR相比,它具有特异性更强、能有效解决PCR污染问题、自动化程度高等特点。文章概述了实时荧光定量PCR技术的原理、优缺点及其在食品微生物检测中的应用与研究进展,并探讨了它的技术发展和应用前景。     

实时荧光定量PCR在食品致病菌及转基因成分检测中应用

与传统定性PCR技术相比,实时荧光定量PCR有更多的优点,其速度快,特异性更强、灵敏度更高、无污染性、自动化水平高等,且能对DNA模板进行定量。本文综述了实时荧光定量PCR技术原理、分类及其应用,并对其存在的问题和发展前景进行了展望。     

Sensitive detection of porcine DNA in processed animal proteins using a TaqMan real-time PCR assay

A TaqMan real-time PCR method was developed for specific detection of porcine-prohibited material in industrial feeds. The assay combines the use of a porcine-specific primer pair, which amplifies a 79?bp fragment of the mitochondrial (mt) 12?S rRNA gene, and a locked nucleic acid (LNA) TaqMan probe complementary to a target sequence lying between the porcine-specific primers. The nuclear 18?S rRNA gene system, yielding a 77?bp amplicon, was employed as a positive amplification control to monitor the total content of amplifiable DNA in the samples. The specificity of the porcine primers-probe system was verified against different animal and plant species, including mammals, birds and fish. The applicability of the real-time PCR protocol to detect the presence of porcine mt DNA in feeds was determined through the analysis of 190 industrial feeds (19 known reference and 171 blind samples) subjected to stringent processing treatments. The performance of the method allows qualitative and highly sensitive detection of short fragments from porcine DNA in all the industrial feeds declared to contain porcine material. Although the method has quantitative potential, the real quantitative capability of the assay is limited by the existing variability in terms of composition and processing conditions of the feeds, which affect the amount and quality of amplifiable DNA.     

Sensitive detection of porcine DNA in processed animal proteins using a TaqMan real-time PCR assay

A TaqMan real-time PCR method was developed for specific detection of porcine-prohibited material in industrial feeds. The assay combines the use of a porcine-specific primer pair, which amplifies a 79?bp fragment of the mitochondrial (mt) 12?S rRNA gene, and a locked nucleic acid (LNA) TaqMan probe complementary to a target sequence lying between the porcine-specific primers. The nuclear 18?S rRNA gene system, yielding a 77?bp amplicon, was employed as a positive amplification control to monitor the total content of amplifiable DNA in the samples. The specificity of the porcine primers-probe system was verified against different animal and plant species, including mammals, birds and fish. The applicability of the real-time PCR protocol to detect the presence of porcine mt DNA in feeds was determined through the analysis of 190 industrial feeds (19 known reference and 171 blind samples) subjected to stringent processing treatments. The performance of the method allows qualitative and highly sensitive detection of short fragments from porcine DNA in all the industrial feeds declared to contain porcine material. Although the method has quantitative potential, the real quantitative capability of the assay is limited by the existing variability in terms of composition and processing conditions of the feeds, which affect the amount and quality of amplifiable DNA.     

实时荧光定量PCR技术在粮油转基因成分检测中的应用

随着转基因技术在谷物育种中的广泛应用,以及转基因粮油及其制品消费的不断增加,加强粮油转基因成分的检测技术的研究极为必要.阐述了实时荧光定量PCR技术在粮油转基因成分检测中的应用,主要从实时荧光定量PCR技术,实验室条件,实验步骤以及应用范围等方面进行了阐述.     

Two quantitative hexaplex real-time PCR systems for the detection and quantification of DNA from twelve allergens in food

According to the EU and Swiss legislation, allergens in food have to be labelled. Consumers, exhibiting allergic reaction, must be able to avoid such food and its products. In order to provide efficient and reliable methods, two novel hexaplex quantitative real-time polymerase chain reaction systems were developed and validated. The first system simultaneously determines DNA contents from cashew, peanut, hazelnut, celery, soy, mustard, whereas the second system determines DNA contents from milk (beef), egg (chicken), almonds, sesame, pistachio and walnut. The two tests exhibited a good specificity and a detection limit of at least 0.1?% for all analytes. This was additionally verified using samples from proficiency tests. Application on samples from the market revealed realistic and useful information about allergen contents. Quantitative results according to weight are not possible yet.     

Targets and methods for the detection of processed animal proteins in animal feedstuffs

Bovine spongiform encephalopathy (BSE), or ‘mad cow disease’, is one of several transmissable spongiform encephalopathies (TSEs) known to affect certain mammals and is spread through the ingestion of infected animal tissue. It is believed that the inadvertent contamination of meat and bone meal (MBM) with infected animal tissue and the subsequent use of this material as a feed supplement contributed to the spread of the disease in cattle. As a result, the use of processed animal proteins (PAPs) in animal feeds is regulated in many parts of the world. Although feed testing is the only definitive means to certify compliance, regulatory compliance often relies solely on paper certification. Recently, rapid methods have become available that can be used by regulators to determine compliance during routine inspections. We describe a rapid, immunochromatographic strip test that can detect 0.1% MBM in animal feed. The test takes 15 min to perform and large numbers of samples can be screened for PAPs simultaneously.     

Rapid detection and identification of Streptococcus macedonicus by species-specific PCR and DNA hybridisation

The aim of this study was to develop a simple and specific method for the rapid detection and identification of Streptococcus macedonicus. The method was based on polymerase chain reaction (PCR) using species-specific primers derived from the 16S rRNA gene. Specific identification was proven on seven S. macedonicus strains, while 16 strains belonging to different lactic acid bacteria species were tested negative. The PCR assay was capable of detecting 100 pg of S. macedonicus DNA, and it was also efficient on single colonies of the bacterium. Furthermore, the same bacterial strains were used for the specificity evaluation of a S. macedonicus species-specific probe. Neither species-specific PCR nor DNA hybridisation experiments could differentiate Streptococcus waius from S. macedonicus, due to the identity of the 16S rRNA gene of the two species, indicating high phylogenetical relatedness. This was further confirmed by the comparative sequence analysis of the 16S-23S rRNA intergenic regions. It was thus clearly demonstrated that S. waius, recently described as a novel Streptococcus species, is phylogenetically identical to S. macedonicus.     

实时荧光PCR定性定量检测混合食用油脂中的花生油成分

为鉴别鉴定花生油在食用油脂中的存在与否及其含量,研究了定性定量检测花生特异性基因片段的分子生物学检测方法。结果表明,生花生需加热处理后再提取DNA才能作为PCR扩增的模板,食用油脂中可提取出用于定性定量PCR的DNA,花生的Arah1基因是具有花生种属特异性的基因,采用实时荧光PCR方法检测Arah1基因片段可定性定量检测混合食用油脂中的花生油成分。     

A papaya-specific gene, papain, used as an endogenous reference gene in qualitative and real-time quantitative PCR detection of transgenic papayas

Genetically modified (GM) papaya lines have been approved for commercialization and are widely cultivated in many countries. As a step towards the development of reliable qualitative and quantitative PCR methods for detecting GM papayas, one papaya (Fructus Caricae Carica papaya L.) species specific gene, papain, was selected and validated as suitable for using as an endogenous reference gene in transgenic papaya PCR detection. In this article, both qualitative and quantitative PCR assays for the papain gene were assayed with 11 different papaya varieties and identical amplification products were obtained with all of them. No amplified fragments could be detected when DNA samples from 18 kinds of other species were used as templates, which demonstrated that this system was specific for papaya. In real-time quantitative PCR analysis, the detection limit was as low as 10 pg of DNA. Southern blot analysis confirmed that the papain gene was two copies in the tested papaya varieties and no allelic variation was testified among these tested papaya varieties. In addition, the common used exogenous genes of the coat protein (CP) and the replicase (RP) were also assayed in qualitative and real-time quantitative PCR. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Wentao Xu and Weibin Bai contributed equally to this work.     

Real-time quantitative PCR of Staphylococcus aureus and application in restaurant meals

Staphylococcus aureus is considered the second most common pathogen to cause outbreaks of food poisoning, exceeded only by Campylobacter. Consumption of foods containing this microorganism is often identified as the cause of illness. In this study, a rapid, reliable, and sensitive real-time quantitative PCR was developed and compared with conventional culture methods. Real-time quantitative PCR was carried out by purifying DNA extracts of S. aureus with a Staphylococcus sample preparation kit and quantifying it in the LightCycler system with hybridization probes. The assay was linear from a range of 10 to 10(6) S. aureus cells (r2 > 0.997). The PCR reaction presented an efficiency of >85%. Accuracy of the PCR-based assay, expressed as percent bias, was around 13%, and the precision, expressed as a percentage of the coefficient of variation, was 7 to 10%. Intraday and interday variability were studied at 10(2) CFU/g and was 12 and 14%, respectively. The proposed method was applied to the analysis of 77 samples of restaurant meals in Valencia (Spain). In 11.6% of samples S. aureus was detected by real-time quantitative PCR, as well as by the conventional microbiological method. An excellent correspondence between real-time quantitative PCR and microbiological numbers (CFU/g) was observed with deviations of < 28%.     

Development of a quantitative real-time PCR assay for the detection of Aspergillus carbonarius in grapes

Aspergillus carbonarius is the main species responsible for the production of ochratoxin A (OTA) in wine grapes. To monitor and quantify A. carbonarious in grapes, a quantitative real-time PCR assay was developed as a possible tool for predicting the potential ochratoxigenic risk. DNA extraction from grape berries was performed by using conventional extraction and clean up through EZNA Hi-bond spin columns. A TaqMan probe was used to quantify A. carbonarius genomic DNA in grape berries samples. An exogenous internal positive control was used to overcome DNA recovery losses due to matrix inhibition. The quantification of fungal genomic DNA in naturally contaminated grape was performed using the TaqMan signal versus spectrophotometrically measured DNA quantities (Log10) calibration curve with a linearity range from 50 to 5 x 10(-4) ng of DNA. A positive correlation (R2=0.92) was found between A. carbonarious DNA content and OTA concentration in naturally contaminated grape samples. This is the first application of TaqMan real-time PCR for identifying and quantifying A. carbonarius genomic DNA occurring in grapes. The rapid DNA extraction method for grapes, together with the commercial availability of reagents and instrumentation, allows to perform a remarkable number of reproducible assays (96-well format) in less than 4 h.     

The detection of commercial duck species in food using a single probe-multiple species-specific primer real-time PCR assay

A real-time PCR assay for the simultaneous detection of Mallard and Muscovy duck is described. Species-specific primers were designed for Mallard or Muscovy duck using the mitochondrial cytochrome b gene sequence. These primer sets were multiplexed with a single duck probe to produce a simple, rapid and robust real-time PCR assay. This assay was shown to be specific for duck compared to a wide range of commercially important meat species and was used for the successful detection of duck meat in complex food matrices. This is the first report of an assay that will detect all species of commercially available duck in commercial products using real-time PCR.     

Detection of banned ruminant-derived material in industrial feedstuffs by TaqMan real-time PCR Assay

A ruminant-specific real-time PCR system was designed and applied for the detection of processed animal protein from ruminants in industrial feedstuffs. The assay includes a primer pair and a TaqMan probe selectively targeting mitochondrial 16S rRNA gene sequences from the ruminant group and another primer-probe set based on the eukaryotic nuclear 18S rRNA gene (positive amplification control). Both ruminant and eukaryotic PCR systems generated short PCR amplicons of 79 and 77 bp, respectively. To evaluate the suitability of the real-time PCR assay for the detection of banned by-products of ruminant origin, 126 feed samples subjected to rendering under current European legislation regulations were analyzed. The assay achieved 100% success in classifying the samples as positive or negative in terms of qualitative ruminant composition, with a detection limit of 0.1%. The quantitative ability of the assay is however restricted by variations in the composition and treatment of the feeds, which affect the amount and quality of amplifiable DNA.