Addition of gold nanoparticles to real-time PCR: effect on PCR profile and SYBR Green I fluorescence

Real-time quantitative polymerase chain reaction (qPCR) is the industry standard technique for the quantitative analysis of nucleic acids due to its unmatched sensitivity and specificity. Optimisation and improvements of this fundamental technique over the past decade have largely consisted of attempts to allow faster and more accurate ramping between critical temperatures by improving assay reagents and the thermal geometry of the PCR chamber. Small gold nanoparticles (Au-NPs) have been reported to improve PCR yield under fast cycling conditions. In this study, we investigated the effect of Au-NPs on optimised real-time qPCR assays by amplifying DNA sequences from genetically modified canola in the presence and absence of 0.9 nM Au-NPs of diameter 12 ± 2nm. Contrary to expectations, we found that Au-NPs altered the PCR amplification profile when using a SYBR Green I detection system due to fluorescence quenching; furthermore, high-resolution melt (HRM) analysis demonstrated that Au-NPs destabilised the double-stranded PCR product. The results indicate that effects on the assay detection system must be carefully evaluated before Au-NPs are included in any qPCR assay. Figure Raw amplification profiles in the presence and absence of gold nanoparticles     

Protein quantification, sandwich ELISA, and real-time PCR used to monitor industrial cleaning procedures for contamination with peanut and celery allergens

In the United States, peanut is one of the main sources of food allergens. Similarly, celery is a common allergenic food in Western Europe. Severe allergic reactions to both foods are common. Unexpected allergic reactions can occur after the consumption of celery- and peanut-free foods as a result of inadvertent cross-contaminations during manufacturing. Therefore, in cooperation with a flavor manufacturer, we monitored the cleaning process of slurry preparation equipment with regard to contaminations of follow-up products with celery and peanut compounds. Washing water samples taken after different cleaning steps and follow-up products were analyzed for the presence of celery and peanut traces with a celery-specific real-time polymerase chain reaction (PCR) and a peanut-specific sandwich enzyme-linked immunosorbent assay (ELISA). PCR and ELISA were compared with a nonspecific protein assay to evaluate whether the detection of protein traces can be a fast and cost-effective method for monitoring the effectiveness of wet cleaning procedures. Additionally, the allergenic potential of the celery and peanut mush, which were used as source material, were measured by a mediator release assay using a rat basophilic leukemia (RBL) cell line. In conclusion, the quantification of total protein in washing water was suitable for monitoring the cleaning process. Our study also revealed evidence that, in cases where wet cleaning is applicable, allergenic traces can be removed with high efficiency.     

Evaluation of different machines used to quantify genetic modification by real-time PCR

Quantification of genetic modification (GM) is often undertaken to test for compliance with the European Union GM labeling threshold in food. Different control laboratories will often use common validated methods, but with different models of real-time PCR machines. We performed two separate ring trials to evaluate the relative precision and accuracy of different types of real-time PCR machines used to quantify the concentration of GM maize. Both trials used dual-labeled fluorogenic probes for quantification. The first ring trial used separate GM and reference assays (a single fluorescence channel), and the second used a combined duplex assay (two simultaneous fluorescence channels). Five manufacturers and seven models--including a 96-well microtiter-plate, rotary, and portable machines--were examined. In one trial, the machine used had a significant effect on precision, but in the other it did not. Overall, the degree of variation due to the machine model was lower than other factors. No significant repeatable difference in accuracy was observed between machine models. It was not possible to use sufficient replication of machine type in each laboratory to examine all sources of variation in this study, but the results strongly indicate that factors other than machine type or manufacturer (e.g., method or laboratory) contribute more to variation in a GM quantification result.     

Nanoliter droplet array for microRNA detection based on enzymatic stem-loop probes ligation and SYBR Green real-time PCR

In this paper, a nanoliter droplet array based on enzymatic stem-loop probes ligation and SYBR Green real-time PCR for quantification of microRNA was developed. By employing T4 RNA ligase 2 instead of T4 DNA ligase, we designed simplified stem-loop probes to perform microRNA-templated DNA ligation and reduced the non-specific ligation of T4 DNA ligase. SYBR green I dye was employed instead of TaqMan probes in present miniaturized real-time PCR systems. Specifically, we optimized the dosage of SYBR Green I dye in nanoliter droplet and verified the performance of this system by detecting synthetic mir-122 with a 6 logs dynamic range (from 1.5 × 10⁵ to 1.5 × 10¹⁰ copies). Linear relationship of the standard curve (R² = 0.9997) and high PCR amplification efficiency (96.83%) were obtained under the optimized conditions. We detected the expression of mir-122 across five mouse tissues and the result was consistent with that TaqMan microRNA assay. We think this miniaturized real-time PCR platform reduced the detection cost considerably, thus showing the great potential to quantitative biology.     

Development and validation of a multiplex real-time PCR method to simultaneously detect 47 targets for the identification of genetically modified organisms

Considering the increase of the total cultivated land area dedicated to genetically modified organisms (GMO), the consumers’ perception toward GMO and the need to comply with various local GMO legislations, efficient and accurate analytical methods are needed for their detection and identification. Considered as the gold standard for GMO analysis, the real-time polymerase chain reaction (RTi-PCR) technology was optimised to produce a high-throughput GMO screening method. Based on simultaneous 24 multiplex RTi-PCR running on a ready-to-use 384-well plate, this new procedure allows the detection and identification of 47 targets on seven samples in duplicate. To comply with GMO analytical quality requirements, a negative and a positive control were analysed in parallel. In addition, an internal positive control was also included in each reaction well for the detection of potential PCR inhibition. Tested on non-GM materials, on different GM events and on proficiency test samples, the method offered high specificity and sensitivity with an absolute limit of detection between 1 and 16 copies depending on the target. Easy to use, fast and cost efficient, this multiplex approach fits the purpose of GMO testing laboratories.

Evaluation of applied biosystems MicroSEQ real-time PCR system for detection of Salmonella spp. in food

Real-time PCR methods for detecting foodborne pathogens offer the advantages of simplicity and quick time-to-results compared to traditional culture methods. In this study, the MicroSEQ real-time PCR system was evaluated for detection of Salmonella spp. in 10 different food matrixes following the AOAC Research Institute's Performance Tested Method validation program. In addition, the performance of the MicroSEQ system was evaluated for the detection of Salmonella in peanut butter as a part of the Emergency Response Validation Program sponsored by the AOAC Research Institute. The system was compared to the ISO 6579 reference method using a paired-study design for detecting Salmonella spp. in raw ground beef, raw chicken, raw shrimp, Brie cheese, shell eggs, cantaloupe, chocolate, black pepper, dry infant formula, and dry pet food. For the peanut butter study, the system was compared to the U.S. Food and Drug Administration's Bacteriological Analytical Manual procedures using an unpaired-study design. No significant difference in performance was observed between the MicroSEQ Salmonella spp. detection system and the corresponding reference methods for all 11 food matrixes. The MicroSEQ system detected all Salmonella strains tested, while showing good discrimination against detection of an exclusivity panel of 30 strains, with high accuracy.     

A novel electrochemical noise sensor applied to detect food safety

A novel electrochemical noise (EN) sensor was elaborately designed to detect the metal residue in energy drinks. By calculating the characteristic parameter, noise resistance R _n, obtained from the EN data, the tin and iron residue can be semiquantitatively evaluated. In addition, R _n was further compared with the inductively coupled plasma mass spectrometer (ICP-MS) results. Accordingly, an interesting relationship was found between the EN data and ICP-MS results. The experimental results reveal that R _n can indirectly reflect the corrosion-induced metal release from the packaging materials; a lower R _n means a higher metal release. This electrochemical sensor has potential applications in evaluating food safety because of its fast, economic and in-situ features.     

Immunoassay kit used to detect the presence of bovine material in processed foods

The Tepnel Bio Kit for the detection of beef in cooked foods was assessed to determine its validity in demonstrating if food being imported into New Zealand contains beef material. The test suffered no interference from the presence of other common nonbovine species meats accepted as food within New Zealand and it detected beef in cooked samples of mixed meats when the proportion of beef in the mixture was >2 or >1%, depending on other meat species present. The documentation supplied with the kit indicates that the specific proteins it measures in cooked beef are stable to 130 degrees C. This was confirmed in the literature when the kit was used to test meat and bone meal cooked to at least 133 degrees C. However, our results showed these proteins to be much less stable when heated to elevated temperatures in moist food under pressure, and samples containing beef ceased to be positive by the immunoassay test after being autoclaved to 121 degrees C. This suggests that the test may not be able to detect even relatively high levels of beef in low-acid canned foods, which are normally retorted under pressure to approximately 121 degrees C.     

Assessment of terahertz spectroscopy to detect antibiotic residues in food and feed matrices

We report the use of terahertz (THz) spectroscopy to explore the spectral properties of eleven antibiotics commonly used in livestock production. Eight of the eleven antibiotics showed specific fingerprints in the frequency range between 0.1 and 2 THz. The main spectral features of two antibiotics (doxycycline and sulfapyridine) were still detectable when they were mixed with three food matrices (feed, milk, and egg powder). These preliminary results indicate that THz spectroscopy could be suitable for screening applications to detect the presence of antibiotic residues in the food industry, with the prospect to allow inspections directly on the production lines. THz spectroscopy is a non-destructive, non-contact, and real-time technique that requires very little sample preparation. Moreover, THz radiation can penetrate plastic and paper, which enables the detection of antibiotics in packaged food.     

Food safety: screening tests used to detect and quantify GMO proteins

GMO protein content in maize flours or fresh crops can be easily assessed by the enzyme-linked immunosorbent assays (ELISA) and immunochromatographic (lateral flow) strip tests commercially available. Therefore, ELISA can be seen as a useful tool for screening, for control purposes and for traceability implementation. In order to highlight the importance of monitoring GMO protein presence in food products and to investigate the performance of representative ELISA commercial kits, we evaluated three commercial kits by measuring the amount of Cry1Ab/Cry1Ac in IRMM certified reference materials. Presented at AOAC Europe/Eurachem Symposium March 2005, Brussels, Belgium     

Evaluation of applied biosystems MicroSeq real-time PCR system for detection of Listeria monocytogenes in food. Performance Tested Method 011002

Increasingly, more food companies are relying on molecular methods, such as PCR, for pathogen detection due to their improved simplicity, sensitivity, and rapid time to results. This report describes the validation of a new Real-Time PCR method to detect Listeria monocytogenes in nine different food matrixes. The complete system consists of the MicroSEQ L. monocytogenes Detection Kit, sample preparation, the Applied Biosystems 7500 Fast Real-Time PCR instrument, and RapidFinder Express software. Two sample preparation methods were validated: the PrepSEQ Nucleic Acid extraction kit and the PrepSEQ Rapid Spin sample preparation kit. The test method was compared to the ISO 11290-1 reference method using an unpaired-study design to detect L. monocytogenes in roast beef, cured bacon, lox (smoked salmon), lettuce, whole cow's milk, dry infant formula, ice cream, salad dressing, and mayonnaise. The MicroSEQ L. monocytogenes Detection Kit and the ISO 11290-1 reference method showed equivalent detection based on Chi-square analysis for all food matrixes when the samples were prepared using either of the two sample preparation methods. An independent validation confirmed these findings on smoked salmon and whole cow's milk. The MicroSEQ kit detected all 50 L. monocytogenes strains tested, and none of the 30 nontargeted bacteria strains.     

Validation of a newly developed hexaplex real-time PCR assay for screening for presence of GMOs in food, feed and seed

For years, an increasing number and diversity of genetically modified plants has been grown on a commercial scale. The need for detection and identification of these genetically modified organisms (GMOs) calls for broad and at the same time flexible high throughput testing methods. Here we describe the development and validation of a hexaplex real-time polymerase chain reaction (PCR) screening assay covering more than 100 approved GMOs containing at least one of the GMO targets of the assay. The assay comprises detection systems for Cauliflower Mosaic Virus 35S promoter, Agrobacterium tumefaciens NOS terminator, Figwort Mosaic Virus 34S promoter and two construct-specific sequences present in novel genetically modified soybean and maize that lack common screening elements. Additionally a detection system for an internal positive control (IPC) indicating the presence or absence of PCR inhibiting substances was included. The six real-time PCR systems were allocated to five detection channels showing no significant crosstalk between the detection channels. As part of an extensive validation, a limit of detection (LOD_abs) ≤ ten target copies was proven in hexaplex format. A sensitivity ≤ ten target copies of each GMO detection system was still shown in highly asymmetric target situations in the presence of 1,000 copies of all other GMO targets of each detection channel. Furthermore, the applicability to a broad sample spectrum and reliable indication of inhibition by the IPC system was demonstrated. The presented hexaplex assay offers sensitive and reliable detection of GMOs in processed and unprocessed food, feed and seed samples with high efficiency.     

Validation of a carnation-specific gene, ANS, used as an endogenous reference gene in qualitative and real-time quantitative PCR for carnations

The validation of the anthocyanin synthase (ANS) gene as a carnation endogenous reference gene applicable both in classical and real-time PCR methods is a prerequisite for the development of PCR assays for genetically modified (GM) carnation detection. This is important due to the fact that GM carnation lines, developed by Florigene Pty Ltd, have been approved for commercialization. In this study, both methods were tested on 14 different carnation cultivars, and identical amplification products were obtained with all of them. No amplification products were observed with samples from 14 other plant species, which demonstrated that the system was specific to carnation. The results of Southern blot analysis confirmed that the ANS gene had a low copy number in the 10 tested carnation varieties. In qualitative and real-time PCR assays, the LOD values of 0.05 and 0.005 ng carnation DNA, respectively, were validated. Moreover, the real-time PCR system was validated with high PCR efficiency and linearity. Thus, the ANS gene had species specificity, low heterogeneity, and low copy number among the tested cultivars. These results provide evidence that the gene can be used as an endogenous reference gene of carnation, as well as in qualitative and quantitative PCR systems.     

A high-throughput microfluidic biochip to quantify bacterial adhesion to single host cells by real-time PCR assay

A high-throughput microfluidic poly-(dimethylsiloxane) biochip was developed to quantify bacterial adhesion to single host cells by real-time PCR assay. The biochip is simply structured with a two-dimensional array of 900 micro-wells, one inlet, and one outlet micro-channels. Isolation of single infected host cells into the individual micro-wells of the biochip was achieved by one-step vacuum-driven microfluidics. The adhered bacterial cells were then quantified by direct on-chip real-time PCR assay with single-bacterium-detection sensitivity. The performance of this microfluidic platform was demonstrated through profiling of the association of a common bacterial pathogen, Pseudomonas aeruginosa, to single host human lung epithelial A549 cells, revealing an adherence distribution that has not been previously reported. This microfluidic platform offers a simple and effective tool for biologists to analyze pathogen–host interaction at the single-cell level without the necessities of fluorescence labeling. The chip can similarly be used for other PCR-based applications requiring single-cell analysis.     

A standard additions method reduces inhibitor-induced bias in quantitative real-time PCR

A method of calibration for real-time quantitative polymerase chain reaction (qPCR) experiments based on the method of standard additions combined with non-linear curve fitting is described. The method is tested by comparing the results of a traditionally calibrated qPCR experiment with the standard additions experiment in the presence of 2 mM EDTA, a known inhibitor chosen to provide an unambiguous test of the principle by inducing an approximately twofold bias in apparent copy number calculated using traditional calibration. The standard additions method is shown to substantially reduce inhibitor-induced bias in quantitative real-time qPCR.     

An evaluation of the capability of a biolayer interferometry biosensor to detect low-molecular-weight food contaminants

The safety of our food is an essential requirement of society. One well-recognised threat is that of chemical contamination of our food, where low-molecular-weight compounds such as biotoxins, drug residues and pesticides are present. Low-cost, rapid screening procedures are sought to discriminate the suspect samples from the population, thus selecting only these to be forwarded for confirmatory analysis. Many biosensor assays have been developed as screening tools in food contaminant analysis, but these tend to be electrochemical, fluorescence or surface plasmon resonance based. An alternative approach is the use of biolayer interferometry, which has become established in drug discovery and life science studies but is only now emerging as a potential tool in the analysis of food contaminants. A biolayer interferometry biosensor was assessed using domoic acid as a model compound. Instrument repeatability was tested by simultaneously producing six calibration curves showing replicate repeatability (n?=?2) ranging from 0.1 to 6.5 % CV with individual concentration measurements (n?=?12) ranging from 4.3 to 9.3 % CV, giving a calibration curve midpoint of 7.5 ng/ml (2.3 % CV (n?=?6)). Reproducibility was assessed by producing three calibration curves on different days, giving a midpoint of 7.5 ng/ml (3.4 %CV (n?=?3)). It was further shown, using assay development techniques, that the calibration curve midpoint could be adjusted from 10.4 to 1.9 ng/ml by varying assay parameters before the simultaneous construction of three calibration curves in matrix and buffer. Sensitivity of the assay compared favourably with previously published biosensor data for domoic acid.     

Comparison of He and BF3 neutron detectors used to detect hydrogenous material buried in soil

Thermal neutrons detectors have been used for a long time and continue to be used to detect hydrogenous material. In this work, BF₃ and ³He detectors ability have been compared with each other to detect Polyethylene (PE) sample that was buried in soil. It was found that neither BF₃ nor ³He could detect PE sample without shields. This research shows that a thickness of 5 cm graphite is suitable shield for ³He detector that has been used to detect buried PE sample.     

ARMS TaqMan real-time PCR for genotyping factor V Leiden mutation in Han Chinese

Factor V Leiden (FV_Leiden) is a missense mutation of 1691 position (G1691A) in exon 10 of FV gene, and being a genetic risk for venous thrombosis. Currently, there are several PCR-based methods for detecting FV_Leiden mutation; however, these methods have disadvantages such as time-consuming, cumbersome steps and potentially hazardous gels. The aims of present study were to develop a simple, time-saving, accurate, and gel-free method, called amplification refractory mutation system (ARMS) TaqMan real-time PCR, for detecting FV_Leiden mutation. We severally designed two specific reverse primers for mutant and wild-type through intentional introduction of mismatched nucleotide at the penultimate 3′ position. Although target amplicon amplification efficiency is reduced, but another corresponding amplicon is almost completely inhibited. Then, specific TaqMan-probe was designed to detect target amplicon. Established method was used to detect 500 unselected samples in Han Chinese, the results showed 499 cases of wild-type and one heterozygote. Afterward, 50 randomly picked wild-type cases and one heterozygote were reexamined by bidirectional DNA sequencing, which is considered as “Gold standard method.” Exhilaratingly, the results detected by the two methods were completely consistent. At last, allelic frequency of FV_Leiden was calculated the in Han Chinese. Given the above results, A FV_Leiden heterozygote has been found in 500 random samples in Han Chinese, and the allelic frequency was 0.1%. In conclusion, the ARMS TaqMan real-time PCR is an ideal detecting system for genotyping FV_Leiden mutation in clinical application, and FV_Leiden mutation exists in Han Chinese despite extremely low prevalence.     

气相色谱-质谱法测定食品接触材料表面印刷油墨中的光引发剂

建立了食品接触材料表面印刷油墨中光引发剂(PIs)二苯甲酮(BP)、4-甲基二苯甲酮(MBP)、对二甲氨基苯甲酸乙酯(EDAB)、N,N-二甲氨基苯甲酸异辛酯(EHDAB)和1-羟基环己基苯基甲酮(Irgacure 184)的气相色谱-质谱(GC-MS)分析方法。样品以乙酸乙酯为萃取溶剂进行索氏提取,萃取溶液经净化分离和富集以后,以5%苯基-95%甲基聚硅氧烷为固定相(DB-5MS),采用选择离子扫描(SIM)方式对上述5种PIs进行了定性和定量分析。5种PIs在5.0～200.0 μg/L范围内的线性关系良好(R2＞0.9995),在2种浓度的添加水平下,回收率为66.7%～89.4%,相对标准偏差＜10%。方法的定量限为0.0017～0.0036 mg/dm2。该方法的样品前处理过程简单,易操作,适用于常规进出口食品接触材料表面印刷油墨中PIs的快速检测。     

Potential use of the activation energy value calculated from the thermoluminescence glow curves to detect irradiated food

We herein report on the calculation of the activation energy (E _a) from the thermoluminescence (TL) glow curves performed by the initial rise method that allows us to discriminate between irradiated and non-irradiated sesame seeds. E _a values of natural TL (0.68 ± 0.03 eV) and gamma-induced TL (never lower than 0.82 ± 0.02 eV) appear as a complementary criterion to be used differentiating between irradiated and non-irradiated foodstuffs with the position and the intensity of the main peak of the TL emission. In addition, E _a values taken from irradiated sesame samples at different doses (1, 5 and 10 kGy) and stored up to 15 months after being processed were compared to a ‘positive’ Spanish blend (i.e. at least one component was commercially irradiated).