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.     

A statistical approach for evaluation of PCR results to improve the practical limit of quantification (LOQ) of GMO analyses (SIMQUANT)

The predominant approach for quantification of genetically modified organisms (GMO) is the application of quantitative real-time PCR. However, for a large number of processed food and feed products, this approach is unsuitable, because they contain low amounts (mass) of amplifiable DNA. Here we present a novel approach, “Single molecule quantification” (SIMQUANT) for GMO quantification of samples with extremely low amounts of DNA. The approach is based on statistics and application of multiple qualitative parallel PCRs. Here the qualitative PCRs were done using real-time PCR setup, but this is not a requirement. The difference is that the quantitative real-time PCR requires that the target copy number exceeds the absolute limit of quantification (LOQ_abs) and provides quantity estimates by extrapolation from a linear regressional relationship between an observed cycle threshold (Ct) value and copy numbers, while with SIMQUANT the template DNA typically contains very few, e.g., one target copy per PCR volume and the quantity is estimated on the basis of observed ratio between positive and negative individual PCRs. The components of this analysis are the numbers of test samples, the size of each sample and the outcome in number and relative ratio of positive and negative test results. The approach results in a statistical estimate of the relative GM concentration based on the probability that one or more amplifiable GM template copies are present in discrete volumes. Thus, the approach is based on the ratio of discrete volumes without or with one or more PCR-amplifiable GM target copies. The approach described here can be used reliably with more than a 100-fold improvement of the practical LOQ (LOQ_pract) compared to real-time quantitative PCR based on standard curves.     

Development and in‐house validation of the event‐specific qualitative and quantitative PCR detection methods for genetically modified cotton MON15985

BACKGROUND: To implement genetically modified organism (GMO) labeling regulations, an event‐specific analysis method based on the junction sequence between exogenous integration and host genomic DNA has become the preferential approach for GMO identification and quantification. RESULTS: In this study, specific primers and TaqMan probes based on the revealed 5′‐end junction sequence of GM cotton MON15985 were designed, and qualitative and quantitative polymerase chain reaction (PCR) assays were established employing the designed primers and probes. In the qualitative PCR assay, the limit of detection (LOD) was 0.5 g kg^?1 in 100 ng total cotton genomic DNA, corresponding to about 17 copies of haploid cotton genomic DNA, and the LOD and limit of quantification (LOQ) for quantitative PCR assay were 10 and 17 copies of haploid cotton genomic DNA, respectively. Furthermore, the developed quantitative PCR assays were validated in‐house by five different researchers. Also, five practical samples with known GM contents were quantified using the developed PCR assay in in‐house validation, and the bias between the true and quantification values ranged from 2.06% to 12.59%. CONCLUSION: This study shows that the developed qualitative and quantitative PCR methods are applicable for the identification and quantification of GM cotton MON15985 and its derivates. Copyright © 2009 Society of Chemical Industry     

A model freed from endogenous reference gene for quantification of genetically modified DNA by real-time PCR. 1. Quantification of DNA from genetically modified organisms in haplo-species materials

This paper is the first part of a serial study investigating a quantification model freed from endogenous reference gene for genetically modified (GM) content by real-time polymerase chain reaction (PCR). The serial study involves two parts: (1) quantitative determination of GM DNA in haplo-species plant samples; (2) quantitative determination of GM DNA in multi-species plant samples. The paper describes a methodology to quantify the GM content in a DNA extract using on one hand real-time PCR to determine the amount of GM targets present and on the other hand absorbance reading at 260 nm to measure the total DNA present in sample. The ratio of both values is expressed as GM percentage. The most prominent dominance of the novel model is that the direct quantitative relation between the initial amount of target template in real-time PCR reaction (X ₀) and the content of GM DNA in tested material (X _SD) is established. Theoretical analysis indicates that the developed quantitative model relieved from the dependence on endogenous reference genes is suitable to quantify the GM content in haplo-species plant sample, in addition, it has the applicability in the quantitative detection of GM content in multi-species GM plant sample. A trail, in which 75 haplo-species GM plant samples were involved, was conducted to validate the suitability of the novel quantification model. The bias varied from 0.00 to 24.00% except a tested sample with lower level of GM content, and the precision expressed as coefficient of variation (CV) was from 2.81 to 25.00%. The limit of quantitation (LOQ) of the quantitative assay was as low as 0.1%. Compared with the previous papers and the performance requirements raised by European Network of GMO Laboratories (ENGL) for analytical methods of GMO testing, the results demonstrated that the established quantification model is a suitable alternative to the more traditional endogenous reference assay in the quantification of GM content in haplo-species plant sample.     

A procedural approach for the identification of sources of uncertainty associated with GM quantification and real-time quantitative PCR measurements

Estimation of measurement uncertainty is important as it provides objective evidence that an assay or instrument is ‘fit for purpose’, and is a requirement for accreditation to ISO17025. Technological developments within the bioanalytical laboratory have progressed rapidly in the past few years. However, the measurement science associated with these technologies has not yet fully matured. The provision of a full measurement uncertainty budget is often difficult because of a lack of standardised approaches and the constraints associated with time, finances and expertise. A procedural approach for the identification of factors that can account for measurement uncertainty in quantification of genetically modified (GM) ingredients using real-time quantitative PCR is presented here. This example illustrates an approach for the identification of factors that can be applied to related assays concerned with GM quantification, can be utilised as part of existing uncertainty approaches, and can provide the basis for making quantitative estimates of uncertainty that will contribute towards a full measurement uncertainty budget.     

Development and in-house validation of a reference molecule pMIR604 for simplex and duplex event-specific identification and quantification of GM maize MIR604

Effective analysis methods for genetically modified organisms (GMOs) using reliable reference molecules as calibrators are necessary for the implementation of labeling policies. While no available detection systems based on the reference molecule have been reported for GM maize MIR604. Here we established the simplex and duplex qualitative and quantitative PCR systems for maize MIR604 employing a reliable new reference molecule pMIR604 as a calibrator, which contains fragments of the revealed 5′ flanking sequence of maize MIR604 and taxon-specific sequence zSSIIb. The limits of detection (LODs) were both 10 copies. The absolute LOD and limit of quantification were confirmed to be as low as 10 and 25 copies of pMIR604 through in-house validation both in simplex and duplex real-time PCR assays. For quantification of practical samples, results from five operators indicated that the biases of the data derived from each participant ranged from 2.00 to 23.00% and from 3.00 to 24.00% in simplex and duplex PCR systems, respectively. The relative standard deviations of the mean values for different GM content maize samples were all within 13.60% both in simplex and duplex analyses. This study showed that the detection systems of GM maize MIR604 using a new reference molecule pMIR604 as a calibrator are applicable for analysis of GM maize MIR604 and suitable for use as a preferable substitute of the reference material derived from plant raw materials.     

Comparison of plasmid and genomic DNA calibrants for the quantification of genetically modified ingredients

Real-time PCR is the method of choice for the quantification of the genetically modified (GM) content of food. Recent EU Commission recommendations advocate expressing the GM content as the percentage of the number of GM target DNA sequences per target taxon specific sequence. The provision of reference materials certified for their copy number content, for example in the form of plasmids, are hence desirable to fulfil this EU recommendation. An inter-laboratory trial was conducted to compare plasmid DNA to the more traditional genomic DNA approach for quantitative PCR calibration in terms of copy numbers, using the model system of Roundup Ready? soya. Data was analysed from 48 randomised real-time quantitative PCR plates under conditions of reproducibility across three international laboratories. Results demonstrated that the specific plasmid DNA used in the laboratory trial provided a suitable alternative to genomic DNA for use as a calibrant in GM quantification. In the current investigation, plasmid calibrants gave equal or better performance characteristics in terms of precision and closeness to the expected value, than their genomic equivalents.     

Real-time multiplex PCR: An accurate method for the detection and quantification of 35S-CaMV promoter in genetically modified maize-containing food

A very sensitive and new real-time multiplex PCR method for the quantification of genetically modified (GM) maize crops in food materials was developed and validated for an ABI Prism 7700 Sequence Detection System. In the assay described, fluorescence-labelled TaqMan probes were chosen to detect the amplified DNA fragments during PCR. In this multiplex approach, maize-specific DNA (zein) and 35S-CaMV promoter-specific DNA fragments are amplified in the same tube. The method was tested for the detection and quantification of the four maize events that are approved in Europe and contain the 35S-CaMV promoter: Bt11, Bt176, Mon810 and T25 maize. Quantification was based on a standard curve prepared from certified maize flour reference material prepared by the Institute for Reference Materials and Measurements. Quantification within the range of the standard curve (0.05-1% GM maize) and up to 100% was possible. Repeatability of the method for each GM maize event was determined; coefficients of variations ranged from 28-40%. In addition, three internal Nestlé laboratories successfully applied this method and comparable results were obtained.     

转基因棉花MON88913转化体特异性定性、定量PCR检测方法

本文以我国批准商业化的转基因耐草甘膦棉花MON88913为研究对象,建立并验证了其转化体特异性定性、定量PCR检测方法.建立的定性PCR方法的检测极限是20个拷贝棉花单倍体基因组DNA,定量PCR方法的检测和定量极限分别是10和20个拷贝棉花单倍体基因组DNA.同时,我们组织了实验室5位研究人员对建立的定量PCR检测方法进行了协同验证.对5个盲样的定量分析结果显示与真实值的偏差介于1.59% 和10.12%之间,完全满足国际标准25%偏差范围的要求,完全可用于转基因棉花MON88913的实际样品检测.     

Simple,sensitive, accurate multiplex quantitative competitive PCR with capillary electrophoresis detection for the determination of genetically modified maize

Legislation in the EU requires that foods containing more than 0.9% of genetically modified organisms (GMOs) should be labelled. To this end, we have developed a simple and accurate capillary electrophoresis multiplex quantitative competitive PCR (ce-mqcPCR) method for event-specific quantification of the five novel GM maize events DAS59122, LY038, MON88017, MIR604 and Event 3272. The method combines the simplicity of constructing multiple competitors in silico with the high resolution and sensitivity of fluorescence capillary electrophoresis and the use of an internal template reference amplicon. The competitors are synthesised commercially and added in equal amounts as a restriction enzyme-digested plasmid insert to the multiplex PCR. Quantification is performed by analysing the relative amounts of GMO and GMO competitor fragment pairs after capillary electrophoresis and correcting for differences in maize DNA by comparing with the internal reference gene amplicon. Since the competitors employ the same primers as their corresponding targets, all existing qualitative multiplex PCRs can in principle easily be converted to quantitative assays without changing primer sets or amplification conditions. The ce-mqcPCR method correctly determined 120 GMO templates in known mixed samples. No false-positive or false-negative signals were obtained.     

Detection and quantification of Salmonella in pure cultures using 5'-nuclease polymerase chain reaction

Advances in detection and quantification assays based on nucleic acids conceivably will revolutionize the ability to quickly and specifically detect and quantify microorganisms in foods. Among these assays, the polymerase chain reaction (PCR) assay and the TaqMan PCR Detection System (Perkin-Elmer) probably are among the most promising. Since a 5'-nuclease PCR renders possible the automated and direct detection and quantification of PCR products (Holland et al., 1991. Proc. Natl. Acad. Sci. USA 88, 7276-7280), microorganisms in foods can be detected and quantified indirectly within a few hours through analysis of the microbial DNA or RNA sequences present. In the present report we have adapted a 5'-nuclease-based kit for the quantification of Salmonella.     

A novel poisson distribution-based approach for testing boundaries of real-time PCR assays for food pathogen quantification

The validation of quantitative real-time PCR systems and above all, proof of the detection limit of this method, is a frequently and intensively discussed topic in food pathogen detection. Among proper sample collection, assay design, careful experimental design, execution of real-time PCR, and data analysis, the validation of the method per se ensuring reliable quantification data is of prime importance. The purpose of this study was to evaluate a novel validation tool for real-time PCR assays, based on the theoretical possibility of the amplification of a single DNA target. The underlying mathematical basis for the work is Poisson distribution, which describes patterns of low particle numbers in a volume. In this context, we focused on the quantitative aspect of real-time PCR for the first time. This allowed for demonstration of the reliable amplification of a lone target DNA molecule and the demonstration of the distinct discrimination between integer molecular numbers when using low initial copy numbers. A real-time PCR assay amplifying a 274-bp fragment of the positive regulatory protein A locus of Listeria monocytogenes was used for this work. Evidence for a linear range of quantification from a single target copy to 10 ng of target DNA was experimentally demonstrated, and evidence for the significance of this novel validation approach is presented here.     

Direct Quantification of Campylobacter jejuni in Chicken Fecal Samples Using Real-Time PCR: Evaluation of Six Rapid DNA Extraction Methods

Direct and accurate quantification of Campylobacter in poultry is crucial for the assessment of public health risks and the evaluation of the effectiveness of control measures against Campylobacter in poultry. The aim of this study was to assess several rapid DNA extraction methods for their effectiveness for the direct quantification (without enrichment) of Campylobacter jejuni in chicken fecal samples using real-time PCR. The presence of inhibitory substances in chicken fecal samples may reduce or even completely impede the PCR amplification process making quantification very difficult. Six rapid DNA extraction methods were compared based on their limit of detection, efficiency, reproducibility, and precision. Standard curves were designed for all the methods tested in order to assess their performance on the direct quantification of C. jejuni in chicken fecal samples. As a result of this study, the Easy-DNA (Invitrogen) method generated lower Ct values, the best amplification efficiency (AE?=?93.2 %) and good precision (R squared?=?0.996). The method NucleoSpin® Tissue was able to detect samples spiked with the lowest Campylobacter concentration level (10 CFU/ml) but the amplification efficiency was not optimal (AE?=?139.5 %). DNA extraction methods Easy-DNA Invitrogen, MiniMAG® and NucleoSpin® Tissue produced good real-time PCR reproducibility generating standard deviations from 0.3 to 0.8 between replicates.     

Quantification of bovine leukemia virus proviral DNA using a low-cost real-time polymerase chain reaction

The detection of bovine leukemia virus (BLV) proviral DNA is an important tool to address whether an animal is infected with BLV. Compared with serological assays, real-time PCR accounts for greater sensitivity and can serve as a confirmatory test for the clarification of inconclusive or discordant serological test results. However, the high cost related to real-time PCR assays has limited their systematic inclusion in BLV surveillance and eradication programs. The aim of the present study was to validate a low-cost quantitative real-time PCR. Interestingly, by using SYBR Green detection dye, we were able to reduce the cost of a single reaction by a factor of 5 compared with most common assays based on the use of fluorogenic probes (i.e., TaqMan technology). This approach allowed a highly sensitive and specific detection and quantification of BLV proviral DNA from purified peripheral blood leukocytes and a milk matrix. Due to its simplicity and low cost, our in-house BLV SYBR quantitative real-time PCR might be used either as a screening or as a confirmatory test in BLV control programs.     

The applicability of digital PCR for the assessment of detection limits in GMO analysis

We investigate the applicability of using digital PCR to estimate absolute limits of detection and quantitation associated with the validation of traditional real-time PCR methods for analysis of genetically modified (GM) ingredients. We also demonstrate the use of dynamic arrays as a precursor in selecting suitable sample dilution levels for accurate copy number assessment using the aforementioned digital PCR. Additionally, we further explore the relevance of digital PCR in accurately quantifying plasmid copy numbers associated with a commercially available Certified Reference Material. The use of digital PCR has the advantage of facilitating absolute single molecule detection, therefore negating the necessity for standards on a calibration curve, and reducing associated matrix effects. The approaches described in this paper enable pre-existing validated protocols to be re-examined, and estimates based on an alternative approach using digital PCR to be used in order to objectively characterise sensitivity limits.     

Application of QUIZ for GM quantification in food

The quantification of GM in samples can be performed without the need for certified reference materials (CRMs) using QUIZ (quantitation using informative zeros). In this work we have applied QUIZ to estimate the contents of RoundUp Ready™ soya and MON810 in processed food containing one or both GMs. Results show good agreement between derived values and known input of GM material and compare favourably with quantitative real-time PCR.     

Laboratory performance study of the quantitative detection method for genetically modified soybeans (roundup ready soybeans 40-3-2)

To investigate important factors affecting the analytical results, a laboratory-performance study was attempted for the Japanese official methods to detect genetically modified (GM) soybeans (40-3-2). Test samples containing 0, 1 and 5% GM soya powder in non-GM soya powder was prepared. A set of 3 test samples was sent to the participating laboratories along with the protocol. The data were collected from all laboratories and statistically analyzed. In the real-time PCR detection method, the average values of the GM 1% and 5% samples were both much lower than the spiked value because the laboratories using a silica-membrane DNA extraction method underestimated the GM value. On the other hand, the laboratories using other extraction methods, such as the CTAB method obtained values close to the spiked value. These results suggest that use of the silica-membrane DNA extraction method may result in underestimation of the GM content in the real-time PCR method. In the ELISA method, the average value of 5% spiked samples appears to be slightly higher than the fortified value. But, overall, it was considered that reported values were close to the spiked level.     

Influence of cocoa components on the PCR detection of soy lecithin DNA

The labelling of genetically modified organisms (GMOs) is mandatory in the EU. To comply with this regulation, the International Standardisation Organisation recently published standards for the extraction of DNA and PCR detection of GMOs. The PCR detection of (GM) soy lecithin in chocolate however is not an easy assignment. For the most part this is due to inhibitors such as polyphenols resulting from the cocoa mass and the low level of soy lecithin present. Many factors however, such as the choice of extraction method, sample size and PCR protocol influence the PCR end result. Therefore, preconditions regarding the quality, quantity and purity of the extracted DNA should be carefully determined to guarantee a successful GMO analysis.     

四种基因定量方法对实时荧光PCR与微滴式数字PCR检测霍乱弧菌基因表达量的分析

基因表达量测定对基因功能解析和探索生命机理具有重大意义,目前实时荧光PCR和微滴式数字PCR技术是进行基因表达量测定的有效方法。本文以低温处理的霍乱弧菌sfs、vcc、Rec A、hly及16S r RNA基因为研究对象,分别用实时荧光PCR和微滴式数字PCR技术对低温处理前后霍乱弧菌的上述基因表达量进行测定,实时荧光PCR实验数据分别采用??CT和ge Norm法进行分析,微滴式数字PCR实验数据分别采用相对定量和绝对定量法进行分析,获得处理组基因表达量相对于对照组的变化倍数;使用多维尺度法来分析四种基因定量方法的数据。结果表明,在本实验条件下,四种基因定量方法的分析结果存在差异;16S r RNA基因表达量发生了变化,不适合作为内参基因;微滴式数字PCR能更直观的给出基因表达量变化的结果,并且基因表达差异相对定量分析的准确度更高。     

A survey of the use of soy in processed Turkish meat products and detection of genetic modification

To screen for possible illegal use of soybeans in meat products, the performance characteristics of a commercial polymer chain reaction (PCR) kit for detection of soybean DNA in raw and cooked meat products were established. Minced chicken and beef products containing soybean at levels from 0.1% to 10.0% were analysed by real-time PCR to amplify the soybean lectin gene. The PCR method could reliably detect the addition of soybean at a level of 0.1%. A survey of 38 Turkish processed meat products found only six samples to be negative for the presence of soybean. In 32 (84%) positive samples, 13 (34%) contained levels of soy above 0.1%. Of soybean positive samples, further DNA analysis was conducted by real-time PCR to detect whether genetically modified (GM) soybean had been used. Of 32 meat samples containing soybean, two samples were positive for GM modification.