Kinetic Outlier Detection (KOD) in real-time PCR

Real-time PCR is becoming the method of choice for precise quantification of minute amounts of nucleic acids. For proper comparison of samples, almost all quantification methods assume similar PCR efficiencies in the exponential phase of the reaction. However, inhibition of PCR is common when working with biological samples and may invalidate the assumed similarity of PCR efficiencies. Here we present a statistical method, Kinetic Outlier Detection (KOD), to detect samples with dissimilar efficiencies. KOD is based on a comparison of PCR efficiency, estimated from the amplification curve of a test sample, with the mean PCR efficiency of samples in a training set. KOD is demonstrated and validated on samples with the same initial number of template molecules, where PCR is inhibited to various degrees by elevated concentrations of dNTP; and in detection of cDNA samples with an aberrant ratio of two genes. Translating the dissimilarity in efficiency to quantity, KOD identifies outliers that differ by 1.3–1.9-fold in their quantity from normal samples with a P-value of 0.05. This precision is higher than the minimal 2-fold difference in number of DNA molecules that real-time PCR usually aims to detect. Thus, KOD may be a useful tool for outlier detection in real-time PCR.     

Validation of kinetics similarity in qPCR

Quantitative real-time PCR (qPCR) is the method of choice for specific and sensitive quantification of nucleic acids. However, data validation is still a major issue, partially due to the complex effect of PCR inhibition on the results. If undetected PCR inhibition may severely impair the accuracy and sensitivity of results. PCR inhibition is addressed by prevention, detection and correction of PCR results. Recently, a new family of computational methods for the detection of PCR inhibition called kinetics outlier detection (KOD) emerged. KOD methods are based on comparison of one or a few kinetic parameters describing a test reaction to those describing a set of reference reactions. Modern KOD can detect PCR inhibition reflected by shift of the amplification curve by merely half a cycle with specificity and sensitivity >90%. Based solely on data analysis, these tools complement measures to improve and control pre-analytics. KOD methods do not require labor and materials, do not affect the reaction accuracy and sensitivity and they can be automated for fast and reliable quantification. This review describes the background of KOD methods, their principles, assumptions, strengths and limitations. Finally, the review provides recommendations how to use KOD and how to evaluate its performance.     

PCR-based assays for the fish pathogen Aeromonas salmonicida. I. Evaluation of three PCR primer sets for detection and identification

In an effort to develop a rapid diagnostic test for the fish pathogen Aeromonas salmonicida, the performance of 2 polymerase chain reaction (PCR) primer sets (AP and PAAS) targeting the fish pathogen A. salmonicida and 1 PCR primer set (MIY) targeting A. salmonicida subsp. salmonicida were evaluated. Initially, the PCR assays were used to screen purified DNA extracted from 308 A. salmonicida isolates. The AP and PAAS PCR tests were demonstrated to be 100% specific for the species A. salmonicida and did not cross-react with any of the non-target organisms (bacterial species other than A. salmonicida) used in this study. The combined sensitivity of the AP and PAAS tests was 99.4% and offered the best coverage in terms of identifying the target organism. The MIY PCR appeared to be 100% sensitive and specific for A. salmonicida subsp. salmonicida. Studies with tissues, spiked with known quantities of bacteria, were conducted to determine the lower detection limit of the PCR tests, and then the ability of these PCR tests to detect A. salmonicida in experimentally infected salmonids was assessed.     

Development of PCR-based methods for detection of Sphaerothecum destruens in fish tissues

Single-round and nested polymerase chain reaction (PCR) tests were developed for amplification of a 434 bp fragment of the small subunit ribosomal RNA (18S rRNA) gene from Sphaerothecum destruens, previously known as the rosette agent, an intracellular parasite of salmonid fishes. Both tests have successfully amplified S. destruens-specific DNA from different isolates of S. destruens but not from related organisms. The limits of detection using the nested PCR test were 1 pg for purified S. destruens genomic DNA and 0.1 fg for plasmid DNA. We conducted 2 experimental transmission studies, consisting of injection or waterborne exposure of juvenile winter-run Chinook salmon Oncorhynchus tshawytscha to spore stages of the parasite. In the injection study, parasite DNA was detected in 100% of kidney samples from exposed fish (n = 83) at 1 and 3 mo post-exposure using nested PCR, versus 98% using microscopic analysis of Gram-stained impression smears made from the kidney. Following waterborne exposure, fish were sampled over the course of a year. From each fish, samples of gill, liver, posterior intestine and kidney were analyzed. S. destruens-specific DNA was detected most often in gill and kidney over the course of the experiment, and 71% (64/90) of the exposed fish were identified as positive for S. destruens using the nested PCR test, versus 16% (14/90) using microscopic analysis of Gram-stained kidney smears. Natural infections in captive broodstock of adult winter-run Chinook salmon, originally diagnosed by examination of Gram-stained kidney smears, were confirmed using the nested PCR test in all fish examined (15/15). Further, the nested test amplified parasite-specific DNA from other tissues in these fish with varying frequencies. This report introduces the first DNA-based detection method for S. destruens, to be used alone as a diagnostic tool or in conjunction with histologic tests for confirmatory identification of the parasite.     

Studies on effective PCR screening strategies for white spot syndrome virus (WSSV) detection in Penaeus monodon brooders.

We re-tested stored (frozen) DNA samples in 5 independent polymerase chain reaction (PCR) replicates and confirmed that equivocal test results from a previous study on white spot syndrome virus (WSSV) in brooders and their offspring arose because amounts of WSSV DNA in the test samples were near the sensitivity limits of the detection method. Since spawning stress may trigger WSSV replication, we also captured a fresh batch of 45 brooders for WSSV PCR testing before and after spawning. Replicates of their spawned egg batches were also WSSV PCR tested. For these 45 brooders, WSSV prevalence before spawning was 67% (15/45 1-step PCR positive, 15/45 2-step PCR positive and 15/45 2-step PCR negative). Only 27 (60%) spawned successfully. Of the successful spawners, 56% were WSSV PCR positive before spawning and 74% after. Brooders (15) that were heavily infected (i.e. 1-step PCR positive) when captured mostly died within 1 to 4 d, but 3 (20%) did manage to spawn. All their egg batch sub-samples were 1-step PCR positive and many failed to hatch. The remaining 30 shrimp were divided into a lightly infected group (21) and a 2-step PCR negative group (9) based on replicate PCR tests. The spawning rates for these 2 groups were high (81 and 78%, respectively). None of the negative spawners (7) became WSSV positive after spawning and none gave egg samples positive for WSSV. In the lightly infected group (21), 6 brooders were 2-step WSSV PCR negative and 15 were 2-step WSSV PCR positive upon capture. However, all of them were WSSV PCR positive in replicate tests and after spawning or death. Four died without spawning. The remaining 17 spawned but only 2 gave egg samples PCR negative for WSSV. The other 15 gave PCR positive egg samples, but they could be divided into 2 spawner groups: those (7) that became heavily infected (i.e. 1-step PCR positive) after spawning and those (8) that remained lightly infected (i.e. became or remained 2-step PCR positive only). Of the brooders that became heavily infected after spawning, almost all egg sample replicates (91 %) tested 2-step PCR positive. One brooder even gave heavily infected (i.e. 1-step PCR positive) egg samples. For the brooders that remained lightly infected after spawning, only 27% of the egg sample replicates were 2-step PCR positive. Based on these results, we recommend that to avoid false negatives in WSSV PCR brooder tests screening tests should be delayed until after spawning. We also recommend, with our PCR detection system, discarding all egg batches from brooders that are 1-step PCR positive after spawning. On the other hand, it may be possible with appropriate monitoring to use eggs from 2-step PCR positive brooders for production of WSSV-free or lightly infected postlarvae. These may be used to stock shrimp ponds under low-stress rearing conditions.     

Sources of variation in the mutagenic potency of complex chemical mixtures based on the Salmonella/microsome assay.

Twenty laboratories worldwide participated in a collaborative trial sponsored by the International Programme on Chemical Safety on the mutagenicity of complex mixtures as expressed in the Salmonella/microsome assay. The U.S. National Institute of Standards and Technology provided homogeneous reference samples of urban air and diesel particles and a coal tar solution to each participating laboratory, along with samples of benzo[a]pyrene and 1-nitropyrene which served as positive controls. Mutagenic potency was characterized by the slope of the initial linear component of the dose-response curve. Analysis of variance revealed significant interlaboratory variation in mutagenic potency, which accounted for 57-96% of the total variance on a logarithmic scale, depending on the sample, strain and activation conditions. Variation among replicate extractions of organic material (required for the air and diesel particles) and among replicate bioassays within the same laboratory was also appreciable. The average potencies for air and diesel particles in laboratories using Soxhlet extracts were not significantly different from those in laboratories using sonication, although there was larger interlaboratory variation for the Soxhlet method. Repeatability (which approximates the coefficient of variation within laboratories) ranged from 18 to 40% for air and diesel particles extracted using sonication, depending on the strain and activation conditions. Repeatability of Soxhlet-extracted air and diesel particles, however, ranged from about 37 to 89% including outliers and from about 11 to 31% excluding outliers. Repeatability of the coal tar sample and the 2 positive controls was in the range 18-34%. Reproducibility (which approximates the coefficient of variation between laboratories) was generally at least twice repeatability, and exceeded 100% for Soxhlet-extracted air and diesel particles, as well as 1-nitropyrene. Reanalysis of the data omitting observations of more than 1500 revertants/plate generally had little effect on these results. Elimination of outlying observations had limited impact, with the exception of Soxhlet-extracted air and diesel particles. In this case, reproducibility of bioassay results was notably improved, due largely to the omission of results for replicate extractions which varied more than 5-fold within one laboratory. Normalization of the log potency slopes for the mixtures by the corresponding slopes for benzo[a]pyrene tended to reduce this variation, although variation was increased after normalization by 1-nitropyrene. Adjustment for the percentage of organic matter extracted from the air and diesel particulate samples had little effect on variation for sonication-extracted particles, whereas variation was reduced for diesel particles and increased for air particles for Soxhlet.     

Species-specific detection of hydrocarbon-utilizing bacteria

Rapid detection and quantitative assessment of specific microbial species in environmental samples is desirable for monitoring changes in ecosystems and for tracking natural or introduced microbial species during bioremediation of contaminated sites. In the interests of developing rapid tests for hydrocarbon-degrading bacteria, species-specific PCR primer sets have been developed for Pseudomonas aeruginosa, Stentrophomonas (Xanthomonas) maltophilia, and Serratia marsescens. Highly variable regions of the 16S rRNA gene were used to design these primer sets. The amplification products of these primer sets have been verified and validated with hemi-nested PCR and with ligase chain reaction (LCR) techniques, and have been applied to the analyses of environmental water samples. These species-specific primer sets were also chosen to amplify in conjunction with a universal set of PCR primers chosen from highly conserved neighboring sequences in the same gene. These multiplex or competitive PCR procedures enable testing with an internal marker and/or the quantitative estimation of the relative proportion of the microbial community that any one of these species occupies. In addition, this universal PCR primer set amplified the same size amplicon from a wide spectrum of procaryotic and eucaryotic organisms and may have potential in earth biota analyses.     

The application of real-time PCR to the identification, detection and quantification of Pyrenophora species in barley seed

A real-time quantitative PCR technique has been used to develop a rapid and sensitive seed health test for Pyrenophora spp. on barley seed. Using the fluorescent reporter dye SYBR Green I for real-time detection of PCR amplification, pathogen DNA extracted from infected seed can be quantified to the picogram level. The amount of Pyrenophora DNA extracted from seed samples of an artificial infection level gradient, constructed by mixing infected and uninfected seed, correlated with good agreement ( r  = 0.931) to percentage infection levels of the same samples measured by agar plate testing. In addition, a correlation of r  = 0.883 was obtained between the two testing methods for naturally infected seed, ranging from 0% to 89% infection. Samples could be quantified to below the 2% voluntary threshold required for deciding on seed treatment. The proposed test was performed in three parts: (i) quantification of Pyrenophora spp. infection using Pyrenophora -specific PCR primers; (ii) test of any negative samples from (i) with barley-specific PCR primers to check the DNA extraction process; (iii) test of positive samples from (i) for the presence of Pyrenophora graminea using P. graminea -specific PCR primers. All PCRs were performed in the LightCycler™ instrument allowing each PCR run and analysis to be completed within 30 min. With the current daily receipt of samples (batches up to 16) the test can be completed in 8 h, compared to 7 days for the traditional agar plate test.     

An in-house real-time polymerase chain reaction: standardisation and comparison with the Cobas Amplicor HBV monitor and Cobas AmpliPrep/Cobas TaqMan HBV tests for the quantification of hepatitis B virus DNA

This study aimed to standardise an in-house real-time polymerase chain reaction (rtPCR) to allow quantification of hepatitis B virus (HBV) DNA in serum or plasma samples, and to compare this method with two commercial assays, the Cobas Amplicor HBV monitor and the Cobas AmpliPrep/Cobas TaqMan HBV test. Samples from 397 patients from the state of São Paulo were analysed by all three methods. Fifty-two samples were from patients who were human immunodeficiency virus and hepatitis C virus positive, but HBV negative. Genotypes were characterised, and the viral load was measure in each sample. The in-house rtPCR showed an excellent success rate compared with commercial tests; inter-assay and intra-assay coefficients correlated with commercial tests (r = 0.96 and r = 0.913, p < 0.001) and the in-house test showed no genotype-dependent differences in detection and quantification rates. The in-house assay tested in this study could be used for screening and quantifying HBV DNA in order to monitor patients during therapy.     

Global DNA Hypomethylation in Liver Cancer Cases and Controls: A Phase I Preclinical Biomarker Development Study

Background: Global genomic DNA hypomethylation is a feature of genomic DNA derived from solid and hematologic tumors in animal models and human carcinogenesis. Global genomic DNA hypomethylation may be the earliest epigenetic change from a normal to a pre-malignant cell. Objectives: To test if global hypomethylation is a good marker for early detection of cancer we used a novel quantification method of 2’-deoxynucleosides to evaluate DNA methylation in liver cancer cases and controls. Methods: Frozen tissue from liver cancer patients and controls were obtained from the Cooperative Human Tissue Network. DNA was extracted using standard methods. Genomic DNA samples were boiled and treated with nuclease P1 and alkaline phosphatase. Global genomic DNA methylation patterns were obtained using HPLC for fraction separation and mass spectrometry for quantification. A two-sample t test was performed using Welch’s approximation for samples with unequal variances. A Wilcoxon rank sum test was also performed. Results: A global genomic DNA methylation index measuring methylated cytidine relative to global cytidine in the genome was significantly lower (p-value = 0.001) for all cases, mean = 2.43 (95% CI, 2.08, 2.78), when compared to controls, mean = 3.55 (95% CI, 3.16, 3.93). Discussion: A correlation between global genomic DNA methylation patterns and type of liver tissue was observed. These results add to the accumulating body of evidence suggesting that global DNA hypomethylation may be a useful biomarker to distinguish between liver cancer cases and controls.     

Reliability of diagnostic techniques forErwinia amylovora, the causative agent of fire blight disease

A total of 20 putative strains of Erwinia amylovora originating from 11 samples of host plants with symptoms of fire blight were analyzed in detail using commercial polyclonal antibodies in immunochemical tests. Fourteen strains reacted negatively in all tests; 6 strains reacted positively with a polyclonal antibody for PTA-ELISA (plate-trapped antigen-enzyme linked immunosorbent assay) at a concentration corresponding to A620 = 0.1, while at A620 readings of 0.01 and 0.001 the results were negative. Five strains reacted positively with a polyclonal antibody for indirect immunofluorescence test at all tested concentrations. Three of those strains were positive in the PCR test with AMSbL and AMSbR primers designed for detection of E. amylovora. In hypersensitivity test in tobacco and in immature pear fruit assay, all putative strains were negative while a known reference strain of E. amylovora gave a typical hypersensitive-reaction response. On a medium with 5% sucrose the reference strain of E. amylovora produced levan while putative strains did not. After modification of the PCR protocol, 3 putative strains reacted as negatives. Optimization of PCR test was achieved by finding the optimum annealing temperature and time for primers. The recommended annealing temperature (49 degrees C) for these primers was increased to 55 degrees C and the annealing time was reduced from 2 min to 30 s. Using the microbial identification system Biolog those 3 strains were identified as Pantoea dispersa (1 strain) and Pantoea agglomerans (2 strains). The strains are supposed to be white variants of the species P. dispersa and P. agglomerans occurring less frequently than the yellow variants. Since there were positive reactions in our immunochemical tests these strains could cause false positives in routine screening of plant samples.     

Comparison of methods for quantification of cytochrome cd(1)-denitrifying bacteria in environmental marine samples

Two PCR primer sets were developed for the detection and quantification of cytochrome cd(1)-denitrifying bacteria in environmental marine samples. The specificity and sensitivity of these primers were tested. Both primer sets were suitable for detection, but only one set, cd3F-cd4R, was suitable for the quantification and enumeration of the functional community using most-probable-number PCR and competitive PCR techniques. Quantification of cytochrome cd(1) denitrifiers taken from marine sediment and water samples was achieved using two different molecular techniques which target the nirS gene, and the results were compared to those obtained by using the classical cultivation method. Enumerations using both molecular techniques yielded similar results in seawater and sediment samples. However, both molecular techniques showed 1,000 or 10 times more cytochrome cd(1) denitrifiers in the sediment or water samples, respectively, than were found by use of the conventional cultivation method for counting.     

Real-time PCR for simultaneous detection and quantification of Borrelia burgdorferi in field-collected Ixodes scapularis ticks from the Northeastern United States

The density of spirochetes in field-collected or experimentally infected ticks is estimated mainly by assays based on microscopy. In this study, a real-time quantitative PCR (qPCR) protocol targeting the Borrelia burgdorferi-specific recA gene was adapted for use with a Lightcycler for rapid detection and quantification of the Lyme disease spirochete, B. burgdorferi, in field-collected Ixodes scapularis ticks. The sensitivity of qPCR for detection of B. burgdorferi DNA in infected ticks was comparable to that of a well-established nested PCR targeting the 16S-23S rRNA spacer. Of the 498 I. scapularis ticks collected from four northeastern states (Rhode Island, Connecticut, New York, and New Jersey), 91 of 438 (20.7%) nymphal ticks and 15 of 60 (25.0%) adult ticks were positive by qPCR assay. The number of spirochetes in individual ticks varied from 25 to 197,200 with a mean of 1,964 spirochetes per nymphal tick and a mean of 5,351 spirochetes per adult tick. No significant differences were found in the mean numbers of spirochetes counted either in nymphal ticks collected at different locations in these four states (P = 0.23 by one-way analysis of variance test) or in ticks infected with the three distinct ribosomal spacer restriction fragment length polymorphism types of B. burgdorferi (P = 0.39). A high degree of spirochete aggregation among infected ticks (variance-to-mean ratio of 24,877; moment estimate of k = 0.279) was observed. From the frequency distribution data and previously published transmission studies, we estimated that a minimum of 300 organisms may be required in a host-seeking nymphal tick to be able to transmit infection to mice while feeding on mice. These data indicate that real-time qPCR is a reliable approach for simultaneous detection and quantification of B. burgdorferi infection in field-collected ticks and can be used for ecological and epidemiological surveillance of Lyme disease spirochetes.     

Standardized ultrasound hepatic/renal ratio and hepatic attenuation rate to quantify liver fat content: an improvement method

Accurate measures of liver fat content are essential for investigating the role of hepatic steatosis in the pathophysiology of multiple metabolic disorders. No traditional imaging methods can accurately quantify liver fat content. [(1)H]-magnetic resonance spectroscopy (MRS) is restricted in large-scale studies because of the practical and technological issues. Previous attempts on computer-aided ultrasound quantification of liver fat content varied in method, and the ultrasound quantitative parameters measured from different ultrasound machines were hardly comparable. We aimed to establish and validate a simple and propagable method for quantitative assessment of liver fat content based on the combination of standardized ultrasound quantitative parameters, using [(1)H]-MRS as gold standard. Totally 127 participants were examined with both ultrasonography (US) and [(1)H]-MRS. Ultrasound hepatic/renal echo-intensity ratio (H/R) and ultrasound hepatic echo-intensity attenuation rate (HA) were obtained from ordinary ultrasound images using computer program. Both parameters were standardized using a tissue-mimicking phantom before analysis. Standardized ultrasound H/R and HA were positively correlated with the liver fat content by [(1)H]-MRS (r = 0.884, P < 0.001 and r = 0.711, P < 0.001, respectively). Linear regression analysis showed ultrasound H/R could modestly predict the amount of liver fat (adjusted explained variance 78.0%, P < 0.001). The addition of ultrasound HA slightly improved the adjusted explained variance to 79.8%. Difference of estimated liver fat contents between different ultrasound machines and operators was reasonably well. Thus, computer-aided US is a valid method to estimate liver fat content and can be applied extensively after standardization of ultrasound quantitative parameters.     

PCR-based quantification of Pneumocystis carinii in in vitro systems

In many laboratories, PCR has become a routine method for the sensitive diagnosis of Pneumocystis carinii in patient samples. In contrast, quantification of fungal numbers in in vitro setups still largely relies on more conventional procedures such as histological stainings. These are time consuming and their applications are limited when dealing with small fungal numbers contaminated with tissue and cellular debris. This study presents a sensitive and rapid method for P. carinii quantification based on PCR analysis that can be easily integrated into standard detection procedures without requiring any major additional steps. P. carinii-specific PCR performed with total DNA extracted from both standard samples with known fungal numbers and experimental samples was quantified relative to PCR products of a standard concentration from a control plasmid added prior to DNA extraction. This measure controlled for variations in DNA extraction and PCR efficiency among the samples to be compared. The correlation between analyzed P. carinii-specific DNA and the actual fungal numbers employed was highly significant.     

Use of real-time PCR to discriminate parasitic and saprophagous behaviour by nematophagous fungi

Entomopathogenic nematodes (EPNs) are important pathogens of soilborne insects and are sometimes developed commercially to manage insect pests. Numerous nematophagous fungal species (NF) prey on nematodes and are thought to be important in regulating natural or introduced EPN populations. However, nematophagy by these fungi in nature cannot be inferred using existing methods to estimate their abundance in soil because many of these fungi are saprophytes, resorting to parasitism primarily when certain nutrients are limiting. Therefore, we developed an assay to quantify NF DNA in samples of nematodes. Species-specific primers and TaqMan probes were designed from the ITS rDNA regions of Arthrobotrys dactyloides, Arthrobotrys oligospora, Arthrobotrys musiformis, Gamsylella gephyropagum and Catenaria sp. When tested against 23 non-target fungi, the TaqMan real-time PCR assay provided sensitive and target-specific quantification over a linear range. The amount of A. dactyloides or Catenaria sp. DNA in 20 infected nematodes, measured by real-time PCR, differed between fungal species (P=0.001), but not between experiments (P>0.05). However, estimates of relative NF parasitism using a bioassay with 20 nematodes infected by either species, differed greatly (P<0.001) depending on whether the fungi were alone or combined in the samples used in the assay. Tests done to simulate detection of NF DNA in environmental samples showed that, for all species, background genomic DNA and/or soil contaminants reduced the quantity of DNA detected. Nested PCR was ineffective for increasing the detection of NF in environmental samples. Indeed, real-time PCR detected higher amounts of NF DNA than did nested PCR. The spatial patterns of NF parasitism in a citrus orchard were derived using real-time PCR and samples of nematodes extracted from soil. The parasitism by Catenaria sp. was positively related to the abundance of both heterorhabditid and steinernematid EPNs. The possible significance of the associations is ambiguous because NF attack a broad range of nematode taxa whereas EPNs are a small minority of the total nematode population in a soil sample. These studies demonstrate the potential of real-time PCR to study the role of NF parasitism in soil food webs.     

Qualitative and Quantitative PCR Methods for Event-specific Detection of Genetically Modified Cotton Mon1445 and Mon531

Based on the DNA sequences of the junctions between recombinant and cotton genomic DNA of the two genetically modified (GM) cotton varieties, herbicide-tolerance Mon1445 and insect-resistant Mon531, event-specific primers and probes for qualitative and quantitative PCR detection for both GM cotton varieties were designed, and corresponding detection methods were developed. In qualitative PCR detection, the simplex and multiplex PCR detection systems were established and employed to identify Mon1445 and Mon531 from other GM cottons and crops. The limits of detection (LODs) of the simplex PCR were 0.05% for both Mon1445 and Mon531 using 100 ng DNA templates in one reaction, and the LOD of multiplex PCR analysis was 0.1%. For further quantitative detection using TaqMan real-time PCR systems for Mon1445 and Mon531, one plasmid pMD-ECS, used as reference molecule was constructed, which contained the quantitative amplified fragments of Mon1445, Mon531, and cotton endogenous reference gene. The limits of quantification (LOQs) of Mon1445 and Mon531 event-specific PCR systems using plasmid pMD-ECS as reference molecule were 10 copies, and the quantification range was from 0.03 to 100% in 100 ng of the DNA template for one reaction. Thereafter, five mixed cotton samples containing 0, 0.5, 0.9, 3 and 5% Mon1445 or Mon531 were quantified using established real-time PCR systems to evaluate the accuracy and precision of the developed real-time PCR detection systems. The accuracy expressed as bias varied from 1.33 to 8.89% for tested Mon1445 cotton samples, and from 2.67 to 6.80% for Mon531. The precision expressed as relative standard deviations (RSD) were different from 1.13 to 30.00% for Mon1445 cotton, and from 1.27 to 24.68% for Mon531. The range of RSD was similar to other laboratory results (25%). Concluded from above results, we believed that the established event-specific qualitative and quantitative PCR systems for Mon1445 and Mon531 in this study are acceptable and suitable for GM cotton identification and quantification.