Uncertainty in Measuring Construct-specific Fragments of Genetically Modified Maize MON863 by Real Time Quantitative PCR

[Objective] The study aimed at evaluating the uncertainty in measuring the construct-specific fragments of genetically modified maize MON863 by real time quantitative PCR.[Method] The content of construct-specific fragments in MON863 samples was determined by real time quantitative PCR,and then the uncertainty of measurement result was evaluated according to the sources of uncertainty like the PCR system,the data processing and the micropipette.[Result] Type A evaluation of uncertainty（uA） in the measurement was 1.7×10^-2;Type B evaluation of uncertainty（uB） was 9.0×10^-4;the combined standard uncertainty（uC） was 1.7×10^-2;the expanded uncertainty（U95） was 0.036 and the finally measured result was 1.08%±0.036.[Conclusion] The main uncertainty of the result measured by real time quantitative PCR came from the randomizing effect in the experimental process.

Establishment of a Real-time Fluorescent Quantitative PCR Assay for Detection of Genetically Modified Maize Line MON88017

In order to improve the standardized technical systems of quantitative analyses for genetically modified organisms （GMOs） and products, ensure bio-safety and reduce ecological risk in China, a real-time fluorescent quantitative PCR assay was established for detection of genetically modified maize line MON88017. The established method was evaluated based on the specificity, sensitivity, accuracy and measurement uncertainty. The results showed that the established method had strong specificity in detection of genetically modified maize line MON88017. 1.50% MON88017 sample was detected with 29 replica- tions. The average measured value （ 1. 541% ） was close to the actual value （ 1.50% ） and the relative deviation was 2.70%. The variation coefficient of the measured value was 0.110 g ; the recovery was 100.00% and the measurement uncertainty was 0. 096. The limit of detection for genetically modified maize line MON88017 with the established method was 5 copies at the 97.5% confidence level. Thus, the real-time fluorescent quantitative PCR assay established in this study exhibited high specificity, accuracy and sensitivity, which could provide technical support for the safety supervision of genetically modified organ- isms and products in China.

Establishment of a Construct-specific Real-time PCR Method for Quantitative Detection of Genetically Modified Maize Line MIR604

In this study, a construct-specific real-time PCR method for quantitative detection of genetically modified maize line MIR604 was established with prim- ers and probes designed based on vector sequence of MIR604 under optimized reaction system and thermal cycling condition. By using the established method, six non-genetically modified crops, genetically modified maize line MIR604 and other non-target genetically modified crops were detected. According to the results, flu- orescence signal could be detected in genomie DNA of MIR604, but other non-genetically modified crops and non-target genetically modified crops exhibited no fluo- rescence signal of MIR604 molecular fragment. Certified reference materials containing 1% MIR604 were detected with the established method and the results indi- cated that the average relative content in the test samples was 1.05%. The sensitivity analysis results indicated that MIR604 nucleic acid fragment with at least five copies could be detected with the established method. In conclusion, the construct-specific real-time PCR method for quantitative detection of genetically modified maize line MIR604 is of high specificity, accuracy and sensitivity, which provides technical support for safety supervision of genetically modified organisms in China.

Development of an Event-specific Quantitative PCR for Genetically Modified Maize (Zea mays) Event NK603

[ Objective ] The aim of this study was to develop a quantitative PCR detection method for genetically modified maize event NK603, so as to provide ba- sis for quantitative analysis of event NK603. [ Methods ] A quantitative PCR detection method for genetically modified maize event NK603 was developed using primers and Taqman probe designed according to the flanking sequence of event NK603, which was then adopted to detect the samples containing 2% NK603 stand- ard （with uncertain quantity of 10% ）. [ Results ] The slope of standard curve ranged between -3.6 and -3.1, and the correlation coefficient was higher than 0. 99. The amplification efficiency of this method reached 100.2%, fallen between 90% and 110%. The detected quantity of the experimental sample was 1.9%, closer to the true quantity （2%）. [ Conclusion] This quantitative PCR detection method for genetically modified maize event NK603 is very precise and can be a- dopted in routine testing analysis.

Expression profiles of Cry1Ab protein and its insecticidal efficacy against the invasive fall armyworm for Chinese domestic GM maize DBN9936

The fall armyworm(FAW)Spodoptera frugiperda,which originated in the Americas,is advancing across China and threatening the nation’s maize crops.Currently,one widely used tool for its control is genetically modified(GM)Bacillus thuringiensis(Bt)maize.Sufficient content of Bt protein in appropriate plant parts is crucial for enhancing resistance against insect pests.In this study,we conducted a systematic investigation of Cry1 Ab levels in Chinese domestic GM maize DBN9936,which has recently obtained a biosafety certificate,and evaluated its efficacy against FAW.Quantification of expression levels of Cry1 Ab,via ELISA,indicated a spatio-temporal dynamic,with significant variation of mean Cry1 Ab,ranging from 0.76 to 8.48μg g-1 FW with the Cry1 Ab protein level ranked as:V6-V8 leaf>R1 leaf>R4 leaf>R1 silk>VT tassel>R4 kernel.Among the nine locations,the Cry1 Ab levels in DBN9936 of the Xinxiang,Langfang,and Harbin fields were significantly lower than those from Wuhan and Shenyang,and were slightly,but not significantly lower than those from the other four fields.Furthermore,the artificial diet-Cry1 Ab mixture and plant tissue feeding bioassays revealed that DBN9936 has high efficacy against FAW.The insecticidal efficacy of different tissues against FAW larvae reached 34-100%with a descending order of lethality as follows:VT leaf>R4 leaf>R1 husk>R1 silk>VT tassel>R4 kernel.Taken together,our results showed that Bt-Cry1 Ab maize DBN9936 has potential as a promising strategy to manage FAW.

Influence of Soil Moisture and Air Temperature on the Stability of Cytoplasmic Male Sterility (CMS) in Maize (Zea mays L.)

Cytoplasmic male sterility (CMS) is a maternally inherited trait that suppresses the production of viable pollen. CMS is a useful biological tool for confinement strategies to facilitate coexistence of genetically modified (GM) and non-GM crops in case where it is required. The trait is reversible and can be restored to fertility in the presence of nuclear restorer genes (Rf genes) and by environmental impacts. The aim of this study was to investigate the influence of the level of irrigation on the stability of CMS maize hybrids under defined greenhouse conditions. Additionally the combination of irrigation and air temperature was studied. Three CMS maize hybrids were grown with different levels of irrigation and in different temperature regimes. Tassel characteristics, pollen production and fertility were assessed. The CMS stability was high in hot air temperatures and decreased in lower temperatures. The level of irrigation had no major effect on the level of sterility. The extent of these phenomena was depending on the genotype of CMS maize and should be known before using CMS for coexistence purposes.