Rapid Detection Of Transgenic Crops Based On DNA Isothermal Amplification

Genetically modified crops (GM) have been generalized and commercially planted in many countries. Although it has brought people enormous economic benefits, potential uncertain risk towards people’s health and environment still exists all over the world. Rapid and reliable detection of GMOs has great significance and matters a lot to the international political and economic interests. Traditional methods for GM detection based on foreign protein assays have obvious inherent drawbacks. While the ones based on introduced DNA assays are limited by the requirements of trained operator and also professional, costly and bulky equipment. To explore portable, rapid and simple methods based on nucleic acid analysis for GM detection, three insect-resistant GM rice Huahui 1, Kefeng 6 and KMD lwere used as plant materials. Cross priming isothermal amplification and loop-mediated-isothermal amplification was employed as the amplification methods. Two novel methods were established in this paper:1) pH-inspired label-free electrochemical method and 2) visual detection method based on phosphate ions assay. After that, a informative strategy based on logical judgement for rapid identification of GM events was investigated, which was further employed for GM rice event recognition on a paper base with visual dots array.Main contents and results are summarized as follows:1) Cross-priming amplification assay targeting for T-nos was established and investigated. A set of six specific primers, recognizing eight distinct regions on T-nos sequence was designed. Real-time CPA was monitored by S YTO 9 and compared with conventional real-time PCR. The results indicated that real-time fluorescent CPA had high specificity and the hmit of detection was about 103 copies of rice genomic DNA. Compared to conventional real-time PCR, real-time fluorescent CPA had a comparable sensitivity, while taken shorter time (save about 10 min). In addition, different contents of GM contaminated rice seeds powder samples were detected for practical application. The result showed real-time fluorescent CPA could detect 0.5% GM contaminated samples at least, which is sensitive enough for the requirements of GM labeling systems in most countries/regions of the world. That is, CPA is shown to be a great alternation of traditional fluorescent PCR with less time and easier equipments needed.2) A portable and label-free pH-mediated electrochemical method for the detection of DNA amplification is described. With protons released as readout, DNA amplifications were detected in real-time or at the end-point. The optimal reaction composition for pH-CPA was 50 mM K+,15 mM NH4+with 3 mM KOH addition. Commercial pH sensor was successfully used for real-time pH-CPA detection. We defined the signal threshold as the point at which amplification was most efficient and the corresponding pH decreased most rapidly. For each pH-CPA reaction, this threshold is reliably decided by identifying the local minimum in the corresponding pH-changes derivative trace. We subsequently defined the "time to threshold" (tth) as the time required for a particular sample reaction to reach the signal threshold. We observed distinct local minima separated by approximately 3 min for pH traces with 10 times difference in the copy number of initial genomic DNA template. Compared with the fluorescent real-time CPA, the testing time of DNA amplification was saved about 20%. Besides, home-made pH-sensitive disposable screen printed electrodes were fabricated and detected CPA at the end-point. The potential peak spacing of positive samples with initial templates about 103 copies of genomic DNA decreased about 28.3 mV after amplification, that is 0.38 pH, which is comparable with the result obtained from commercial pH sensor (approximately 0.35 pH). In conclusion, protons released during DNA amplification can be detected electrochemically as readout for real-time or at the end-point. Coupled with isothermal amplification technology, it is shown to be portable and easy to use. Benefit from the feature of electrochemical detection methods, it also can be easily integrated into a miniature device and thus has greater potential of application in many different areas.3) A visual method based on Pi assays for DNA amplification was established. The feasibilities of this strategy for different amplification systems use were systematically studied. A disposable contamination proof strip was also designed. Results indicated that dNTP template-independent decomposition was the only source of non-specific Pi and the main factor affected such process is reaction temperature. Non-specifically Pi generated is about 13% of the initial dNTPs in PCR reation (40 complete PCR cycles) and 2% of the initial dNTPs in CPA reaction (63℃ for 1 h). For the specific Pi generation study, positive samples with PPase treatment in CPA assay generated about 2.86 mM Pi, which was nearly 36 times of that in the positive samples without PPase treatment or 53 times of that in the negative samples. In PCR assays, about 0.5 mM Pi has been produced in positive samples comparing to about 0.15 mM in negative ones. That was, about 87.5% of initial dNTPs were used for DNA amplification and 3.36% decomposed template-independently in CPA assay, which were 22.3% and 18.6% correspondingly in PCR. In addition, we also found that with only 15 min of CPA amplification (103 copies of gDNA as target), a distinct difference in color between positive and negtive samples can be observed visually, saving up about 50% time required in electrochemical method described in Chapter 3. Besides, this Pi induced visual detection methods was further proved have the potential to be used in other kinds of isothermal amplification system. Thus, a yes/no answer for DNA isothermal amplification can be obtained visually with this phosphate ions-based method described. It can maximize the advantage of isothermal amplification technology to be used on site in resource-limited areas.4) An informative strategy based on logical judgement for rapid identification of GM events was described. Three common insect-resistant GM rice (Huahui 1, Kefeng 6 and KMD 1) were used as plant materials. A set of target gene was first screened to cover all of these three events. LAMP was taken as a typical example in this study. After amplification, samples were treated with Mo-Sb-Vc coloration reagent. Taking every blue readout as 1 and colorless readout as 0, each sample can get a serial of number such as "1101". On the basis of sequenced numbers obtained, the exact events of testing samples can be identified. The results have shown that for a simultaneous tracking of sps, Nos, CaMV35S and Cry 1Ac, an incubation duration of 35 min is needed.0.5% contaminated samples can be detected visually and specifically. Intensive processing procedure can hardly affect the performance of this LAMP-based Pi-tracking detection method. To further explore the possible application of this strategy described, a paper-based colorimetric assay was conducted. Whatman grade 1 Chr chromatography paper was chosen to be the matrix and pretreated with Pi coloration reagents in an array pattern. Amplified samples were added onto the paper base and there would be blue dots array appeared as a result. GM events can be then obtained accordingly. In conclusion, we have described a fundamentally different strategy to identify insect-resistant GM rice, which is informative, rapid, ready to use and easy to be updated. It is providing new ways for multiple target analysis in a wide area.