Study Of Visual Detection Technology Based On Loop-mediated Isothermal Amplification For Genetically Modified Organisms

Loop-mediated isothermal amplification(LAMP) is rapid, high sensitivity and specificity in nucleic acid amplification under isothermal conditions, and had been used widely in many fields.However, the high sensitivity and amplification efficiency made it easy to bring contamination,so researchers studied out various ways to avoid opening tubes and made it simpler, visual and time saving when testing the products of LAMP after finishing the amplification, such as turbidimetric method, HNB, wax pill dye, and the agarose gel electrophoresis, turbidimetric method, HNB and SYBR Green I were the technology that popular.These existing methods either need opening tubes or indicators were hard to get, or two colors of before/after LAMP were the same system. To perfect the existing methods and find more easy, visual ways, our work and outcomes were as follows:(1) LAMP-ACBK detection method: In this study, a new closed-tube colorimetric method for monitoring LAMP with a novel metal indicator was developed. Firstly, Acid Chrome Blue K(ACBK), the metal indicator was evaluated in the initial LAMP reaction combined with various combination of reaction ingredient, such as reaction buffer, dNTPs mixture, primers mixture or Mg2+, respectively. This procedure demonstrated that the solution color of the LAMP reaction with ACBK can be changed from red to blue, as the concentration of Mg2+ decreasing in the reaction solution. Subsequently, the LAMP with ACBK for detection of 35 S was optimized and established. Further, the specificity of the new established colorimetric assay employing ACBK in LAMP reaction for 35 S was tested with diverse transgenic events come from different crops,and the sensitivity threshold of the assay was absolute 100 copies for transgenic rice genomic DNA and 50 ng 0.1% DNA from rice, soybean, rape, and maize, respectively. Finally, the applicability of the LAMP assay was further validated successfully using practical maize samples. All the detection results can be easily discerned either via naked-eye or using UV-vis spectroscopy.(2) LAMP-G-quadruplex peroxidase detection method: In this study, a new closed-tube colorimetric method based on the green of G-quadruplex peroxidase assay for monitoring LAMP was developed. Firstly, four groups of primers that were(FIP/BIP, FLP/BLP),(FIP/BIP-G4,FLP/BLP),(FIP/BIP, FLP/BLP-G4),(FIP/BIP-G4, FLP/BLP-G4) with other primers were added in LAMP reaction mix, respectively, and finished the amplification under 63 °C(12, 24, 36, 48,60) min, 85 °C 2 min. This procedure demonstrated that the reaction efficiency of LAMP withprimers(FIP/BIP-G4, FLP/BLP-G4) was lower than the other groups, and reactions containing(FIP/BIP, FLP/BLP-G4) had the smallest influence. Subsequently, the LAMP with G-quadruplex peroxidase for detection of 35 S was optimized and established. Further, the specificity of the new established colorimetric assay employing G-quadruplex peroxidase in LAMP reaction for35 S was tested with diverse transgenic events come from different crops, and the sensitivity threshold of the assay was about 32 ng/μL for transgenic rice genomic DNA and 50 ng 0.1%DNA from rice, soybean, rape, and maize, respectively. Finally, the applicability of the LAMP assay was further validated successfully using practical maize samples. All the detection results can be easily discerned via naked-eye.(3) LAMP-Strip detection method: In this study, a new simple, sensitive and visual detection method of LAMP production based on the strip technology was established. The primers FIP and BIP of LAMP was modified by FMA and Biotin, respectively, so we gained FIP-5’6-FAM and BIP-5’Biotin, other primers were as same. After the LAMP amplification, we can read the result in 2-3 min by putting the stripes into the finished LAMP reaction. Then the LAMP followed by the strip for detection of 35 S and TNOS was optimized and established.Further, the specificity of the new established employing strips in LAMP reaction for 35 S and TNOS was tested with diverse transgenic events come from different crops, and the sensitivity threshold of the assay for detecting 35 S and TNOS was about 5.4, 27.2 ng/μL respectively for transgenic rice genomic DNA.