Method for analyzing result of loop-mediated isothermal amplification

Abstract

The invention relates to a method for analyzing a result of loop-mediated isothermal amplification (LAMP), aiming at establishing a new method for analyzing the result of LAMP reaction, in which qualitative and quantitative information of a target gene is obtained by determining the change of electric conductivity of an LAMP solution; the method comprises the working steps: (1) thin wall tubes are inserted into a working sensor of an inductive conductivity gauge to serve as LAMP reaction tanks; (2) the value of electric conductivity of mixed solution before and after the biochemical reaction in every reaction tank is determined on line; and (3) the result of LAMP reaction can be deduced from the change of electric conductivity of each reaction unit so as to represent target DNA information. The method has the characteristics of being simple, convenient and fast, high in sensitivity, simple and convenient in operation, simple and inexpensive in instrument and equipment and low in expenditure, and can realize biological genetic testing at a low cost. The method can be extensively applied to the fields such as food and drug quality control, environmental monitoring, clinical diagnosis, genetically modified organism and discrimination of products thereof and the like.

Description

Technical field: [0001] The invention belongs to the gene detection technology, specifically, it reflects the biochemical reaction process by on-line monitoring the change of the conductivity of the loop-mediated isothermal gene amplification (loop-mediated isothermal amplification, LAMP) reaction solution of the array, and then obtains the qualitative analysis of the target gene. , quantitative information. Background technique: [0002] The widely researched and applied LAMP gene detection technology uses 4 different specific primers to identify 6 specific regions of the target gene, and performs amplification reactions under isothermal conditions without thermal denaturation of templates and long-term temperature cycles. Superiority (see literature Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T: Loop-mediated isothermal amplification of DNA, Nucleic Acids Res 2000, 28(12): E63.). Compared with the traditional PCR amplification technology, LAMP ...

AI Technical Summary

Problems solved by technology

[0006] 1. Directly observe the generated magnesium pyrophosphate precipitate (see literature Enosawa M, Kageyama S, Sawai K, Watanabe K, Notomi T, Onoe S, Mori Y, Yokomizo Y: Use of Loop-Mediated Isothermal Amplification of the IS900Sequence for Rapid Detection of Cultured Mycobacterium avium subsp.Paratuberculosis, J.Clin.Microbiol.2003, 41, 4359-4365), or observe the color change after adding a chromogenic agent (see literature Zhang Q, Shi C, Huang J, Jia K, Chen X, Liu H: Rapid diagnosis of turbot reddish body iridovirus in turbot using the loop-mediated isothermal amplification method, J.Virol.Meth.2009, 158, 18-23)——The disadvantage of this method is that subjective errors are inevitable

[0007] 2. Determination of magnesium pyrophosphate precipitation method by turbidimeter (see literature Mori Y, Kitao M, Tomita N, Notomi T: Real-time turbidimetry of LAMP reaction for quantifying template DNA, J.Biochem.Biophys.Methods.2004, 59, 145-157), - the disadvantage of this method is that it increases the investment of expensive instruments, and has strict requirements on the transparency of each relevant reagent solution

[0008] 3. Electrophoresis method (see literature Ihira M, Yoshikawa T, Enomoto Y, Akimoto S, Ohashi M, Suga S, Nishimura N, Ozaki T, Nishiyama Y, Notomi T, Ohta Y, Asano Y: Rapid Diagnosis of Human Herpesvirus 6 Infection by a Novel DNA Amplification Method, Loop-Mediated Isothermal Amplification, J.Clin.Microbiol.2004, 42(1), 140-145) - the disadvantage of this method is that it takes a long time and is inefficient

[0009] 4. Fluorescence quantitative PCR instrument for fluorescence quantitative detection (see literature Cai Zhejun, Feng Jiexiong, Zhu Shenghe, Nucleic acid loop-mediated isothermal amplification technology, International Journal of Laboratory Medicine, 2006, 27, 1092-1096) - the disadvantage of this method is that it greatly increases It reduces the cost of use and is not conducive to the miniaturization design of the instrument

[0010] 5. Hybrid biosensor method (see literature Sun Wei, Gao Hongwei, Zhong Jianghua, Qin Peng, Jiao Kui, Ring-mediated isothermal amplification-a method for electrochemical detection of Listeria monocytogenes labeled with nano-cadmium sulfide, patent publication number: 101260424 and literature Ahmed M, Hasan Q, Hossain M, Saito M, Tamiya E: Meat species identification based on the loop mediated isothermal amplification and electrochemical DNA sensor, Food Control, 2010, 21, 599-605) - the disadvantage of this method is The sensor is cumbersome to make and can only be used once

[0011] To sum up, the current amplification result analysis method is the key factor restricting the faster development and promotion of LAMP