Development Of A Novel Multiplex Loop-mediated Isothermal Amplification (mLAMP) Method Based On Invasive Reaction For Detection Of Nucleic Acid Sequence Variation

The alternation of nucleic acid sequence causes changes in gene sequence, resulting in significant impacts on the occurrence, development and treatment of diseases. Various ways such as SNP, mutation, methylation, insertions and deletions have been found valuable in the sequence variations. Traditional detection techniques like sequencing, allele-specific PCR, digital PCR and cascade enzyme signal amplification method have some drawbacks which are low sensitivity, high cost and the inevitability of expensive instruments that limits their clinic application.Loop-mediated isothermal amplification (LAMP) is a highly sensitive isothermal technology offering simple, rapid and cost-effective way for nucleic acid amplification. However, the complex structure of its product leads to difficulty in the analysis of multiple targets. Thus, the applification of conventional multiple LAMP detection based on enzyme digestion, fluorescent label or Tm value is restricted by cumbersome operation, high cost and limited length of amplicons.Invader assay is a highly specific method for sequence analysis. Combined with molecular beacon in previous work, we have established a cascade enzyme signal amplification method (CESA) whose specificity is high enough to detect 0.1% somatic mutant molecule out of wild-type ones, thus, suitable for the analysis of nucleic acid sequence variation. However, such method requires expensive equipment for fluorescence detection of molecular beacon and the detection limit can only reaches 1 fM.Gold nanoparticle probes, widely used for genetic detection in recent years, are oligonucleotide probes modified with colloidal gold through a covalent Au-S bond. Coupled with invader assay, we successfully developed the probe and established a method for visualized detection of the signal molecule produced by invader reaction, consequently satisfied the requirement of fluorescence detection.This subject firstly established LAMP method whose sensitivity is high enough to detect 10 copies/tube target molecules. And then coupled the method with invader assay to achieve high specificity, we set up a novel method for high sensitive and specific visualized detection through gold nanoparticle probes. Finally, by increasing the number of LAMP amplification, a visualized detection method with multiple isothermal amplification and specific identification was established. It is a universal method for the analysis of nucleic acid sequence difference with several advantages pursued by traditional methods such as high sensitivity, high specificity, low cost and easy to observe.