A Universal Biosensor For Multiplex DNA Detection Based On Hairpin Probe Assisted Cascade Signal Amplification

Pharmacogenomics, also known as genomic medicine or genome pharmacology, is a new discipline to study how the genome and gene mutation affect absorption and metabolism, efficacy and adverse reaction of drugs in vivo, aiming to guide discovery and rational administration.According to WHO statistics, one third of the global death patients died from irrational drug use, and the gene polymorphism is the ultimate factor for individual differences in drug response. Cytochrome P450enzymes (CYP) based biocatalysis is one of the most important system for drug and toxicant metabolism. CYP belongs to beta cytochrome superfamily protease, with a haem prosthetic group. CYP participates in various metabolism and transformation process for a wide variety of substrates, such as toxic substances, carcinogen and about90%clinical medicine. CYP2D6is one of the most important enzyme in CYP, accounting for20%in drug metabolism by CYP. The polymorphism of CYP2D6gene results in the difference of drug metabolism among races and populations, so rapid analysis for polymorphism of C YP2D6genes is of great significance to guide rational medication.Traditional method for analysis of gene polymorphism isgenerallybased on polymerase chain reaction (PCR), such as restriction fragment length polymorphism analysis (RFLP), allele specific amplification (ASA), real-time quantitative PCR and DNA sequencing. Although the sensitivity of these technologies are high, yet they require expensive instruments and professional staff, which limit the application in developing countries and remote areas lack of medical resources.Currently the isothermal nucleic acid amplification technologies, such as nucleicacid sequence based amplification(NASBA),loop-mediated isothermal amplification (LAMP), helicasedependent isothermal DNA amplification (HDA) have emerged as substitutes for PCR based technology, which not only shorten the diagnosis time, but also lowered the requirement for equipment. However, these techniques also involve some problems. For instance, NASBA technique is only suitable for RNA analysis and contains multiple enzyme reaction steps; LAMP demands a complex primer design and the helicases in HDA are susceptible to be inhibited by samples. These problems have affected the stability and practicability of theisothermal nucleic acid amplification technologies.In this research, we construct a lateral flow biosensor for distinguishmultiple genes based on enzyme assisted isothermal DNA amplification. We employed this method to genotype the polymorphism site of gene CYP2D6*4. The whole process of analysis is15minutes and the sensitivity is100aM with the linear range from10fM to100nM. Compared with the traditional PCR based analysis method, this approach possess the advantages of simple operation, fast reaction, high sensitivity, low cost and instrument independence, and can be used in hospital for POCT (Point of care test) and personalized medicineguidance.