Study On The New Method Of Isothermal Double Stranded Nucleic Acids Amplification And Detection

Nucleic acids amplification technology is a hot area of biochemical analysis, and many methods based on nucleic acids amplification for detection has been developed. Polymerase chain reaction (PCR) and its derived technologies need repeated heating and cooling processes, which greatly depend on complicated instrument. So, they have some limitations when applied in the field of rapid detection. It is very important that the development of isothermal nucleic acid amplification are developed for simple, rapid, sensitive and accurate detection. In this paper, based on traditional strand displacement amplification, a method for double stranded nucleic acids amplification and detection in isothermal conditions was established, and the non-specificity amplification appeared in this type of reaction was studied.In this paper, coupled with nicking enzyme and polymerase, a new method for double stranded nucleic acids isothermal amplification and detection was established. This method realized that the new single-stranded DNA (ssDNA) target generation process occurred at a single temperature with next amplification reaction in closed and isothermal system, no need of additional heat-denature procedures. The method was simple, rapid and sensitive. The pBluescript Ⅱ KS (+) plasmid (referred to PBS plasmid) DNA was used as target and detected by this method. the result showed a good linearly relationship with the concentration range from 1 fmol to 1 zmol. The method showed good anti-jamming in complex system. In order to verify the practical application of this method, we conducted the detection of vibrio parahaemolyticus(VP) by this method. The 10-14 mol/L the genome DNA of VP could be detected to show the practical application of this method.In order to further improve the sensitivity of the method to detect VP,-the feasibility of molecular beacons used in this method was demonstrated. In the same time, the non-specificity amplification caused by primers containing the recognition site of nicking enzymes based on previous research was further studied, which would provide a basic data for future experiments.