New Technology Study On DNA Isothermal Amplification Methods For The Detection Of Vibrio Parahaemolyticus

In recent years, the development of nucleic acid amplification technology has been widely used in the field of life science and related fields, and plays very important roles. Polymerase chain reaction(PCR) need repeated thermal denaturation and greatly depends on the complicated instrument, which limits its application in point-of-care testing. Thus, it is very important to develop simple, rapid, sensitive and accurate detection methods of isothermal nucleic acid amplification for nucleic acid detection. Here, we reported two simple, rapid and sensitive detection methods of isothermal nucleic acid amplification.In chapter II, based on the principle of proximity effect, we developed a new isothermal nucleic acid amplification method for DNA detection. Firstly, we designed two kinds of DNA detection methods. We chose the special sequence of Vibrio parahaemolyticus as a detection target. In addition, the length and concentration of primers were optimized. Under the optimum conditions, different concentrations of target DNA was detected, and 0.1 fmol target could be well identified from the blank.The time corresponding to the maximum slope in the fluorescence curve as well as the point of inflection(POI) was linearly related to the negative logarithmic(lg) value of the target amount in the range from 100 fmol to 0.1 fmol. Thus, we constructed a method which was easy-operated, rapid and low dependence of the instrument.In chapter III, we presented a new isothermal amplification method of double-stranded DNA based on a double-stranded fluorescence probe. The method successfully used nicking endonuclease and polymerase to linearly switch dsDNA target to ssDNA. The double-stranded fluorescent probes hybridized with ssDNA and triggered amplification reaction. The method could detect the pBluescript II KS(+)plasmid with a detection limit of 2.3 amol. We have verified that this method had a high specificity, and could effectively distinguish one-base mismatch. This isothermal amplification method for dsDNA detection was simple, rapid, strong anti-jamming,and highly specific. With these advantages, this method will provide a new platform for the detection of pathogenic microorganism, such as vibrio parahemolyticus and vibrio cholerae.