Study On New Technology Of The Isothermal Detection For Double-stranded DNA And Its Application In Marine Vibrio Detection Research

Vibrio Parahemolyticus(V.Parahemolyticus)is a kind of important pathogenic bacteria causing food poisoning.V.Parahemolyticus is the main foodborne pathogenic bacteria in coastal area at present,and it widely exists in fish,shrimp,crab,shellfish and other seafood.Therefore,efficient and rapid detection of V.Parahaemolyticus is of great significance for food safety,disease surveillance and other fields.In order to detect V.Parahemolyticus more simply and rapidly,we have developed three kinds of isothermal nucleic acid amplification reaction in this paper.First of all,we reported exponential strand-displacement amplification(SDA)based on nicking enzyme and DNA polymerase.However,the results showed that the presence of nicking enzyme could lead to non-specific amplification.Therefore,in order to avoid non-specific amplification caused by nicking enzyme,we utilized loop-mediated isothermal amplification(LAMP)for detection of V.Parahaemolyticus,which is an isothermal nucleic acid amplification method with no need of nicking enzyme and only requires a DNA polymerase.The results showed that the reaction had good specificity and sensitivity for detection of V.Parahaemolyticus,and the detection limit was as low as 10 aM.We found that betaine could not promote the reaction,but reduce the efficiency of the reaction in the course of the detection of V.Parahaemolyticus by LAMP.Owing to betaine used in the reaction system at the beginning of LAMP reported,it has been widely used in isothermal nucleic acid amplification reaction.It isa common nucleic acid reaction additive.So,we further studied the mechanism of betaine reducing isothermal nucleic acid amplification reaction.The results showed that betaine could hinder the effective collision between complementary single-stranded DNA(ssDNA),and decrease association rate constant K1,thereby reducing the reaction efficiency.In this work,0.8 M betaine made LAMP efficiency drop to approximately 1%.We have verified that betaine used as a common additive in isothermal nucleic acid amplification reactions could slow the reaction efficiency instead of accelerating,and the mechanism of molecular barrier also has been clarified.This finding therefore suggested that it was time for betaine to migrate from isothermal nucleic acid amplification reactions,which would not only simplify reaction system and lower experimental cost,but also increase the reaction efficiency to shorten reaction time.This finding would greatly speed up the development of isothermal nucleic acid amplification methods,enabling it to be more appropriate for implementation of point-of-care testing(POCT).In addition,we established a new isothermal nucleic acid amplification reaction that requires only a DNA polymerase,which is denaturation bubble-mediated strand exchange amplification(SEA).In this paper,we used SEA to detect V.Parahaemolyticus,and the results showed that the detection limit could reach 10 pM.Additionally,SEA reaction had high specificity and strong anti-jamming capability.The betaine could decrease association rate constant K1,hinder the hybridization with complementary ssDNA,so we speculated that the additive which could increase dissociation rate constant K2 can promote double-stranded DNA(dsDNA)unwinding.Therefore,we further focused on the effect of additives on DNA strand exchange reaction(SER).And the results showed that pullulan,betaine,dithiothreitol,D-trehalose almost have no positive effect in DNA strand exchange reaction;hexadecyl trimethyl ammonium bromide(CTAB),dimethyl sulfoxide(DMSO),formamide,PEG 200,PEG 600,PEG 2000,PEG 6000 can promote the rate of DNA strand exchange reaction,especially 20% formamide,30% PEG 200,40%DMSO,which have the great effect on DNA strand exchange reaction.It has been proved that PEG 200 can unwind dsDNA,increase the dissociation rate constant K2,and promote the efficiency of DNA self-assembly reaction.Therefore,our finding will be very useful to the development of nucleic acid reaction,such as DNA devices,DNA circuits,DNA origami and amplifiers.