| Genus Vibrio is a class of bacteria widely found in seawater and seafood.Among them,more than a dozen are pathogenic bacteria which can cause human disease.In these pathogens,Vibrio cholerae,Vibrio parahaemolyticus and Vibrio vulnificus cause the highest mortality rate.Vibrios in seafood or other foods can cause a series of gastrointestinal diseases like vomiting,diarrhea,or sepsis and even death through human intake or wound infection.The culture of eating raw seafood is deep-rooted,providing the harzardous way for Vibrio and other pathogenic baceteria to threaten human health.With the spread of raw-food culture,the reports of Vibriosis cases caused by Vibrio also draw attention.The rapid detection and control of Vibrio have become a particularly important way to inherit the food culture and maintain human safety and health.Traditional PCR method has been limited by itself because of its sensitivity and time-consuming problems.More and more researchers are looking for more suitable methods.Gold nanoparticles(Au NPs)whose different optical properties can be obtained by synthesizing different sizes and shapes are a kind of material with quantum dots.In recent years,gold nanoparticles have been introduced into the biosensors to solve practical problems and made great progress.In this paper,the first analysis of the correlations between the components that belongs to LAMP/RPA’s reaction system and gold nanoparticles and looking for the best reaction conditions were done.For the first time,the detection of Vibrio cholerae was carried out based on recombinase polymerase amplification,and gold nanoparticles were introduced into the reaction.A set of colorimetric methods which are fast,low-cost,sensitive and reliable were built up.The main research results are as follows:1.Compared with the qPCR,RPA method is found to be more sensitive,fast and reliable and could quantify Vibrio cholerae in the seafood without relying on expensive instruments.The detection limit of this method was 5 copies within 10 minutes and is highly reproducible in eight independent test experiments.Based on the lolB gene,the specificity of RPA is highly conserved within the Vibrio cholerae species.The applicability of RPA in the food industry was verified by testing 45 shrimp samples.In summary,the RPA method is a sensitive,rapid detection method that can even quantify Vibrio cholerae in seafood.2.Based on the RPA amplification reaction of Vibrio cholerae,a colorimetric method based on RPA and AuNPs was established.Firstly,it was found that 103 copies of plasmid DNA in RPA reaction system were amplified after 6 min while amplified at 5.3 min in AuNPs-RPA reaction system,indicating that AuNPs can accelerate the initiation of the reaction and increase the reaction rate of RPA.Secondly,the interactions between protein components/primers and gold nanoparticles in RPA were described,which provides a theoretical basis for stabilizing AuNPs.We observed the color change after the amplification was completed,and we found that the different loading orders and whether the primer incubation of the AuNPs was done would affect the color results.If the primers were incubated with AuNPs and then added the enzymes,the protective effect on AuNPs would be kept at the highest level,and there is no difference between tthe color of the experimental and control groups.Enzyme inactivation caused the changes in the structure of the enzyme.Amounts of AuNPs added into the system then would involve in salt-induced aggregation.The precipitation phenomenon of experimental and control groups happened after adjusting the pH to the isoelectric point of the enzymes.Incubation overnight of the primers and probe with AuNPs before adding enzymes resulted in red in both the experimental and control groups.The results show that the enzymes play a protective effect and the role of the macromolecules is strong protection and adsorption in the space.To achieve qualitative visual results,we need to add the enzymes after the primers.The final result of the color is blue in positive tests,purple-red in negative ones.The mechanism is that the recombinase entrains the primers to form primers-enzyme polymer,making the primers and enzymes both lose the AuNPs protection function.Despite the amplicons were produced,double-stranded DNA can not compensate for the protective function.The failure of amplification in control groups makes the binding of recombinase and primers or their dynamic separation and release.In summary,gold nanoparticles can increase the reaction rate of RPA,qualitatify,reduce the time for end-point analysis and reduce experimental costs for users of RPA basic kits.3.The study of Au NPs-LAMP system based on SH-modified primers included the effect of ssDNA,ds DNA,dNTP and Bst DNA polymerase on AuNPs.The results showed that ssDNA,dsDNA and d NTP could stabilize the dispersion of AuNPs,while Bst DNA polymerase had non-specific adsorption,and AuNPs were clustered together by Bst DNA polymerase.Followed by the establishment of the method includes a more special way to add AuNPs to improve the stability of AuNPs and the convenience of detection.The primers were diluted with AuNPs to form red primers,and the SH-modified primers were self-assembled with AuNPs,eliminating the cumbersome anchoring steps.The optimum reaction conditions were as follows: temperature parameter setting range was 61-65 ℃;time range was 30-65 min;16 nm-diameter AuNPs concentration was 2.3 nM,11.5 nM,23 nM,46 nM.The optimum reaction conditions were obtained at 63 ℃ for 55 min and Au NPs 23 nM.Finally,the colorimetric method was applied to the detection of Vibrio parahaemolyticus,and its minimum detection limit was determined.The LOD of quatitative result was 200 pg in 55 min.Positive and negative results could be based on the color of products whether red or blue.4.The unmodified AuNPs based on PEG steric hinders were involved in the LAMP colorimetric assay.The mechanism of reducing the probability of collision between gold nanoparticles and increasing the spatial stability is proposed,and the experimental results were verified.The concentration of PEG was optimized from 9% to 39%.The results show that 39% was the optimized concentration.Based on these two principles,we compared PEG-AuNPs method with SH primers-AuNPs LAMP and found that the color changes were reversed,i.e.the positive results were blue while the negative results were red.PEG-AuNPs method can be detected semi-quantitatively at 100 fg in 55 min at 63 ℃,which is more sensitive than SH primer-AuNPs.Therefore,PEG-AuNPs method can be a semi-quantitative visualization method.Although it is aimed at the specific detection of Vibrio,it can be applied in other pathogens or other nucleic acid targets detection.So it can provide efficient means for the basic laboratory or testing platform,and can provide technical support for food-borne pathogens control and food safety monitoringOn the whole,the isothermal nucleic acid amplification reaction system of gold nanoparticle material can adapt well to the requirement of detection getting rid of large instruments.So,it has good application prospect in visual inspection.In the meantime of,it can also improve the reaction rate and decrease the cross contamination. |
