Application Of Nucleic Acid Signal Amplification Strategy Based On G-quadruplex In Salmonella Detection Methods

Salmonella(SALM)is a common food-borne pathogen that can causes human and animal diseases.Food poisoning events are possibly exploded when poultry and livestock served as the source of infection and food is used as the transmission medium.Therefore,rapid detection of SALM is the key to ensure food safety.In this study,based on nucleic acid assisted signal amplification strategy,three detection methods for Salmonella were established with the help of G-quadruplex structure and functional characteristics,combined with nucleic acid amplification technology.The main researches are described as follows:(1)Application of As-PCR amplification technology based on G-quadruplex for visual detection of SalmonellaThe reverse complementary sequence of G-quadruplex was labeled at the 5’end of the restriction primer of the asymmetric PCR(As-PCR).When it was exhausted in the reaction system,a large number of single-stranded DNA(ssDNA)products tagged with G-quadruplex sequence were amplified by non-restriction primers with high concentration.The purified products bound with hemin would form deoxyribozyme(DNAzyme)with peroxidase activity,and catalyze hydrogen peroxide(H2O2)and 2,2’-diazo-bis(3-ethylbenzothiazolyn-6-sulfonic acid)diamine salt(ABTS2-)to change color from colorless to green,which realized the visual detection of Salmonella by the cascade amplification strategy combining As-PCR with DNAzyme activity.This method had a good linear relationship between the logarithm of Salmonella genomic DNA concentration and 421 nm absorbance in a range of 2 pg/μL-258 ng/μL.The regression equation was y=0.0691x+0.3085(R2=0.9729).The detection limit was 35 CFU/mL for the artificially contaminated milk samples.(2)Application of PCR amplification technology based on G-quadruplex for visual detection of SalmonellaThe reverse complementary sequence of G-quadruplex was additionally labeled at the 5’ end of upstream and downstream primers of PCR.A large number of double stranded DNA(dsDNA)products containing G-quadruplex sequence were obtained through specific identification and amplification of target genes.After purification and denaturation,the products were transformed into colorimetric signal with ABTS2-and H2O2 in the presence of hemin and K+,so as to achieve the purpose of nucleic acid signal amplification for visual detection of Salmonella by the combination strategy of PCR amplification and DNAzyme activity.Under the optimized reaction system,there was a good linear relationship between the logarithm of Salmonella genomic DNA concentration and the absorbance value at 421 nm.The regression equation was y=0.1299x+0.2179(R2=0.9945)with a linear range of 0.07～771.6 ng/μL.The detection limit was 18 CFU/mL for the artificially contaminated milk samples.(3)Application of PCR-RCA double amplification based on G-quadruplex for detection of SalmonellaThe reverse complementary sequence of G-quadruplex was inserted into a dumbbell circular DNA template for rolling circle amplification(RCA)amplification.By the double amplification of PCR-RCA,and the enhancement of thioflavin T(ThT)fluorescence signal specifically combined with G-quadruplex,the detection of Salmonella by PCR-RCA technology based on G-quadruplex was established.A good linear relationship was set up between the logarithm of Salmonella genomic DNA concentration and the fluorescence signal intensity at 486 nm.The regression equation was y=2.101x+2.8723(R2=0.9925)within a linear range of 17 fg/μL～1.7 ng/μL.The detection limit was 5 CFU/mL for the artificially contaminated Salmonella milk samples.In summary,by using three nucleic acid amplification methods,the genomic DNA of Salmonella was specifically identified and the G-rich sequences were amplified.With the help of the deoxyribozyme properties of G-quadruplex and the enhanced fluorescent dye properties,the nucleic acid signals were amplified and converted to realize the quantitative detection of Salmonella.These strategies charactered with high effective,specific and sensitive provides a new method and technical support for the rapid detection of food-borne pathogens.