Based On SRCA-NEMA-G-quadruplexes Visual And Electrochemical Biosensor For Detection Of Listeria Monocytogenes In Food

Listeria monocytogenes is a foodborne pathogen that causes bacterial foodborne illness,which widespreads in a variety of foods.Infection with this pathogen can lead to sepsis,meningitis,gastroenteritis and even spontaneous abortion,which can be life-threatening in severe cases.Therefore,the development of a rapid and effective method for the detection of Listeria monocytogenes is of great practical significance.In this study,saltatory rolling circle amplification(SRCA)and nicking enzyme mediated amplification(NEMA)were combined to realize highly sensitive visual detection of Listeria monocytogenes by G-quadruplex,and further combining the SRCA-NEMA-G-quadruplex with electrochemical biosensor to realize the quantitative detection of Listeria monocytogenes.It can satisfy the actual requirements of laboratories under different conditions for detection.The main research contents and research results are as follows:(1)The conservatism of hlyA gene was higher by Genbank comparison,so the hlyA gene was selected as the target gene for Listeria monocytogenes detection.The target sequence containing Nb.BsmI specific nicking site was selected,and SRCA primers were designed and screened,and SRCA-NEMA amplification system was established.At the same time,the hairpin DNA(hpDNA)was designed which containing stem riched guanine and loop was complementary to part amplicons of SRCA-NEMA reaction.First,a large number of tandem repeats of double-stranded DNA(dsDNA)containing nicking sites were amplified by SRCA reaction.The SRCA products can be specifically recognized and cleavaged by Nb.BsmI.The SRCA-NEMA reaction was carried out under the action of Bst DNA polymerase to generate a large number of single-stranded DNA(ssDNA),which is complementary to the loop part of hpDNA.The hairpin structure was opened,the G-quadruplex was formed under the action of Hemin and K+.After adding ABTS and H2O2,the color changes of ABTS were visible by the naked eye under the action of G-quadruplex,and the positive result was dark green.On this basis,the visual detection method of SRCA-NEMA-G-quadruplexe for Listeria monocytogenes was established by optimizing the reaction temperature,the ratio of Bst DNA polymerase to Nb.BsmI,and the cleavage time.The experimental results showed that under the optimal reaction system,different concentrations of Listeria monocytogenes genomic DNA were detected,and the visual limit of detection for genomic DNA was 5.4 fg/μL.Different concentrations of pure Listeria monocytogenes cultures were detected with visual detection limit of 8 CFU/mL.In the specific test,only 10 strains of Listeria monocytogenes had significant color changes(dark green)and showed positive result.The color of 20 strains of non-Listeria monocytogenes showed no obvious change and showed negative results,indicating that the method had good specificity.(2)On the basis of the established SRCA-NEMA-G-quadruplex detection method,combined with electrochemical biosensors,the sensitivity of detection was further improved,and quantitative detection method for Listeria monocytogenes was established.The hydrosulfuryl modified hairpin DNA(HS-hpDNA)was fixed on the surface of glassy carbon electrodes(GCE)modified gold nanoparticles(AuNPs)by Au-S bonds,and based on SRCA-NEMA-G-quadruplex electrochemical biosensor was constructed for detecting Listeria monocytogenes by using G-quadruplex as signaling molecule.The results showed that under the optimal response system,different concentrations of Listeria monocytogenes genomic DNA were detected,and the limit of detection for genomic DNA was calculated to be 2.13 fg/μL.Different concentrations of Listeria monocytogenes pure cultures were detected,and the limit of detection for pure cultures was calculated to be 3 CFU/mL.In the specific test,only 10 strains of Listeria monocytogenes had obvious changes in current signal and showed positive results.The current signal of 20 strains of non-Listeria monocytogenes showed no significant change and showed negative results,indicating that the method had good specificity.In the repeatability test of electrochemical biosensors,6 electrodes showed stable signal output with relative standard deviation(RSD)of 3.37%.In the electrochemical biosensor stability test,the working electrode was stored at 4℃ for 14 days,and taken out on the 1st,3rd,5th,7th and 14th days for follow-up tests,and the current signal was maintained at 97.7%,96.5%,94.7%and 91.0%of the initial value of the first day,respectively.The results showed that the electrochemical biosensor had good stability.(3)The visual method and electrochemical biosensor constructed in this paper were used to detect 50 actual samples,and compared with GB 4789.30-2016 and real-time fluorescence quantitative PCR(RT-qPCR),and the experimental results showed that the relative sensitivity of the computational visualization method and the electrochemical biosensor was 100%,the relative specificity was 97.8%,and the compliance rate was 98%.The recovery rate of SRCA-NEMA-G-quadruplex electrochemical biosensor was 91.4%-11 1.1%,and RSD was 2.1%-3.2%.Therefore,the electrochemical biosensor constructed in this study was more accurate and reliable,and had good practical application value.In summary,visualization method and electrochemical biosensor for the detection of Listeria monocytogenes were established based on SRCA-NEMA-G-quadruplex,which have the advantages of high sensitivity and specificity.At the same time,the replacement of common ferrocene(Fc)signaling molecules with G-quadruplex greatly reduces the detection cost.This method can use the naked eye to observe the color change to achieve qualitative detection,and it can also use the current signal change to achieve quantitative detection.The visual qualitative detection is more suitable for relatively backward laboratories or relatively scarce resources of grassroots testing institutions,and the quantitative detection method can be applied to relatively good conditions of laboratories or testing institutions.This study provides new approach for the detection of Listeria monocytogenes and provides new idea for other foodborne pathogens,which has promoting effect for ensuring food and public health safety.