Research On Detection Method Of Luminescent Functional Nucleic Acid Of Salmonella In Food Based On Nucleic Acid Amplification Technology

Objectives Salmonella is a common food-borne pathogen.At present,the real-time nucleic acid detection method of Salmonella usually requires the addition of fluorescent dyes or dual-labeled fluorescent probes,which increases the complexity of the amplification system.Fluorescence self-quenching luminescent functional nucleic acid can realize the conversion of fluorescent signal only through a single label primer,and can realize the integration of primer and probe.Combining the fluorescence self-quenching luminescent functional nucleic acid with nucleic acid amplification technology to realize the amplification of the detection signal,construct two new Salmonella real-time nucleic acid amplification detection methods based on the fluorescent self-quenching luminescent functional nucleic acid,and investigate the detection performance of the new method.It provides new options for rapid quantitative detection of Salmonella,accurate tracking of the source of food poisoning incidents,and timely prevention of the spread of pathogenic bacteria.Methods The study was based on variable temperature nucleic acid amplification techniques and constant temperature nucleic acid amplification techniques,respectively,and combined self-quenching luminescent functional nucleic acids into the two amplification techniques to achieve real-time detection of Salmonella.1 Based on variable temperature polymerase chain reaction(PCR),four pairs of common specific genes of Salmonella(invA,hut,hns,gyrB)were screened for the specific primers,and the linear primers were functionalized to form fluorescent self-quenching primers for real-time detection of the products using fluorescent signals.The primer concentrations(500 nmol/L,400 nmol/L,300 nmol/L,200 nmol/L,100 nmol/L)and the annealing temperatures(50°C,52°C,54°C,56°C)of the innovative real-time fluorescence quantitative PCR(IRT-PCR)were investigated.The optimal primer concentrations and annealing temperatures were selected for subsequent experiments.The detection limit,quantification limit,linear range of detection,specificity and other indexes of the method were evaluated.To further demonstrate the feasibility of the IRT-PCR method,the experimental method was used for real sample detection to examine the precision and accuracy of the method.2 Based on constant temperature recombinase polymerase amplification(RPA),five pairs of linear primers were obtained by screening against five common specific genes of Salmonella(invA,hilA,hut,hns,gyrB),respectively,in order to investigate the ability of the method to detect real-time amplification products,the primers were functionalized to form self-quenching primers with fluorescent groups.The effects of primer concentrations(120nmol/L,100 nmol/L,80 nmol/L,60 nmol/L)and reaction temperatures(36℃,39℃,42℃,45℃)on the amplification results were investigated,and the optimal primer concentrations and reaction temperatures were selected for innovative real-time fluorescence quantitative RPA(IRT-RPA).The parameters of the method such as limit of quantification,limit of detection,sensitivity and specificity were examined.To further validate the feasibility of the IRT-RPA for Salmonella detection,the method was performed on real samples for Salmonella spiking,and the precision and accuracy of the method were demonstrated.Results 1 The amplification curves and agarose gel electrophoresis indicated that the study successfully screened for hairpin-shaped single-labeled fluorescent self-quenching primers targeting the Salmonella-specific invA gene.The primers were amplified best at a concentration of 400 nmol/L and an annealing temperature of 54°C.Under the optimal conditions,the combination of fluorescent self-quenching primers with PCR reaction could achieve IRT-PCR detection of Salmonella samples.The detection limit of the method was2 CFU/mL,the quantification limit was 10 CFU/mL,and the linearity was good between10~1～10~5 CFU/mL(R~2>0.99),and no amplification curve appeared for any non-specific amplification.The recoveries of Salmonella spiking assays in all three food matrices ranged from 85.81%to 125.29%,and the precision ranged from 0.08%to 0.92%.The amplification curves and agarose gel electrophoresis results showed that the experiment successfully screened and obtained single-labeled hairpin fluorescent self-quenching primers targeting Salmonella-specific hilA gene.The optimal primer concentration was120 nmol/L and the optimal reaction temperature was 39℃.Under the optimal reaction conditions,the combination of the single-labeled fluorescent self-quenching primers with RPA could achieve real time detection of Salmonella samples with the detection limit of 4CFU/mL and the quantification limit of 10 CFU/mL,showing good linearity between10~1～10~5 CFU/mL(R~2>0.99)and non-specific amplification curves.When the method was used for the real samples,the recoveries of the method were between 66.29%and 138.95%and the precision was between 0.39%and 2.02%.Conclusions 1 A single-labeled hairpin fluorescent self-quenching primer was designed,and the self-quenching primer was combined with PCR detection technology to realize the IRT-PCR method applied to the detection of Salmonella,which can complete the quantitative detection of Salmonella within 1 hour,with low detection limit of 2 CFU/mL,the method can realize the integration of primer and probe,simple,accurate,high sensitivity,and low detection cost.2 The IRT-RPA amplification method based on hairpin-shaped single-labeled fluorescent self-quenching primers was established and successfully applied to the real-time detection of Salmonella,with the detection limit as low as 4CFU/mL and the detection time of 30 min,the amplification system is simple,and the method is rapid,sensitive and highly accurate.Figure 22;Table 9;Reference 151...