| Rapid identification and detection of foodborne pathogens is of great significance for ensuring food safety.High-resolution melting(HRM)analysis technology is widely used for rapid DNA identification and analysis of foodborne pathogenic bacteria because of simple operation and low cost.However,melting curves can overlap with each other,making it difficult to distinguish accurately between target DNA fragments of pathogens to be tested when the melting temperatures(Tm)are similar.In order to overcome this limitation,four types of ddNTP were introduced into the short-strand DNA extension process in this study,and the chain termination products of each base pair were successfully distinguished by HRM technology.On this basis,the determination method of melting for short-strand DNA sequence information was established to achieve the identification and detection of four foodborne pathogens simultaneously.The contents and results were detailed as follows:(1)Determination of short DNA single base extension reaction by highresolution melting.Using a series of chemical synthesis of short strands of DNA of different lengths,it was verified that DNA fragments with single base length differences could be distinguished by HRM.Moreover,single base DNA fragments catalyzed by KF polymerase can also be distinguished by HRM.HRM determination method for single base extension reaction was established.The results showed that,single FAM at 5’ end(G base quenching)was more economical and effective for the short strand DNA melting analysis compared with the fluorescent dye and the double-labeled fluorescent probe.At optimized DNA concentration(0.2 μM)and melting rate(0.05℃/s),HRM analysis can distinguish short strands of DNA with single base differences accurately ranging from 11-23 nt.By optimizing the concentration of KF polymerase and dNTP,the HRM method could identified four kinds of base extension primers and accomplish the detection of single base extension reaction.(2)Determination of short strand DNA sequence information by highresolution melting method.Furthermore,four kinds of ddNTP were introduced into the polymerase extension reaction for chain termination reaction and HRM analysis.A method for the determination of short DNA sequence information by HRM was established.The results showed that under the optimized polymerase concentration and dNTP/ddNTP ratio,both KF polymerase and sequenase could obtain the four base chain termination reaction melting peaks,but the performance of sequenase was better than that of KF polymerase.The type of base of the chain termination reaction can be known according to the added ddNTP,and the length and position of the chain termination base can be determined according to the Tm of the melting peak,so that the sequence information of short strand DNA can be accurately determined.The results were consistent with the denatured gel electrophoresis experiment.The HRM determination performance of short strand DNA length of 25 nt was significantly better than that of 39 nt,which was in line with expectations.(3)High-resolution melting differential detection of DNA sequences of foodborne pathogens.To verify the practical application potential of the method,PCR amplification products of four foodborne pathogens with similar Tm values were selected for differential detection using the HRM sequence information determination method.The results showed that the differences of the amplification products Tm of the four foodborne pathogens were small(～0.2℃)and could not be directly identified by HRM analysis,but their HRM curves were significantly different after the addition of ddNTP.Furthermore,the softwares were used for data analysis in peak segmentation fitting.The Tm of the melting peak was analyzed statistically by the software of peak fitting and multivariate linear discrimination.The results showed that the introduction of any one of the four ddNTP could achieve accurate identification of four single bacteria,but only introduction of ddCTP could achieve accurate identification of six double bacteria mixtures.The introduction of two or three kinds of ddNTP can realize the identification of ten kinds of single and double bacterial mixtures.The HRM method of short strand DNA sequence information established in this study is simple and rapid,and can be completed by conventional fluorescent PCR without gel electrophoresis experiment.In addition to the rapid detection of foodborne pathogens,this method could be can also be widely used in food authenticity,gene modification detection,environmental pollution monitoring and other fields. |
