Rapid Detection Of Pathogens Based On Pyrophosphate Analysis

In recent years,the number of zoonotic diseases has continued to increase.Food poisoning incidents occurre frequently.The rapid detection of pathogenic bacteria becomes an essential guarantee for people’s health and diseases prevention.Current pathogen detection methods,such as isolation and culture methods,immunological analysis and molecular microbiological detection methods,are widely used in the field of food hygiene and clinical medicine.However,these methods have some deficiencies,such as prolonged detection cycles,cumbersome operations,and high costs.In this study,we proposed a novel method for rapid detection of pathogens based on pyrophosphate analysis.Amplification primers were designed for specific gene fragments of pathogens.Then the amplification(PCR)reaction was performed using nucleic acid molecules of Salmonella.The concentration of specific pathogens was detected by analyzing the chemiluminescent signal of pyrophosphates in PCR products or derived single-stranded DNA template from PCR products.The correlation between the amount of pyrophosphate of the PCR products and the initial DNA template content had been confirmed.The rapid detection technique based on pyrophosphate analysis includes two steps: firstly,the nucleic acid molecules in the sample were extracted,then the PCR reaction of specific fragments of pathogenic bacteria was carried out.The pyrophosphates in PCR products was converted to visible light catalyzed by ATP sulfate and luciferase,and the initial DNA template content was estimated based on the signal intensity of pyrophosphates.The experimental results showed that the detection limit of this method was 104 DNA copies.Salmonella was detected for 25 samples of chicken eggs and 25 samples of chicken meat provided by the Yangzhou Poultry Institute.The result of positive detection rate was consistent with the results obtained by the Institute using isolation and culture method at a rate of 92%.Secondly,the single-stranded DNA templates were obtained from PCR products modified by biotin.The DNA templates were used to produce pyrophosphates by incorporating four nucleotides in the polymerization reaction.Experimental results showed that the detection limit of this method was 10 DNA copies.Analysis of false-negative samples of pyrophosphate in the direct measurement of the PCR product indicated that the pyrophosphate assay derived from the single-stranded PCR product could be used to detect food samples containing a lower copy number of Salmonella.In general,the rapid detection technology of pathogenic bacteria based on pyrophosphate analysis has the advantages of short detection time,easy operation,low cost and high sensitivity.It can be applied to rapid detection of pathogenic bacteria in various fields.