Establishment Of HAND System–based Multiplx Real-Time SYBR GREEN I PCR For Detection Of 15 Food-borne Pathogens

In recent years,the incidence of foodborne diseases remains high and is an increasingly serious public health problem all over the world.WHO statistics show the part of foodborne diseases caused by pathogenic microorganisms was more than 50%.There is a critical need for the development and integration of sensitive and efficient methods for foodborne pathogen detection.Traditional microbiological detection and identification methods for foodborne pathogens are well-known to be time-consuming and painstaking as they are increasingly being perceived as lacking to meet the demands of rapid food testing.To prevent the spread of infectious diseases,ensure the food safety,and to protect public health,there is an ever-increasing demand for more rapid,simple and efficient methods of foodborne pathogen detection.Multiplex real-time SG-PCR added more than two pairs of primers and excessive SYBR in one reaction.When SYBR specifically incorporates a DNA double strand,it can emit a fluorescent signal,which is not incorporated into the DNA double strand,does not emit a fluorescent signal,ensuring the increase of the fluorescent signal is combined with the increase of the PCR product.Finally,the observation and determination of the results by the Tm value of the dissolution curveThe 15 foodborne pathogens(enter pathogenic E.coli,enter toxigenic E.coli,enter invasive E.coli,enter hemorrhagic E.coli,enter aggregative E.coli,Yersinia enterocolitica,Y.pseudotuberculosis,Campylobacter coli,Campylobacter jejune,V.Parahemolyticus,V.cholera,Salmonella enteritis,Plesiomonas shigellosis’ s,Aeromonas hydrophila,S.Flexner)were used as model strains.We found multiplex real-time SYBR Green I test for simultaneous detection of 15 food pathogens.The following conclusions were got:(1)We found a multiplex real-time SYBR Green I test based on HAND system for simultaneous detection of 15 food pathogens to be high specificity,good sensitivity,high reproducibility and stability,(2)Using a verification method,we vitrified heat-unstable parts(Taq DNA polymerases,dNTP,primers,dye)onto the cap of a reaction tube.The experimental results show the verification reagent was stable can be preserved at room temperature(25℃)for 1 to 2 years.We successfully employed this technology to the multiplex real-time SG-PCR tests.The experimental results show the activity of the reagent is not effected by vitrification,overcoming the limits because of cold-chain transport and low temperature storage,(3)For the rapid extraction of complex samples,a nucleic acid extraction solution with chelex-100 and tritonx-100 as the main cracking units was developed.To evaluate its effect on nucleic acid extraction of 15 foodborne pathogens.The results showed that it only needs to be lysed at 90 °C for 10 min,which is suitable for multiplex real-time SYBR Green.It has the characteristics of simple operation,good cracking effect,no influence on subsequent reaction,and it is not affected by complex samples such as stools.and food,In conclusion,we established a rapid detection method for simultaneous detection of 15 food pathogens by combining HAND system with vitrification technology,which has good specificity and sensitivity.It solves the problems of tedious and complex nucleic acid extraction and adding samples,as well as the inactivation of reagents in non-low temperature environment.