Botulism Type A And Type B Based On Droplet Digital PCR Establishment Of Clostridium Detection Method And Clinical Application Research

Botulinum poisoning is a deadly paralytic disease caused by botulinum toxin,which seriously endangers human and animal health.Botulism is an infectious disease threatening public safety.It is characterized by rapid onset and high mortality.Botulinum toxin is a protein neurotoxin produced by anaerobic botulinum(C.botulinum)grown under anaerobic conditions.It is deadly to humans and animals,and is the strongest exotoxin known.In recent years,food-borne botulism incidents have been increasing.Canned foods,fermented feeds,and vacuum-packaged foods can produce anaerobic conditions,which can lead to the growth of botulinum bacteria and contaminate food.There are early reports of animal botulism incidents abroad.Up to now,more than 20 countries and regions around the world have reported botulism cases,and more than 16 provinces in my country have reported animal botulism incidents.According to the different antigen serotypes of Clostridium botulinum,it is generally believed that Clostridium botulinum can be divided into seven types,from type A to type G,with the latest type H being discovered.The epidemic botulinum in China is mainly type A and type B.The condition of botulinum poisoning is very dangerous,and the clinical symptoms are not typical and difficult to be diagnosed.The lack of understanding of botulism often leads to misdiagnosis and delays the best treatment opportunity.This happens frequently.Therefore,it is more important to accurately identify botulism in the early stage of onset.The traditional method of detecting botulism is to first cultivate clinical samples through laboratory flora,and then use biological detection methods for detection.However,this detection method is not only complicated in operation,but also has a long detection cycle,usually over 10 days,which is not conducive to clinical diagnosis of botulism and timely control of the risk of botulism.Therefore,the establishment of a fast,efficient and accurate detection method for Clostridium botulinum is an urgent problem to be solved.Experimental Purpose clinical sample stool was used as the detection matrix to establish a droplet digital PCR detection method for the detection of Clostridium botulinum type A and B for the first time,and to evaluate the method’s superiority in the diagnosis of Clostridium botulinum infection Shortcomings and the exploration of the actual clinical sample testing capabilities.Experimental methods Use clinical test methods to isolate and identify botulinum strains in clinical samples,compare the conservative sequences of botulinum type A and type B toxin genes,and design specific primers and probe pairs for botulinum gene sequences.The designed primers and clinical isolates of Clostridium botulinum were used to construct a plasmid standard for Clostridium botulinum toxin gene types A and B,which were used to construct the droplet digital PCR method and continuously optimize the dd PCR reaction conditions.Use plasmid standards to linearly analyze the sensitivity of the droplet digital PCR method,the specificity of the primer probe and the reproducibility of the experimental detection method,and compare the results of the fluorescent quantitative PCR method to analyze the advantages and disadvantages of this digital PCR detection methods.At the same time,the newly-built droplet digital PCR detection method was used to detect the simulated soil clinical specimens containing type A and type B Clostridium botulinum spores for verification,and the detection results of the fluorescence quantitative PCR method were compared to evaluate the clinical detection ability of the method established in this experiment.Finally,use known positive clinical samples to verify the practicality of constructing clinical samples for digital PCR.The results showed that 29 strains of Clostridium botulinum positive were isolated from the environmental samples,including 2 strains of serotype A and 27 strains of serotype B,using conventional laboratory methods,including animal experiments,culture and detection experiments,isolation and purification experiments,gram staining microscopy,and 16SRNA detection.When using the dd PCR method to detect plasmid standards,both A and B plasmid standards showed distinctly separated yin and yang droplets,and no positive droplets appeared in all the control strain amplification systems used.The optimal conditions for dd PCR optimization were determined.The annealing temperature of the botulinum toxin gene reaction system type A was 57.3℃,the optimal concentration of upstream and downstream primers was900nm/L,and the optimal concentration of probes was 250nm/L.The best annealing temperature for type B botulinum is 57.3°C,the best primer concentration is 800 nm/L,and the probe concentration is 350 nm/L.The lower detection limit of the established dd PCR method for Clostridium botulinum type A/B is very low,and the limit of quantification for Clostridium botulinum type A is 0.42 copies/ul.The limit of quantification for Clostridium botulinum type B is 0.44/ul.Experimental results show that when the concentration of Clostridium botulinum genomic DNA ranges from 98 copies/ul to 9.8×10~5 copies/ul,the linear relationship is good,and the concentration of Clostridium botulinum genomic DNA ranges from 29 copies/ul to 2.9×10~5 copies/ul,the linear relationship is good,and it has good repeatability.The coefficient of variation(CV)of 4 gradient concentrations selected in the same batch is less than 8%.The dd PCR detection results in this simulated sample are consistent with the fluorescence quantitative PCR detection results,and are two orders of magnitude more sensitive than the fluorescence quantitative PCR detection.The accuracy of clinical sample test results is 100%,which is consistent with the laboratory bacterial culture method test results,which proves that it has strong clinical practicability.Experimental conclusions Preliminarily proved that the constructed dd PCR method is efficient,sensitive and specific,and can be used for rapid detection of type A and type B Clostridium botulinum.This method can be used as an effective tool to detect the accurate load of Clostridium botulinum.The results show that this method can be used for the diagnosis of clinical Clostridium botulinum infection,and it can also be used as an effective means for future clinical disease monitoring of Clostridium botulinum infection.