| Aquatic pathogenic microorganisms are extremely contagious and can cause widespread pollution of the aquaculture environment,leading to deleterious effects on the life activities of aquatic organisms and thus indirectly endangering human health.In addition,aquatic diseases caused by pathogenic microorganisms are one of the most important causes which lead to economic loss of aquaculture industry.Consumers’demand for aquatic products is increasing with the improvement of living standards,and the requirements for food safety are also increasing.Therefore,rapid,sensitive and accurate identification of pathogenic microorganisms in aquatic animals and water environment is essential to prevent and control disease occurrence,reduce economic losses in aquaculture and interrupt the spread of foodborne diseases in humans.Based on the above research background,this thesis constructs a hybridization chain reaction(HCR)cascade signal amplification platform for the ultrasensitive and specific detection of pathogenic bacteria(E.coli,S.aureus),and further be used for the detection of aquatic animal pathogens.The following research work has been carried out around the above objectives.(1)Construction of hybridization chain reaction cascade amplification platform.A couple of specific primers for the variable sequence of target bacterial 16S r RNAs were designed,and used to obtain amplified target single-stranded DNAs(AT-ss DNA)through a PCR.This was the first primary nucleic acid amplification.Then cascade signal amplification was achieved after HCR reaction triggered by AT-ss DNA,and thus improved the sensitivity of this method.Hairpin structure used in work was labeled with fluorescent group(FAM).The FAM fluorescent signal accumulates with increasing reaction time of HCR,and the final fluorescence intensity is proportional to the concentration of the target bacteria,which can be used for qualitative and quantitative detection of pathogenic bacteria.In this method,the bacteria are lysed by repeated freezing and thawing method to release RNA without nucleic acid purification kit,which not only simplifies the operation process and reduces the risk of sample contamination,but also saves the cost.In addition,the proposed strategy can be used for ultra-sensitive and specific detection of pathogenic bacteria(E.coli,S.aureus)in solution:the detection limit down to 0.49 CFU/m L for Escherichia coli(E.coli),0.31 CFU/m L for Staphylococcus aureus(S.aureus)in buffer,and a linear range from 1 to 1×10~7CFU/m L for E.coli,1 to1×10~6CFU/m L for S.aureus.It is expected to be used for the analysis of actual samples of aquatic pathogens for the extremely wide linear range and ultra-high sensitivity.(2)Application of hybridization chain reaction(HCR)cascaded amplification platform in the detection of pathogenic bacteria in aquatic products.Based on the above research,the platform was applied to the detection of pathogens in aquatic products.A series of serum samples containing different concentration gradients of pathogen were prepared by adding bacteria to the serum.The fluorescence spectrum showed that the calibration curve obtained from the detection of pathogenic bacteria in serum samples is nearly the same as that in the solution,proving the good anti-interference ability of the method for the detection of pathogenic bacteria in complex environments(such as serum).In addition,the tilapia pathogen infection model was constructed,and the method was further successfully applied to the detection of pathogenic bacteria in the blood and different organs(large intestine,gallbladder,heart,liver,ren,gill and skin)of sick tilapia.Finally,the accuracy of the method was verified by standard plate counting method.In summary,the cascaded amplification platform can not only achieve ultra-sensitive detection of pathogens,but also display good anti-interference and detection accuracy in complex samples,which shows great potential in the detection of marine pathogenic microorganisms and prevention and control of aquatic products. |
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