Detecting Pathogenic Microorganisms In Foods With Resonance Energy Transfer

Food-borne diseases caused by pathogenic microorganisms are the world's most important food safety issue.Food production is a long and complicated process,and there are many possibilities for contamination by pathogenic microorganisms.Common pathogenic microorganisms are bacteria,virus and fungi.African swine fever(ASF)is an acute hemorrhagic serious infectious disease caused by the African swine fever virus(ASFV)infection of pigs.The continued spread of ASF not only affects the meat supply of residents,but also affects the safety of the global meat supply.However,there are few studies on the direct detection of ASFV in foods.Detection of ASFV in daily food can trace the source of the virus,monitor the virus more extensively,and further reduce economic losses.Staphylococcus aureus is the most common food-borne pathogen and one of the representatives of Gram-positive bacteria,which can cause many serious infectious diseases.Commonly found in milk,meat,egg,fish and their products,and it can cause systemic infectious diseases such as purulent infection,pneumonia,and even sepsis.Therefore,the construction of a convenient,rapid and effective method for detecting Staphylococcus aureus is of great significance for the effective monitoring,prevention and control of food safety.This work is based on a simple,fast and sensitive resonance energy transfer technology for the detection of ASFV and Staphylococcus aureus in foods.Part I:In this section,we reported a fluorescence resonance energy transfer(FRET)DNA biosensor based on quantum dots(QDs)and gold nanoparticles(Au NPs)for ASFV specific gene detection based on hybridization between single-stranded DNA.ss-DNA-QDs(probe 1)was synthesized by biotin-ss-DNA and streptavidin-QDs based on the specific affinity of streptavidin and biotin,ss'-DNA-Au NPs(probe 2)was synthesized by SH-ss'-DNA and Au NPs(probe 2)with salt aging method.In the absence of target DNA,ss-DNA-QDs(Probe 1)will hybridize with ss'-DNA-Au NPs(Probe 2)making the donor(QDs)and the acceptor(Au NPs)close to each other and trigger FRET effect,and the fluorescence of QDs was quenched by Au NPs.However,in the presence of target DNA,the target DNA competed with Probe 2 to bind Probe 1,which resulted in the destruction of FRET and recovery of the fluorescence of QDs.The developed biosensor can achieve rapid detection for ASFV target DNA with the detection of limit of 0.72?M in 1.25 h,and was verified in pork,ham sausage,and pork dumplings with recoveries from 82.00%to 108.00%and variation coefficients of 0.02-0.15%.This study proposed a new,simple and rapid method to detect ASFV gene fragment in food.Part II:In this section,Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated proteins system 13a(CRISPR/Cas13a)has the specific activity of cutting single-stranded RNA,easy operation and other advantages.Chemiluminescence resonance energy transfer(CRET)is a sensitive detection technology which does not require external light source excitation and no background signal interference.Therefore,this work combined CRISPR/Cas 13a with CRET to sensitively and simply detect S.aureus in food.First,aptamer-c RNA hybrid double-stranded system was synthesized by the principle of base complementary pairing.Aptamer recognized S.aureus in the sample and released c RNA from the aptamer-c RNA hybrid double-stranded system.CRISPR/Cas 13a recognized the target c RNA,CRET probe(Au NPs-RNA₁-HRP)which modified horseradish peroxidase(HRP)and single-stranded RNA was trans-cut(Au NP-RNA₁ was synthesize by Au NPs and SH-RNA₁-biotin through the freezing method,and then Au NPs-RNA₁-HRP was synthesized by Au NP-RNA₁and streptavidin-HRP through the specific binding force of streptavidin and biotin),resulting in CRET was destroyed and the chemiluminescence signal was enhanced after the substrate was added.The above-mentioned sensing strategy realized the quantitative detection of S.aureus within 1 h.It showed a good linear relationship in the concentration range of 10-10⁵cfu/m L,and its detection of limit was 7 cfu/m L.When this strategy was used to detect S.aureus in drinking water and milk,the recovery rate was90.07%-105.5%,and the variation coefficients was 2.20-6.80%,indicating that the combination of CRISPR/Cas 13a and CRET proposed in this paper can be used in quickly and sensitively detecting S.aureus in actual food samples.On the basis of this principle,an immunochromatographic test strip was designed to achieve rapid on-site detection of S.aureus,and the results can be captured directly via mobile phone.The test strip can detect S.aureus(10 cfu/m L)within 10 minutes,and10² cfu/m L S.aureus can be detected in drinking water and milk.This work combined CRISPR/Cas13a,CRET and immunochromatographic technology to establish a rapid visual detection method for S.aureus,in order to provide a reference for further research in the field of rapid and real-time monitoring of pathogenic microorganisms.