Research On The Rapid Identification Of Common Meat Derived Components Based On The Principles Of Nucleic Acid Amplification

Meat food is an important part of human diet.The safety of meat products is directly related to people’s health and life safety.The continuous occurrences of meat safety events in recent years have made people fully aware of the importance for establishing accurate and effective methods with regard to supervision of meat quality.However,the current detection methods are still stayed in laboratorial usages,and only few methods are available for rapid,simple,and on-site detection of meat derived components.Taking the current status of meat product detection into account,in this paper,we have combined nucleic acid amplification technologies including polymerase chain reaction(PCR),loopmediated isothermal amplification(LAMP),and recombinase polymerase amplification(RPA)with popular sensing technologies such as fluorescence analysis,fluorescence polarization(FP),lateral flow strip(LFS),and surface-enhanced Raman spectroscopy(SERS)to establish simple,fast,and sensitive new methods for detection of common meat derived components.The main contents are as follows:(1)Detection of horsemeat component in beef by PCR method based on the functional design of primers.The ends of PCR primers are designed with a unique stemloop structure that is labeled with a pair of fluorophore and quencher.Initially,the fluorophore and quencher on the stem-loop are close to each other,resulting in a low fluorescence signal.In the presence of horsemeat component,functionalized primers will be participated in the amplification process,resulting in the opening of the stem-loop structures at the end of primers and in turn the recovery of fluorescence signal.Along this line,the more the horsemeat component presented,the stronger the fluorescence signal will be collected.Under optimal conditions,the detection limit of this method for adulterated horsemeat in beef is 0.04%(w/w).In addition,the detection of processed beef samples further confirmed the practical application potential of this method.Compared with traditional PCR,this method has obvious advantages in detection efficiency and fully highlights its characteristics of rapid detection and easy operation.(2)Detection of chicken component in beef by fluorescence polarization strategy based on single-stranded binding protein enhancement.A single-stranded binding protein(SSB)enhanced FP strategy is developed for sensitive detection of chicken component by integrating the traditional PCR technology with FP technology.After the extraction of genome templates,PCR amplification is first performed using fluorophorelabeled primers specific to chicken.Then,SSB is added to the amplified products to make the single-stranded primers fully combine with SSB.In the absence of chicken component,the fluorophore-labeled primers can firmly bind to the subsequently added SSB and the movement of the fluorophores are restricted,so the FP signal is very high.In the presence of chicken component,the fluorophore-labeled primers are consumed during the amplification process and cannot bind to the subsequently added SSB,leading to a decreased FP signal.And,the more the chicken component presented,the lower the measured FP signal,thus establishing the correlation between the adulteration proportion of chicken and the FP signal.Under optimal conditions,the detection limit of this method for adulterated chicken in beef is 0.035%(w/w).This method emphasizes the application of FP technology in the rapid analysis of PCR products and highlights uniqueness of using SSB to significantly improve FP signal.Compared with the traditional detection methods based on electrophoresis and fluorescence recovery,this method has the advantages of convenient design,simple operation,and rapid detection.(3)Rapid detection of pork component in beef by fluorescence polarization method based on loop-mediated isothermal amplification.An ideal platform that integrates the isothermal amplification property of LAMP technology and the rapid detection feature of FP technology is constructed by skillfully applying fluorophorelabeled primers to the reaction system of LAMP.The fluorophore-labeled primers have smaller molecular weight,ensuring the attached fluorophores fast rotated for a limited FP signal.In the presence of pork component,primers with lower molecular weight are amplified and converted into products with larger molecular weight.This effect caused the restriction of the free rotation of fluorophores.Therefore,the FP signal is significantly enhanced.Under optimal conditions,as low as 0.01%(w/w)of adulterated pork in beef can be detected by this method.Compared with traditional detection methods that require complex analysis processes,this strategy has the advantages of simplicity,sensitivity,rapidness and on-site detection.Importantly,this unique method is also available for the processed meat products.We expect that this study can provide a new perspective for the establishment of methods for food safety detection.(4)Multiple PCR-based lateral flow strip method for simultaneous detection of chicken,duck and pork components.A visual strategy for simultaneous detection of multiple meat components in meat products is developed by integrating multiple PCR with LFS technology.The ends of three primer sets for chicken,pork and duck are labeled with different nucleic acid tags,respectively,thereby enabling the amplified products with different nucleic acid tags at two ends.When LFS is used to analyze the amplified products,one end of the amplified products can be hybridized with the recognition probes on the gold nanoparticles(Au NPs),while the other end can be hybridized with the capture probes on the corresponding test line,thus fixing Au NPs to the corresponding test line and turning it red.Importantly,each adulterated component corresponds to one test line,respectively.Therefore,the simultaneous and visual detection of three adulterated components can be realized through one reaction.Taking advantages of the high efficiency of PCR technology and the simplicity of LFS technology,the detection limits of this method for chicken,duck and pork are 0.006%(w/w),0.004%(w/w)and 0.007%(w/w),respectively.(5)Simultaneous detection of duck,chicken,and pork components by lateral flow strip method based on recombinase polymerase amplification.A new method for simultaneous detection of duck,chicken and pork components in meat products is developed by combining RPA technology with LFS technology.In the amplification system of RPA,we introduced three pairs of end-functionalized primers that are specific to duck,chicken and pork respectively,enabling the simultaneous amplification of three target genes.In the presence of adulterated components,the double-stranded products labeled at both ends can be obtained by amplification.With the bridging effect of doublestranded products,the Au NPs can be fixed on the corresponding test line and make it red.This method highlights the advantages of isothermal amplification and multiple amplification of RPA technology,and emphasizes the application potential of combination of RPA technology with LFS technology in meat adulteration detection.Under optimal conditions,the detection limits of this method for chicken,duck and pork are 0.009%(w/w),0.005%(w/w)and 0.004%(w/w),respectively.(6)Simultaneous detection of multiple meat components in beef by lateral flow strip method based on surface-enhanced Raman spectroscopy.A new method for detection of multiple meat components in meat products is proposed by combining SERS technology with LFS technology using the amplified products of RPA as medium.To simplify the design of LFS,three kinds of Raman tags are used as the markers of three kinds of meat,and only one test line is used to capture three kinds of amplified products simultaneously.When the test line is colored,it can be proved that there are target components in the samples,and then the specific components of meat products can be analyzed by Raman spectroscopy.This method is developed on the basis of the research of(5),focusing on the combined application of RPA technology,LFS technology and SERS technology in meat component detection.Under optimal conditions,the detection limits of this method for chicken,duck and pork are 0.006%(w/w),0.005%(w/w)and0.009%(w/w),respectively.