| Most meat adulteration is economically motivated adulteration(EMA),which is not only a kind of consumption fraud,but also causes health risks and religious violation,etc.The resulting consumer distrust will also affect the order of the meat industry and international trade.Accurate and sensitive detection method of meat adulteration is an important technical support to ensure the authenticity of meat,among which DNA-based technology has a high reliability due to its species specificity.DNA biosensing technology has high sensitivity and simplicity and it is more suitable for single-stranded DNA recognition than for double-stranded DNA because the recognition function depends on the complementary sequence between the target and the corresponding nucleic acid probe.In addition,the content of target DNA is low in the complex matrix of food samples,so the existing DNA sensing technology is still insufficient in the signal transduction of trace target DNA.Clustered regularly interspaced short palindromic repeats,Clustered regularly interspaced short palindromic Repeats,CRISPR)and CRISPR-associated nuclease(Cas)realize double or single stranded nucleic acids by guiding RNA specific recognition,and CRISPR-Cas 12a can integrate specific recognition,efficient signal conversion and amplification of target DNA.It provides a new idea for fast,sensitive and portable detection of meat adulteration.In this study,a novel DNA biosensor technology was established based on CRISPR-Cas12a system,which was successfully applied to the detection of duck adulteration,and provided a reference for the detection of trace nucleic acids in complex substrates.1.DNA templated silver nanocluster(DNA-AgNCs)fluorescent nanomaterials were synthesized using G-rich DNA sequences as a probe to construct a fluorescent nucleic acid biosensor based on CRISPR-Cas 12a for the adulteration detection of duck meat.The target DNA activates CRISPR-Cas 12a to trans-cleavage the DNA-AgNCs,resulting in a substantial fluorescence reduction.The target DNA can be quantitatively analyzed according to the difference of fluorescence intensity.By optimizing the main conditions of DNA-AgNCs synthesis,such as DNA template,molar ratio of DNA/Ag+,DNA concentration and incubation time,the linear detection range of the proposed method was 10 pM-1 μM,and the limit of detection(LOD)was as low as 1.9 pM,realizing specific and sensitive detection of duck meat adulteration.2.CRISPR-Casl2a-mediated liposome signal amplification technology was constructed combined with Surface enhanced Raman spectroscopy(SERS)and naked eye detection for the nucleic acid detection in duck meat adulteration.Liposomes modified with biotin were prepared by reverse-phase evaporation method.The liposomes could be linked to biotin-streptavidin via bio-ssDNA based on biotin-Streptavidin affinity.Target DNA activates the trans-cleavage of CRISPR-Cas12a,resulting in changes in bio-ssDNA binding function and capture for liposome.After the liposome was broken,the packaged Raman reporter and colorimetric reporter released,which could cause changes in SERS signal and colorimetric signal of the system,and was used for quantitative analysis of target DNA.After optimizing the parameters of L-a-phosphatidylcholine,molar ratio of cholesterol to biotin and concentration of signal molecules for liposome synthesis,SERS detection of target DNA was realized in the range of 1 fM-10 nM,LOD was 100 aM,and LOD of naked eye detection was 10 pM.3.Construction of CRISPR-Cas12a triggered cascade signal amplification SERS/naked-eye dual-mode nucleic acid biosensor for nucleic acid detection in duck meat adulteration.Target DNA can activate Cas12a protein to trans-cleave ssDNA,the initiator in DNA strand displacement reaction(TSDR).The cleavage resulted that TSDR reaction failed to initiate and the trigger chain of hybrid chain reaction(HCR)reaction was retained,which triggers HCR reaction to form a large amount of long double-stranded DNA and DNA enzyme with catalytic function(GQH DNAzyme).GQH DNAzyme could catalysis cysteine(Cys)and 2,Catalysis of 2,2’-azino-bis(3-ethylbenzothiazoline 6-sulfonic acid(ABTS)to produce strong SERS signal difference and colorimetric signal.It can be used for quantitative analysis of target DNA.After optimizing experimental conditions,such as the amount of TSDR initiator,the concentration of 4-NTP in gold nanoparticles functionalization,and the concentration of GQH DNAzyme substrate Cys,the SERS detection of target DNA was realized in the linear rangeof 100 aM-10 nM with the LOD of 34.9 aM,and the naked eye detection LOD was 1 pM.The developed methods have advantages in rapid detection,portability and sensitivity respectively,displaying high accuracy,high sensitivity and good specificity,showing great application prospects in the detection of trace meat adulteration. |
