| Staphylococcus is one of the common pathogenic bacteria,and it is also an important clinical pathogenic bacteria,the produced enterotoxins and hemolytic toxins cause major harm to human health.Therefore,it is significant to strengthen the early screening and prevention of Staphylococcus,especially Staphylococcus aureus and its related ST types.Molecular diagnostic technology is used for detection of food-borne pathogens due to its high detection efficiency,high specificity and sensitivity.The specificity and quantity of molecular targets are the most important factors limiting molecular diagnostic technology.Therefore,it is of great significance to build a efficient molecular target mining platform and establish a corresponding point-of-care testing(POCT)method.In this paper,a biochemical numerical identification system of common staphylococcus was constructed by using the principle of numerical identification.A molecular target mining platform for common Staphylococcus was constructed by using pan-gene analysis,and obtained specific molecular targets of genus,common species and ST-type of S.aureus,and a fluorescence quantitative PCR method was established;The CRISPR/Cas12a-based fluorescence assay,naked eye colorimetric method and lateral flow platform were constructed based on the independently expressed As Cas12a protein.The main research content and results of this thesis are as follows.1)Development and application of biochemical numerical identification system for common StaphylococcusBased on the team’s Staphylococcus resource library,10 biochemical reactions were successfully screened for the identification of 22 common Staphylococcus,A database of positive probability of biochemical reactions was established through biochemical experiments,and then corresponding identification software was developed.The numerical identification method was verified by using 300 Staphylococcus isolates,and the identification accuracy rate reached 90%.2)Construction of the mining of molecular targets platform of pathogenic Staphylococcus and corresponding quantitative detection methodsBased on NCBI Staphylococcal whole-genome sequence,combined with pan-genome analysis,a molecular target mining platform for Staphylococcus genus,common species and S.aureus multilocus sequence typing(MLST)was established.On this basis,1 genus-target of Staphylococcus,19 species-targets of four common Staphylococcus strains and 6ST-targets of S.aureus three important ST were obtained.The q PCR method for detection of S.aureus,S.epidermidis,S.haemolyticus and S.hominis were established using com FA,group＿14348,group＿26190 and group＿26478 targets,respectively.The limits of detection(LOD)in spiked chicken samples were 5.2×10~3CFU/g,2.85×10~4CFU/g,3.17×10~4CFU/g,2.95×10~1CFU/g.The q PCR method for detection of S.aureus ST7,ST188 and ST398 also were established using group＿10498,group＿9419 and group＿9943 targets,respectively.The LODs in spiked milk samples was 8.6×10~2CFU/m L,1.2×10~2CFU/m L and 6.4×10~3CFU/m L.The above q PCR methods all showed good specificity.The established q PCR method was used for the detection of 115 actual samples,showing results consistent with traditional detection methods.3)Preparation of Cas12a protein and establishment of fluorescent-based CRISPR/Cas12a method for detection of S.aureusThe prokaryotic expression system of Cas12a protein was constructed,and the As Cas12a protein was prepared by construction of prokaryotic expression system.The shelf life of the protein was 6 months,and the cleavage activity was better than the commercial Lba Cas12a protein.With the help of the target ds DNA activated"trans-cleavage"ss DNA characteristic of CRISPR/Cas12a,a conventional CRISPR/Cas12a fluorescence detection system based on nucleic acid amplification was established.The whole detection process needs 40 minutes,and shows good specificity.The detection limits in pure culture and spiked samples were 5.1×10~2CFU/m L and 5.1×10~3CFU/m L,respectively,which were 1-2 orders of magnitude higher than those of conventional PCR methods.To further reduce the cost,a GO-CRISPR/As Cas12a method was proposed based on a fluorescence post-quenching strategy by exploiting the strong FRET performance between GO and ss DNA fluorescent probes.The results showed that GO-CRISPR/Cas12a showed good specificity to the target bacteria,and the sensitivity was consistent with the traditional fluorescent CRISPR/Cas12a method.But the detection signal-to-noise ratio was decreased.In the actual sample detection,the detection results of the conventional CRISPR/As Cas12a method and the GO-CRISPR/As Cas12a method were consistent with the traditional methods.4)Establishment of CDA-ECP naked eye colorimetric assay for detection of S.aureus ST398To further improve flexibility and reduce instrument dependence,the Au NP probe naked-eye analysis strategy was combined with the CRISPR/Cas12a system,a enhanced colorimetric platform based on the CRISPR/Cas12a system and advanced DNA-Au NP probe(CDA-ECP)method for the detection of S.aureus ST398 was proposed.The method adopts the transient,ultra-low temperature liquid nitrogen freezing method for preparation of DNA-Au NP probe.The"trans cleavage"ss DNA property of CRISPR/Cas12a was used to cleavage the ligated DNA probe,the degraded ligated DNA fragments cannot bind to the DNA on the surface of Au NP probe so that the Au NP probe solution cannot aggregate and remains bright red,and final identification based on color or absorbance change.The results showed that CDA-ECP had good specificity for target bacteria and strong anti-interference ability for common non-target bacteria.With in 50 min,the LODs of pure culture and spiked samples were 6.4×10~2CFU/m L and 5.8×10~3CFU/g,respectively.By transferring the reaction solution to a 96-well plate and measuring its absorbance using an microplate reader.The CDA-ECP method can be used for high-throughput detection of 24food samples,and the detection results are consistent with standard MLST method.5)Establishment of CRA-LFB lateral flow platform for convenient detection of S.aureusIn order to realize on-site diagnosis based on CRISPR/Cas12a system,a low-cost and convenient CRISPR/Cas-recombinase-assisted amplification based fluorescent lateral flow biosensor(CRA-LFB)was developed.The T and C lines of the lateral flow strip were labeled with the capture DNA probe and streptavidin-modified BSA,respectively,and CRISPR/Cas12a cleavage the ss DNA binding probe,when the sample added to test strip,read the fluorescent band to complete the rapid detection.The whole detection time is 70 min,and it has good specificity for target bacteria and strong anti-interference ability for common non-target bacteria.The method obtained the LODs in pure culture and spiked beef samples were 5.4×10~2CFU/m L and 1.31×10~3CFU/g in,respectively.In summary,a biochemical numerical identification system of common staphylococcus was constructed,which effectively made up for the shortage of domestic staphylococcus numerical identification.Established a molecular target mining platform for pathogenic staphylococcus,and obtained a batch of Staphylococcus-related molecular targets with independent intellectual property rights.The As Cas12a protein was prepared,and a novel molecular diagnostic platform for CRISPR/Cas12a based on nano-quenched fluorescence biosensor,Au NP probe naked-eye colorimetric biosensor and lateral flow strategies was established,which greatly reduced the detection cost and dependence on instruments,and broadened the CRISPR/Cas system.The application in the field of molecular diagnosis provides new ideas for Staphylococcus risk identification and rapid detection. |
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