Application Omic Analysis Combined With SPR Sensors In The Study Of Rapid Detection For Foodborne Microorganisms

In recent years,food poisoning caused by food-borne pathogens and reports of spoilage during food shelf life caused by spoilage organisms have frequently occurred.,which has become a food safety issue that needs urgent solution.Therefore,how to perform real-time detection of microorganisms quickly and effectively has become the key to solve the problem.Due to the complexity of the food system,the food-borne microorganisms also have a variety of ways of survival,such as food contamination caused by the excessive number of E.coli colonies,or food poisoning caused by the production of toxic microorganisms under certain conditions,and events such as food spoilage caused by specific genes in certain types of microorganisms..At present,The detection methods of foodborne microorganisms are limited by the problems of expensive equipment,time-consuming and low-sensitivity detection.Therefore,in this study,the food-borne pathogens Staphylococcus aureus 10071 and 12513,Cronobacter sakazakii BAA 894 and s-3,and Lactobacillus acetotolerans BM-LA14527 were used to sequence and compare their genome and transcriptome information.The detection targets for different characteristics of each microorganism were selected.Based on RCA amplification principle,an RCA reaction system was established for each target,and an SPR sensor detection system was further constructed based on the established RCA amplification method to achieve fast food-borne microorganisms.Accurate,high-throughput real-time detection.1.In this experiment,2 S.aureus,2 C.sakazakii,and 1 L.acetotolerans were used.De novo sequencing of the whole genome of S.aureus and C.sakazakii was performed using the Illumina Hiseq 2500 sequencing platform.The SOAPdenovo assembly software and RAST,GeneMarks and other genetic prediction annotation tools were used to acquire genomic sequences with better assembly results,including 226 scaffolds of S.aureus 10071,84 scaffolds of 12513,and 135 scaffolds of C.sakazakii BAA 894 were obtained.1,734 scaffolds for s-3.It has been predicted that S.aureus 10071 and 12513 have 2603 and 2503 CDS respectively,while C.sakazakii BAA 894 and s-3 have 3980 and 2420 CDS respectively.At the same time,based on databases such as COG and KEGG,the COG function classification and KEGG metabolic pathway enrichment of each gene sequence were annotated using the BLAST tool.2.RNA-seq was used to sequence transcriptomes of 2 C.akazakii biofilms cultured for 24 h.Based on the previous study of our laboratory and GenBank database,the transcriptomes of S.aureus under different concentrations of antibiotics were obtained.The transcriptome of L.acetotolerans under special proliferation conditions.3.Using comparative genomics and transcriptomics technologies,including analysis of homologous sequences among various microbial genomes,and prediction and annotation of homologous sequences based on databases such as NR,VFDB,Two 2513 homologous sequences of S.aureus were obtained,and 2026 homologous sequences of two strains of C.sakazakii were obtained.An analysis of the annotations revealed that there were 70 virulencerelated genes in the homologous sequence of S.aureus and 12 drug-resistance genes with a similarity of 100,and there were 247 virulence-related genes in C.sakazakii homologous sequences.Drug resistance genes were not found.Further analysis of the annotated results of homologous sequences combined with the specificity of the sequences themselves and the causes of the related food safety events resulted in the acquisition of the enterotoxin genes seb,sec,exfoliative toxins eta and drug-associated mecA genes of S.aureus.Also the specific sequence ESA₀2130,the ompA gene associated with pathogenicity of C.sakazakii,and the rancidity-resistance gene horA caused by L.acetotolerans were all used as detection targets.4.Padlock probes were designed for the selected detection targets.According to the principle of isothermal amplification,an RCA amplification system for each target was established,and the specificity was tested.It was found that the established RCA detection system has a high specificity.Then based on RCA amplification system established SPR sensor detection system.Using SPR four-well chips,simultaneous detection of the 3 toxin-associated genes of S.aureus was found to be positive.The results were consistent with the results of “Omics” analysis and RCA detection,confirming the feasibility of comprehensive highthroughput detection of microbial-related characteristics using “Omics” analysis technology.At the same time,ESA₀2130 and ompA genes were detected for C.sakazakii,using 10071 as a negative control and double distilled water as a blank.The results showed that the reaction system had higher specificity.Analysis of the reaction system and reaction process revealed that the RCA-SPR detection system is only 6 μL and the reaction time is less than 20 minutes,in which the detection time is greatly shortened,the consumption of reaction reagents is greatly reduced,and real-time detection can be realized at the same time.