Research On Virulence Genes Of Foodborne Bacillus Cereus And The Naked Eye Detection Method Based On APCR

Bacillus cereus is an opportunistic foodborne pathogen and can be widely distributed in air,soil,sewage,raw and cooked food.B.cereus may cause two types of gastrointestinal disease,namely,emesis and diarrhea.The type of emesis is caused by cereulide,while the type of diarrhea is caused by complex enterotoxins.At present,there is no clear B.cereus set limit value in China’s food safety standards,which makes it impossible to control B.cereus in the food production process.Therefore,to ensure food safety,it is necessary to explore the potential toxicity of B.cereus in food,and to develop a rapid and accurate detection method for monitoring of B.cereus.In this study,we investigated the distribution of 2 types of virulence genes and the relative transcription levels of 4 kinds of enterotoxin genes on different food matrices,and the AuNPs colorimetric method based on asymmetric PCR(aPCR)was developed for the detection of B.cereus.The content per seal is described as follows:In the first chapter,the research progress of B.cereus and aPCR were reviewed.In the second chapter,the virulence genes distribution and the relative transcription levels of the enterotoxin genes on different food matrices were investigated.The PCR method was used to detect the virulence genes in 132 B.cereus isolates from various food products obtained in Jiangxi province,China.The results showed that 2 strains carry the ces B gene and the detection rate was 1.5%,130 strains carry the enterotoxin gene and the detection rate was 98.5%.The detection rate of the cytK,hblD,nheA,and entFM genes in all B.cereus isolates carrying enterotoxin gene was 71%,43%,92%,and 99%,respectively.Besides,125 strains carry at least 2 enterotoxin genes and the detection rate was 96%,including34 strains carry 2 enterotoxin genes,44 strains carry 3 enterotoxin genes,and 47 strains carry all of the four enterotoxin genes.Based on the analysis of the distribution of virulence genes,the relative transcription levels of the enterotoxin genes on different food products were also analyzed.The growth curves of B.cereus in different matrix were obtained by detecting the concentration of B.cereus in different growth periods.RNA isolation was drawn at post-exponential phase.The relative transcrip levels of 4 kinds of enterotoxin genes in rice,noodles,milk powder and rice noodles were assayed by reverse transcription quantitative PCR with gatB_Yqey included as the reference gene.The results showed that the relativetranscription level of enterotoxin gene was correlated with the strain,gene type,and matrix.The relative transcription levels of the four enterotoxin genes were significantly different.For cytK and nheA,the relative transcription levels were influenced by food matrix and strains;For hblD and entFM,the relative transcription levels were largely influenced by the food matrix,most of which were downregulated in four food matrices.In the third chapter,a rapid,highly sensitive and simple method for detection of viable emetic B.cereus by coupling PMA-aPCR and AuNPs colorimetric assay was developed.The B.cereus was first subjected to PMA treatment to eliminate the false positive result of dead bacteria.Then,aPCR was used to generate a large amount of amplified single-stranded DNA(ss DNA).These ssDNA amplicons wrapped around AuNPs resulting from the interaction between nitrogen atoms of the exposed bases in ssDNA and the nanoparticles.In this manner the salt-induced aggregation of AuNPs was prevented,and then the color of the solution was measured by the naked eye and UV-vis spectroscopy.The results showed that PMA could effectively inhibit the PCR amplification of DNA from 107 CFU/mL dead cells,and had no effect on DNA from viable cells.In addition,both ssDNA and double-stranded DNA(dsDNA)could stabilize AuNPs in high salt solution,however,the protective effect of ssDNA is significantly better than that of dsDNA.The limit of detection of this PMA-aPCR-AuNPs colorimetric assay for viable emetic B.cereus reached as low as9.2×101 CFU/mL in pure culture and 3.4×102 CFU/mL in spiked milk,and the proposed method also exhibited excellent discrimination against 8 common pathogenic bacteria in milk.