基于致病菌特有毒力基因的食源性细菌病原体高通量毒力筛查体系的建立及应用

In the face of the threat of newly emerged and unknown foodborne pathogens in future, to acquire the virulence and recombination of pathogens is critically. However, with the in-depth study of genomic information and gene functions, some identified virulence factors were also discovered to be encoded in the genomes of nonpathogenic bacteria, which seriously interfere with and restrict the establishment of high-throughput screening system for the virulence and recombination of pathogens. In our previous study, 1988 identified virulence genes were collected and 620 genes were identified as pathogen-specific virulence genes. We also found that the use of pathogen-specific virulence genes tor detect and identify new pathogens would be more accurately to reflect the virulence and recombination of pathogens..To detect and identify the virulence and recombination of unknown food-borne bacterial pathogens successfully, our obtained foodborne pathogen-specific virulence genes were predicted by the method of orthologs prediction in the newly collected nonpathogenic bacteria. Next the isothermal probes were achieved by the isothermal probe-design algorithm based on the targeted pathogen-specific virulence genes and the probe sets that would detect and identify different virulence genes were constructed. With the combination of whole genome amplification, the oligonucleotides microarray was designed to detect and identify the known virulence,to obtain the virulence and recombination of unknown food-borne bacterial pathogens in the future.

面对未来食品中可能出现的未知新型病原体威胁，获取病原体的毒力与毒力重组信息将成为关键所在。但是，随着基因组信息和基因功能的深入研究，一些已被鉴定的毒力因子被同样发现在非致病菌中，这严重干扰并制约了高通量致病菌毒力筛查体系的建立。前期我们通过对1988个致病菌毒力基因系统分析，获得了620个致病菌特有毒力基因，发现：对于新病原体的检测鉴定，利用致病菌特有毒力基因分析能更准确的反应致病菌的毒力与毒力重组。.本研究在前期获得食源性细菌病原体特有毒力基因的基础上，利用直系同源基因鉴定方法对特有毒力基因在扩展构建的非致病菌菌株中进行进一步比对分析，然后利用设计算法在目标特有毒力基因上设计出特异性等温寡核苷酸探针，分析获得能够检测鉴定不同病原体毒力基因的组合探针集，制备芯片，然后结合全基因组扩增技术，发展用于检测已知细菌毒力基因的高通量检测芯片，以便达到对食品中未知病原细菌的毒力筛查与重组确认。

伴随着食品中出现未知新型病原体的可能性在不断增大，同时这些新出现的病原体可能在毒力、重组以及耐药性方面具有新的特点，必将给公共安全保障带来极大的威胁。面对未知新型病原体的威胁，获取病原体的毒力与毒力重组信息将成为关键所在。前期我们通过对1988个致病菌毒力基因进行系统分析，获得了620个致病菌特有毒力基因，进一步分析发现：对于新病原体的检测鉴定，利用致病菌特有毒力基因分析能更准确的反应致病菌的毒力与毒力重组。.因此，本研究以食品中典型的细菌性病原体为研究对象，利用直系同源基因预测方法对包含24 株食源性细菌病原体的372个毒力基因在非致病菌菌株库中进行比对分析，获得了食源性细菌病原体特有毒力基因；建立大规模预测病原菌特有已知功能模体的方法，得到了食源性细菌病原体特有已知功能模体；建立预测致病菌特有毒力基因保守序列的识别算法，得到了食源性细菌病原体特有毒力基因保守序列数据；利用基于致病菌特有毒力基因保守序列的特异性等温探针设计算法在病原体上设计出特异性等温寡核苷酸探针。在预测获得各种食源性细菌病原体数据的基础上，构建了食源性致病菌特有毒力基因数据库、食源性致病菌特有功能模体数据库、食源性致病菌特有毒力基因保守片段序列数据库以及食源性致病菌特异性等温探针数据库。建立了“致病菌特有毒力基因-特有功能模体-特有毒力基因保守序列-特异性等温探针”的病原体筛查检测体系，在分析获得能够检测鉴定不同病原体毒力基因的组合探针集的基础上，合成部分目的检测基因的检测探针，研制了用于检测已知食源性致病菌毒力基因、菌种特异性基因的基因检测芯片，建立并优化了全基因组基因扩增的技术方法，对比了扩增基因的3种荧光标记方法，建立了基因芯片与全基因组扩增技术组合检测病原体的方法，确定了最佳实验条件并建立相应的标准化操作流程，检测了部分病原体单个及混合样本，结果显示：各个检测探针特异性和敏感性均良好，无交叉反应。