基于高通量测序技术的实验动物沙门氏菌检测方法的建立与评价

目的建立基于高通量测序技术的实验动物沙门氏菌检测方法,应用于实验动物沙门氏菌的检测。方法提取小鼠粪便DNA样本,分别针对16S r DNA区域、23S r DNA区域、16S~23S r DNA IS、23S~5S r DNA IS、gyr B优选区域设计通用引物,对各个引物进行测试分析,优化扩增条件并建库,利用Illumina高通量测序技术检测区分42个样本中的沙门氏菌,评价该方法的特异性和稳定性。结果筛选发现沙门氏菌的菌种分类优选区域为gyr B基因,gyr B基因引物序列为FP5’-AACCACCGCAATCAGACCTT3’,FP5’-AGCCACGAAACCTTCACYA-3’。对引物进行优化,确定最佳扩增条件及样品上样量并正式建库,通过高通量测序和序列分析能检测出42个样品中极微量的沙门氏菌,检测方法稳定,灵敏度高,检测限可达0~102的cfu。结论本实验利用高通量测序技术,建立了一套完整检测实验动物沙门氏菌的生物体系,能检测出实验动物体内极微量的沙门氏菌,检测方法稳定性好,灵敏度高,可沿用至其他种类病原微生物的检测。

Establishment and evaluation of a high throughput sequencing technology for detection of Salmonella in laboratory animals

Objective To establish a detection method of Salmonella in laboratory animals based on a high-throughput sequencing technology, and to apply it in detection of Salmonella in laboratory animals. Methods DNA samples were extracted from mouse feces. Universal primers for 16S rDNA, 23S rDNA, 16S-23S rDNA, 23S-5S rDNA region, gyrB preferred area were designed, respectively. Each primer was tested and analyzed to determine the best amplification conditions and build a database. Forty-two samples of Salmonella were assayed by Illumina high-throughput sequencing technology and evaluated the specificity and stability of this method. Results The species preferred region of Salmonella was gyrB gene region. The primers for gyrB gene were FP5''-AACCACCGCAATCAGACCTT3'' and FP5''-AGCCACGAAACCTTCACYA-3''. The primers were optimized and determined, through a high-throughput sequencing, and the sequence analysis detected very small amount of Salmonella in the 42 samples, indicating that this detection method is stable, highly sensitive, and the limit of detection reached to 0-10² CFU. Conclusions We have established a complete detection system for detection of Salmonella in laboratory animals based on a high-throughput sequencing technology, This system can detect trace amounts of Salmonella in laboratory animals, and this detection method is stable and highly sensitive, which can be also used in detection of other kinds of pathogenic microorganism in laboratory animals.