OrthoANI联合MALDI-TOF MS在胎儿弯曲菌鉴定与分型中的应用  

Application of OrthoANI combined with MALDI-TOF MS in identification and typing of Campylobacter fetus

作　　者：李松 屈平华 姚妍娇 陈茶 李启伟 邓光远 许振杰 陈富 Li Song;Qu Pinghua;Yao Yanjiao;Chen Cha;Li Qiwei;Deng Guangyuan;Xu Zhenjie;Chen Fu(Department of Clinical Laboratory Medicine,Guangdong Provincial Hospital of Chinese Medicine,Guangzhou 510006,China)

机构地区：[1]广东省中医院广州中医药大学第二附属医院检验科,510006 [2]广州市天河区冼村街社区卫生服务中心

出　　处：《中华检验医学杂志》2018年第8期583-588,共6页Chinese Journal of Laboratory Medicine

摘　　要：目的 建立新型弯曲菌—＂胎儿弯曲菌龟亚种（C.fetus subsp.testudinum）＂质谱超级图谱库,为临床提供新的检测平台.方法 对收集的建库菌株采用Illumina二代测序平台2000/miSeq进行高通量全基因组测序,JAVA 8运行环境,进行直系同源基因的全基因组平均核苷酸一致性（average nucleotide identity,OrthoANI）分析;对收集的实验菌株、标准菌株及对照菌株进行质谱分析,建立＂C.fetus subsp.testudinum＂的超级图谱并验证.结果 OrthoANI分析得出：建库菌株与模式菌株C.fetus subsp.testudinum03-427的OrthoANI值为：99.30%~99.96%,而其与C.fetus subsp.venerealisNCTC10354、C.fetus subsp.fetus82-40的OrthoANI值在91.05%~92.26%.参考OrthoANI≥95%作为同一菌种的判定依据,故将建库菌株最终鉴定为＂胎儿弯曲菌龟亚种＂.新建的超级图谱库鉴定准确率100%（与基因测序一致）,可信度区间为：82.3%~99.9%,同一菌株鉴定重复性100%.结论 基于高通量测序及全基因组学分析的微生物分类新＂金标准＂为实验室质谱自建库提供了理想的参考价值,而建立准确、客观的＂C.fetus subsp.testudinum＂质谱超级参考图谱,为临床快速诊断胎儿弯曲菌感染提供了新的平台.Objective Establishing the mass spectrum library of a new Campylobacter- ＂C. fetus subsp, testudinum＂ for rapid species identification in clinical microbiology laboratory. Methods [llumina second generation sequencing platform 2000/miSeq was used to carry out high flux genome sequencing for the strains which were collected to establish mass spectrum library. The analysis oforthologous average nucleotide identity （OrtboANI） between collected strains and reference strains was performed at JAVA 8 operation environment. Then, the mass spectrums ofcollected strains andreferenee strains were acquired using MALDI- TOF MS. And the mass spectrum library of C. fetus subsp, testudinum, were established and verified. Results The OrthoANI analysis showed that the OrthoANI value of the collected strains and the reference strain C. fetus subsp, testudinum03 - 427 was 99.30% - 99.96% , while the OrthoANI values of collected strains and C. fetus subsp, venerealisNCTC10354 orC. fetus subsp, fetus82 - 40 were 91.05% - 92.26%. With reference to OrthoANI ≥ 95% as the basis for the determination of the same strain, the strains which collected to establish mass spectrum library was finally identified as ＂ C. fetus subsp, testudinum＂. The identification accuracy rate of the mass spectrum library was 100% （ consistent with gene sequencing）, and the confidence interval was 82. 3% - 99.9% , identification of the same strain is 100% reproducible. Conclusions The new＂ gold standard＂ based on high throughput sequencing and total genome analysis has provided the ideal reference value for the establishment of mass spectrum library. And the accurate and objective reference spectrum of the＂ C. fetus subsp, testudinum＂ provides a new platform for the rapid diagnosis of fetal Campylobacter infection.

关 键 词：胎儿弯曲杆菌 菌血症 基因组学 高通量核苷酸序列分析 光谱法 质量 基质辅助激光解吸电离 细菌分型技术 

分 类 号：R446.5[医药卫生—诊断学] R714.5[医药卫生—临床医学]