摘要
目的建立细菌感染多重核酸检测试剂参考品并制定其质量标准,为相关产品的质量评价提供依据。方法选择34种可导致中枢神经系统、血液、呼吸道、肠道以及生殖道感染的致病性细菌或真菌,培养富集后应用全自动细菌鉴定及药物敏感分析系统VITEK2进行鉴定。鉴定正确的菌株,应用梯度稀释法测定浓度后进行分组混合成高浓度样本。应用全基因组第二代测序技术验证混合样本的组成情况和特异性。选择基于不同原理的多重核酸检测试剂进行协作标定,根据标定结果确定参考品的质量标准,并对参考品的稳定性进行考察。结果本参考品由28种细菌和6种真菌混合成7支混合参考品,编号为M1~M7,其中细菌的终浓度约为1×10~8~1×10~9 CFU/ml,真菌的终浓度约为1×10~6~1×10~7 CFU/ml。经全基因组第二代测序技术验证,参考品的组成正确且无交叉污染情况。分别应用基于PCR-熔解曲线、等温扩增以及靶向扩增第二代测序技术的多重核酸检测试剂对参考品进行检测,结果显示本参考品经过适当稀释后能够对试剂的准确性、特异性、重复性以及最低检出限等性能指标进行评价,且参考品各混合菌株间无交叉反应。稳定性研究结果表明反复冻融3次不影响本参考品的稳定性。结论本研究建立了细菌感染多重核酸检测试剂参考品并制定其质量标准,能够应用于相关参考品的质量评价。
Objective To establish reference material of multiplex nucleic acid assays for identification of bacterial infection and formulate relevant quality standards,thus providing evidence for quality assessment of related products.Methods Thirty-four pathogenic bacteria and fungi associated with central nervous system,blood,respiratory,intestinal and reproductive tract infections were selected for further culture and enrichment,and were identified by using fully automated microbial identification system and drug sensitive analyzer VITEK2.The gradient dilution method was used to determine the concentration of identified correct strains,then all strains at a high concentration were grouped and mixed as reference material candidates.The composition and specificity of mixed candidates were verified by using the whole genome sequencing.Collaboration study was conducted using multiplex nucleic acid assays based on different detection principles to determine the quality standards of reference material.Finally,the stability of the reference material was evaluated.Results Total 34 pathogenic bacteria and fungi,including 28 bacteria and 6 fungi,were mixed as 7 reference materials coded M1-M7(final concentration of bacteria was 1×108-1×109 CFU/ml and final concentration of fungi was 1×106-1×107 CFU/ml).The whole genome second-generation sequencing technology was applied to verify that the reference product was composed correctly and there was no cross-contamination.Reference product was tested with multiplex nucleic acid assays based on PCR-melting curves,isothermal amplification and targeted-amplification second-generation sequencing technologies.The results showed that detecting the suitably diluted reference material could be used to evaluate the multiplex nucleic acid assays,such as accuracy,specificity,reproducibility and limit of detection,and there was no cross-containment among mixed strains.The stability study showed that 3 times freeze-thawing cycles did not impact the stability of reference material.Conclusions The reference material and its quality standards have been established and it can be used for quality evaluation of multiplex nucleic acid assays for identification of bacterial infection.
作者
刘东来
周海卫
石大伟
沈舒
田亚宾
张春涛
LIU Dong-lai;ZHOU Hai-wei;SHI Da-wei;SHEN Shu;TIAN Ya-bin;ZHANG Chun-tao(Division II of In Vitro Diagnostics for Infectious Diseases,Institute for In Vitro Diagnostics Control,National Institutes for Food and Drug Control,Beijing 100050,China)
出处
《传染病信息》
2018年第3期209-214,共6页
Infectious Disease Information
基金
中国食品药品检定研究院中青年发展研究基金(2016C1)
关键词
细菌感染
多重核酸检测试剂
参考品
bacterial infection
multiplex nucleic acid assay
reference material
