细菌感染多重核酸检测试剂参考品的建立
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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 1×10~8—1×10~9 CFU/ml and final concentration of fungi was 1×10~6—1×10~7 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.
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 1×10~8—1×10~9 CFU/ml and final concentration of fungi was 1×10~6—1×10~7 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.
引文
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[16]田亚宾,周海卫,刘东来,等.水痘带状疱疹病毒Ig G抗体检测试剂国家参考品的研制[J].中国生物制品学杂志,2017,30(4):408-411.
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[21]Food and Drug Administration.Class II special controls guideline:gastrointestinal microorganism multiplex nucleic acid-based assays for detection and identification of microorganisms and toxin genes from human stool specimens[EB/OL].[2015-11-02].https://www.fda.gov/downloads/Medical Devices/Device Regulationand Guidance/Guidance Documents/UCM470559.pdf.
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[23]Montag L,Thomas,Stoermer,et al.Report on the international validation study on bacteria standards(transfusion-relevant bacterial strain panel)and proposal for a validation study for enlargement of the transfusion-relevant bacterial strain panel:proceedings of the World Health Organization Biologicals Unit&WHO Expert Committee on Biological Standardization,Geneva,Switzerland,2010[C].Geneva:World Health Organization,2010.
[24]Salimnia H,Fairfax MR,Lephart PR,et al.Evaluation of the filmarray blood culture identification panel:results of a multi-center controlled trial[J].J Clin Microbiol,2016,54(3):687-698.
[25]Buss SN,Leber A,Chapin K,et al.Multicenter evaluation of the biofire filmarray gastrointestinal panel for etiologic diagnosis of infectious gastroenteritis[J].J Clin Microbiol,2015,53(3):915-925.
[2]Prakash VP,Leblanc L,Alexanderscott NE,et al.Use of a culture-independent gastrointestinal multiplex PCR panel during a shigellosis outbreak:considerations for clinical laboratories and public health[J].J Clin Microbiol,2015,53(3):1048-1049.
[3]Lazcka O,Del Campo FJ Munoz FX.Pathogen detection:a perspective of traditional methods and biosensors[J].Biosens Bioelectron,2007,22(7):1205-1217.
[4]Yang S,Rothman RE.PCR-based diagnostics for infectious diseases:uses,limitations,and future applications in acute-care setti[J].Lancet Infect Dis,2004,4(6):337-348.
[5]Scheler O,Glynn B,Kurg A.Nucleic acid detection technologies and marker molecules in bacterial diagnostics[J].Expert Rev Mol Diagn,2014,14(4):489-500.
[6]Chamberlain JS,Gibbs RA,Ranier JE,et al.Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification[J].Nucleic Acids Res,1988,16(23):11141-11156.
[7]Li Y,Luo D.Multiplexed molecular detection using encoded microparticles and nanoparticles[J].Expert Rev Mol Diagn,2006,6(4):567.
[8]Subramony A,Zachariah P,Krones A,et al.Impact of multiplex polymerase Chain reaction testing for respiratory pathogens on healthcare resource utilization for pediatric inpatients[J].J Pediatr,2016,173:196-201.
[9]赵焕英,薛冰,方霄,等.聚合酶链反应-高分辨率熔解曲线技术在下呼吸道细菌鉴定中的应用[J].微生物与感染,2015,10(5):308-314.
[10]刘妙娥,冯慧艳,王立军.多种呼吸道病原微生物快速筛查技术的建立[J].中国医学工程,2015,10:126-127.
[11]刘宁伟,刘威,黄留玉.多重核酸检测技术研究进展[J].生物技术通讯,2016,27(4):596-600.
[12]李林海,陈丽丹,肖斌,等.宏基因组测序在感染性疾病病原体检测中的应用[J].传染病信息,2018,31(1):15-18.
[13]Food and Drug Administration.Nucleic acid based tests-multiplex panel[EB/OL].[2018-04-09].https://www.fda.gov/Medical Devices/Productsand Medical Procedures/In Vitro Diagnostics/ucm330711.htm.
[14]刘东来,张岩,李玉华,等.寨卡病毒核酸检测试剂应急参考品的建立[J].中国病毒病杂志,2017,(5):343-349.
[15]周海卫,沈舒,石大伟,等.第二代甲型H1N1流感病毒核酸检测试剂参考品的建立[J].中国病毒病杂志,2016,1:45-49.
[16]田亚宾,周海卫,刘东来,等.水痘带状疱疹病毒Ig G抗体检测试剂国家参考品的研制[J].中国生物制品学杂志,2017,30(4):408-411.
[17]国家食品药品监督管理总局.体外诊断试剂注册管理办法[EB/OL].[2014-07-30].http://www.sda.gov.cn/WS01/CL0053/103757.html.
[18]Goldman E,Green LH.Practical handbook of microbiology[M].3rd Ed.USA:CRC Press,Taylor and Francis Group,2015:864.
[19]Food and Drug Administration.Class II special controls guidance document respiratory viral panel multiplex nucleic acid assay[EB/OL].[2009-10-09].https://www.fda.gov/downloads/medicaldevices/deviceregulationandguidance/guidancedocuments/ucm180324.pdf.
[20]Food and Drug Administration.Highly multiplexed microbiological medical countermeasure in vitro nucleic acid based diagnostic devices[EB/OL].[2014-08-27].https://www.fda.gov/downloads/Medical Devices/Device Regulationand Guidance/Guidance Documents/UCM327294.pdf.
[21]Food and Drug Administration.Class II special controls guideline:gastrointestinal microorganism multiplex nucleic acid-based assays for detection and identification of microorganisms and toxin genes from human stool specimens[EB/OL].[2015-11-02].https://www.fda.gov/downloads/Medical Devices/Device Regulationand Guidance/Guidance Documents/UCM470559.pdf.
[22]Food and Drug Administration.Class II special controls guideline:multiplex nucleic acid assay for identification of microorganisms and resistance markers from positive blood cultures[EB/OL].[2015-05-27].https://www.fda.gov/ucm/groups/fdagov-public/@fdagov-meddev-gen/documents/document/ucm448520.pdf.
[23]Montag L,Thomas,Stoermer,et al.Report on the international validation study on bacteria standards(transfusion-relevant bacterial strain panel)and proposal for a validation study for enlargement of the transfusion-relevant bacterial strain panel:proceedings of the World Health Organization Biologicals Unit&WHO Expert Committee on Biological Standardization,Geneva,Switzerland,2010[C].Geneva:World Health Organization,2010.
[24]Salimnia H,Fairfax MR,Lephart PR,et al.Evaluation of the filmarray blood culture identification panel:results of a multi-center controlled trial[J].J Clin Microbiol,2016,54(3):687-698.
[25]Buss SN,Leber A,Chapin K,et al.Multicenter evaluation of the biofire filmarray gastrointestinal panel for etiologic diagnosis of infectious gastroenteritis[J].J Clin Microbiol,2015,53(3):915-925.