人脲原体核酸检测试剂盒国家参考品的制备

Preparation of national reference for detection kit for human ureaplasma

目的 制备人脲原体核酸检测试剂盒国家参考品。方法 培养人脲原体两大生物群14个标准血清型菌株,培养液灭活后经颜色改变单位（color change unit,CCU）法与PCR相结合的方法进行定值,组成能覆盖脲原体所有表型及基因型的14份阳性参考品P1-P14（浓度为106CCU/ml）及14份检测限参考品L1-L14（浓度为104CCU/ml）;选择6份能排除脲原体核酸检测试剂盒非特异性或交叉反应的样品组成阴性参考品N1-N6;选择生物群1的微小脲原体（ureaplasma parvum,UP）1和UP14作为重复性参考品R1、R2（浓度分别为105和104CCU/ml）。用5家公司生产的脲原体核酸检测试剂盒对参考品进行验证,确定准确性、特异性、检测限、重复性、稀释线性的性能指标;并考察参考品在不同条件下（2～8℃放置7 d,37℃放置3、7、12 d,-20℃及常温反复冻融5次）的稳定性。结果 支原体各培养液经CCU法测定,浓度均在106～107CCU/ml之间,以CCU法测定浓度为靶值,绝对偏差均在依0.5个数量级以内。4家公司生产的不同试剂盒检测参考品,在准确性、特异性、检测限及重复性上均符合要求;在稀释线性上,最低稀释浓度为102CCU/ml的样品只有2家可以检出,其他几家的稀释线性相关系数以103～1064个浓度计算,r值均〉0.990 0;1家（RNA检测）检测R2的重复性,CV为5.5%。经不同条件处理的参考品的稳定性有轻微变化,但均处于设定的可接受范围内。结论 制备了人脲原体核酸检测试剂盒国家参考品,该参考品可满足人脲原体核酸分型、定性及定量检测要求,经多家实验室进行验证,可用于国内大多数试剂盒的性能评价及临床实验室质量评价。

Objective To prepare the national reference for detection kit for human ureaplasma nucleic acid. Methods Fourteen standard strains of 14 recognized serovars of two biovars of human ureaplasma were cultured and quantified by color change unit （CCU） and polymerase chain reaction （PCR） to constitute 14 positive references P1- P14 （106 CCU/ml） covering all the phenotypes and genotypes and 14 detection limit references L1- L14 （104 CCU/ml ）. Six samples without non-specific or cross reactions with ureaplasma nucleic acid were screened to constitute negative references N1 - N6. Ureaplasma parvum （UP） 1 and 14 of biovar 1 were selected as reproducibility references R1 and R2 （at concentrations of 105 and 104 CCU/ml） respectively. The references were verified for performance indexes such as accuracy, specificity, detection limit, reproducibility and dilution linearity by using the detection kits for five manufacturers, and evaluated for stabilities under various conditions （storage at 2 - 8 ℃ for 7 d, 37℃ for 3, 7 and 12 d, and 5 cycles of freeze-thawing at -20 ~C and room temperature）. Results The concentrations of various mycoplasma cultures were 10^6 - 10^7 CCU/ml, with an absolute deviation of less than ＋ 0. 5 CCU/ml. The accuracy, specificity, detection limit and reproducibility of references tested by the detection kits from four manufacturers met the relevant requirements. However, the sample at a minimum concentration of 10^2 CCU/ml was only detected by the kits from two manufacturers, while the correlation coefficients of dilution linearity determined by the kits from other manufacturers were calculated as four concentrations from 10^3 to 10^6 CCU / ml, with r values of more than 0. 990 0. The CV of test results for reproducibility of R2 by the detection kit for RNA from one manufacturer was 5. 5%. The stabilities of references after treatment under various conditions showed slightly changes within the designed acceptable range. Conclusion The national reference for detection kit for human ureaplasma nucleic acid was prepared, which met the requirements for genotyping as well as quantitative and qualitative detections of human ureaplasma nucleic acid, and might be used for the evaluation of performances of most domestic detection kits and quality control of clinical laboratories.