吴龙飞、许会艳负责实验操作、论文撰写，于常鸿、陈心怡、李俊强、张素梅、张龙现负责论文修改。
收稿日期: 2025-12-08
修回日期: 2026-01-15
网络出版日期: 2026-04-02
基金资助
国家“十四五”重点研发计划(2023YFD1801000);河南省科技攻关项目(242102110007);国家标准样品研复项目(S2024057)
Preparation and assessment of qualitative reference materials for detection of Cryptosporidium parvum nucleic acid
Received date: 2025-12-08
Revised date: 2026-01-15
Online published: 2026-04-02
Supported by
National Key Research and Development Project of the 14th Five-year Plan(2023YFD1801000);Henan Provincial Science and Technology Research Project(242102110007);National Reference Material Research and Restoration Project(S2024057)
目的 按照GB/T 15000《标准样品工作导则》所规定的标准样品制备流程和工艺制备微小隐孢子虫核酸检测用标准样品。方法 通过犊牛传代获取微小隐孢子虫虫株C1HN（亚型为ⅡdA19G1）卵囊，纯化后提取DNA并测序，溯源性检验正确后分装保存，通过微小隐孢子虫巢式PCR和实时荧光定量PCR（qPCR）对制备的标准样品进行均匀性检验、冻融稳定性检验、短期和长期稳定性检验，通过多家单位合作的方式联合定值。采用SPSS 27.0软件进行统计学分析，数据采用单因素方差分析和配对样本t检验。结果 PCR结果显示，扩增片段大小约为830 bp，与国标检测方法结果一致。测序结果显示，该片段与C1HN虫株上的2号、7号、8号染色体上的SSU rRNA片段序列一致性为99.65%，与感染犊牛虫株完全一致，亚型为ⅡdA19G1。均匀性检验结果显示，样品Ct值为15.00 ± 0.36，组间均方为0.17，组内均方为0.11，差异无统计学意义（F = 1.49，P > 0.05）。冻融稳定性检验结果显示，样品反复冻融第0、3、7、14天的Ct值别为15.67 ± 0.25、15.68 ± 0.22、15.69 ± 0.27、15.71 ± 0.27，差异无统计学意义（t = 0.00、0.31、0.50、0.71，均P > 0.05）。短期稳定性检验结果显示，4 ℃下样品储存第0、1、2、3、4、5、10天的Ct值分别为16.48 ± 0.28、16.48 ± 0.25、16.49 ± 0.28、16.50 ± 0.25、16.53 ± 0.26、16.56 ± 0.25、16.57 ± 0.24，差异无统计学意义（t = 0.00、0.04、0.34、0.46、1.56、1.60、1.84，均P > 0.05）；25 ℃下样品储存第0、1、2、3、4、5、10天的Ct值分别为16.20 ± 0.18、16.21 ± 0.14、16.21 ± 0.13、16.22 ± 0.14、16.23 ± 0.14、16.24 ± 0.14、16.25 ± 0.15，差异无统计学意义（t = 0.00、0.17、0.28、0.47、0.61、1.09、1.40，均P > 0.05）；37 ℃下样品储存第0、1、2、3、4、5、10天的Ct值分别为16.21 ± 0.17、16.22 ± 0.16、16.23 ± 0.16、16.24 ± 0.17、16.26 ± 0.18、16.25 ± 0.18、16.26 ± 0.19，差异无统计学意义（t = 0.00、0.33、0.56、1.10、1.20、1.34、1.89，均P > 0.05）。长期稳定性结果显示，-20 ℃下样品储存第0、30、90、180、270、360天的Ct值分别为15.67 ± 0.11、15.67 ± 0.21、15.67 ± 0.21、15.67 ± 0.25、16.67 ± 0.24、15.67 ± 0.23，差异无统计学意义（t = 0.00、0.01、0.11、0.23、0.37、0.39，均P > 0.05）；第180、360天巢式PCR结果显示，特异性条带大小均约为830 bp，测序结果与先前一致。9家单位出具的标准样品复核报告均证明制备的标准样品可作为微小隐孢子虫核酸检测阳性样品。结论 制备的标准样品达到了GB/T 15000《标准样品工作导则》的要求，溯源性可靠、均匀性良好、稳定性符合要求、定值结果正确。
吴龙飞 , 许会艳 , 于常鸿 , 陈心怡 , 李俊强 , 张素梅 , 张龙现 . 微小隐孢子虫核酸定性标准样品的制备及评估[J]. 中国寄生虫学与寄生虫病杂志, 2026 , 44(2) : 260 -265 . DOI: 10.12140/j.issn.1000-7423.2026.02.016
Objective To prepare reference materials for detection of Cryptosporidium parvum nucleic acid according to procedures and processes for preparation of reference materials specified in the national criteria GB/T 15000 Directives for the work of reference materials. Methods A large number of oocysts of the C. parvum C1HN strain (subtype IIdA19G1) were obtained through calf passage, and purified, and genomic DNA was extracted from C. parvum oocysts using DNA extraction kits. Then, the source of oocyst DNA was traced. Once the traceability of the extracted DNA was verified, DNA samples were aliquoted and cryopreserved. The homogeneity, freeze-thaw stability, and short- and long-term stability of C. parvum oocyst DNA samples were evaluated with nested PCR and quantitative Real-time PCR (qPCR) assays, and the constant values were determined collaboratively by multiple institutions. All statistical analyses were performed using the software SPSS 27.0, and differences of means were tested for statistical significance with one-way analysis of variance (ANOVA) and paired-samples t test. Results The PCR amplification fragment was approximately 830 bp in size, which was consistent with the results detected by the national standard. Sequencing revealed that the SSU rRNA gene fragments on chromosomes 2, 7, and 8 of this strain exhibited 99.65% sequence identity with those of the C1HN strain, indicating complete concordance with the infecting strain, and the subtype was identified as ⅡdA19G1. Homogeneity tests showed that the Ct value of the samples was 15.00 ± 0.36, with a mean square of 0.17 between groups and 0.11 within groups, indicating no statistically significant difference (F = 1.49, P > 0.05). Freeze-thaw stability tests showed that the Ct values of the samples were 15.67 ± 0.25, 15.68 ± 0.22, 15.69 ± 0.27, 15.71 ± 0.27 on days 0 (t = 0.00, P > 0.05), 3 (t = 0.31, P > 0.05), 7 (t = 0.50, P > 0.05), and 14 following repeated freeze-thaw cycles (t = 0.71, P > 0.05), respectively. Short-term stability tests showed that the Ct values were 16.48 ± 0.28, 16.48 ± 0.25, 16.49 ± 0.28, 16.50 ± 0.25, 16.53 ± 0.26, 16.56 ± 0.25, 16.57 ± 0.24 following storage at 4 ℃ for 0 (t = 0, P > 0.05), 1 (t = 0.04, P > 0.05), 2 (t = 0.34, P > 0.05), 3 (t = 0.46, P > 0.05), 4 (t = 1.56, P > 0.05), 5 (t = 1.60, P > 0.05), and 10 days (t = 1.84, P > 0.05); 16.20 ± 0.18, 16.21 ± 0.14, 16.21 ± 0.13, 16.22 ± 0.14, 16.23 ± 0.14, 16.24 ± 0.14, and 16.25 ± 0.15 following storage at 25 ℃ for 0 (t = 0.00, P > 0.05), 1 (t = 0.17, P > 0.05), 2 (t = 0.28, P > 0.05), 3 (t = 0.47, P > 0.05), 4 (t = 0.61, P > 0.05), 5 (t = 1.09, P > 0.05), and 10 days (t = 1.40, P > 0.05); and 16.21 ± 0.17, 16.22 ± 0.16, 16.23 ± 0.16, 16.24 ± 0.17, 16.26 ± 0.18, 16.25 ± 0.18 and 16.26 ± 0.19 following storage at 37 ℃ for 0 (t = 0.00, P > 0.05), 1 (t = 0.33, P > 0.05), 2 (t = 0.56, P > 0.05), 3 (t = 1.10, P > 0.05), 4 (t = 1.20, P > 0.05), 5 (t = 1.34, P > 0.05), and 10 days (t = 1.89, P > 0.05), respectively. Long-term stability tests showed that the Ct values were 15.67 ± 0.11, 15.67 ± 0.21, 15.67 ± 0.21, 15.67 ± 0.25, 16.67 ± 0.24 and 15.67 ± 0.23 following storage at -20 ℃ for 0 (t = 0.00, P > 0.05), 30 (t = 0.01, P > 0.05), 90 (t = 0.11, P > 0.05), 180 (t = 0.23, P > 0.05), 270 (t = 0.33, P > 0.05), and 360 days (t = 0.39, P > 0.05), respectively. Nested PCR assays of samples stored for 180 and 360 days amplified specific bands with approximately 830 bp in size, and sequencing results were consistent with those previously obtained. All participating institutions provided verification reports confirming that the prepared reference materials might be used as a qualitative positive control for detection of C. parvum nucleic acid. Conclusion The prepared reference material meets the requirements specified in the national standard GB/T 15000 Directives for the work of reference materials with reliable traceability, good homogeneity, stability in compliance with specifications, and accurate constant values.
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