目的 研发鹿胎DNA阳性质控品,组装检测试剂盒。方法 提取鹿胎及其伪品DNA,设计通用鹿引物并进行PCR扩增,克隆种属特异性目的片段,构建鹿胎DNA阳性质控品并组装DNA检测试剂盒,进行试剂盒性能实验并应用于市售鹿胎样品检测。结果 应用美国国家生物技术信息中心(NCBI)分析测序比对结果,正确率为100%,在526 bp处可见明显条带,与目的片段相符,可作为鹿胎阳性质控品。阳性质控品灵敏性最低检测限为10 ng·μL-1,经多次反复冻融的试剂盒,仍可确保PCR检测结果的稳定性。试剂盒应用于对市售鹿胎及伪品的检测,PCR检测结果与预期相符。结论 构建鹿胎阳性质控品作为电泳分析时的阳性对照,结果更加准确稳定,适用于对鹿胎及伪品的快速鉴定,保障对鹿胎质量的有效监管。
Abstract
OBJECTIVE To develop a deer fetus DNA positive control sample and assemble a test kit. METHODS Deer fetal DNA and its fake DNA were extracted, universal deer primers were designed and PCR amplified, species-specific target fragments were cloned, deer fetal DNA positive quality control products were constructed, and a DNA detection kit was assembled. The performance test of the kit was carried out and applied on commercially available deer fetus samples. RESULTS The sequencing comparison results were analyzed by NCBI. The accuracy rate was 100%. A clear band was found at 526 bp, which was consistent with the target fragment. The constructed sample can be used as a deer fetal positive control sample. The minimum detection limit of the sensitivity of the positive control was 10-1 ng·μL-1. After repeated freeze-thaw cycles, the stability of the PCR test results was proved. The test results of the commercially available deer fetuses and fakes using the kit were in line with expectations. CONCLUSION The constructed deer fetal positive control can provide more accurate and stable results for electrophoretic analysis and is suitable for the rapid identification of deer fetuses and fakes, which guarantees effective supervision of deer fetus quality.
关键词
鹿胎 /
阳性质控品 /
DNA检测试剂盒 /
分子生物学 /
鉴别真伪
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Key words
deer fetus /
positive control /
DNA detection kit /
molecular biology /
authenticity
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中图分类号：
R284
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参考文献
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脚注
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基金
吉林省科技发展计划项目资助(20200404152YY,20200403047SF,20160204004NY,20190303047SF);吉林省科技创新中心建设项目资助(20190902018TC);北华大学研究生创新计划资助(北华研创合字[2019]016);吉林省重点科技成果转化项目资助(20170307001YY)
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