艾滋病模型中关键指标 SIV DNA 绝对定量微滴式数字 PCR 技术的创新应用

doi:10. 3969 / j.issn.1671-7856. 2020. 05. 005

首页 > 过刊浏览>2020年第30卷第5期 >27-30,53. DOI:10. 3969 / j.issn.1671-7856. 2020. 05. 005

艾滋病模型中关键指标 SIV DNA 绝对定量微滴式数字 PCR 技术的创新应用
DOI:
                        10. 3969 / j.issn.1671-7856. 2020. 05. 005
                    
作者:
                        张丽丽张丽丽
中国医学科学院医学实验动物研究所,北京协和医学院比较医学中心,国家卫健委人类疾病比较医学重点实验 室,国家中医药管理局人类疾病动物模型三级实验室,新发再发传染病动物模型研究北京市重点实验室, 北京 100021
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陆佳涵陆佳涵
中国医学科学院医学实验动物研究所,北京协和医学院比较医学中心,国家卫健委人类疾病比较医学重点实验 室,国家中医药管理局人类疾病动物模型三级实验室,新发再发传染病动物模型研究北京市重点实验室, 北京 100021
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薛 婧薛 婧
中国医学科学院医学实验动物研究所,北京协和医学院比较医学中心,国家卫健委人类疾病比较医学重点实验 室,国家中医药管理局人类疾病动物模型三级实验室,新发再发传染病动物模型研究北京市重点实验室, 北京 100021
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丛 喆丛 喆
中国医学科学院医学实验动物研究所,北京协和医学院比较医学中心,国家卫健委人类疾病比较医学重点实验 室,国家中医药管理局人类疾病动物模型三级实验室,新发再发传染病动物模型研究北京市重点实验室, 北京 100021
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魏 强魏 强
中国医学科学院医学实验动物研究所,北京协和医学院比较医学中心,国家卫健委人类疾病比较医学重点实验 室,国家中医药管理局人类疾病动物模型三级实验室,新发再发传染病动物模型研究北京市重点实验室, 北京 100021
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作者单位:中国医学科学院医学实验动物研究所,北京协和医学院比较医学中心,国家卫健委人类疾病比较医学重点实验 室,国家中医药管理局人类疾病动物模型三级实验室,新发再发传染病动物模型研究北京市重点实验室, 北京 100021
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中图分类号:R-33
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The establishment and application of an absolute quantitative method for analyzing SIV DNA via droplet digital PCR

Author:

ZHANG Lili
ZHANG Lili
Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences (CAMS);Comparative Medicine Center, Peking Union Medical College(PUMC);NHC Key Laboratory of Human Disease Comparative Medicine; Key Laboratory of Human Diseases Animal Models, State Administration of Traditional Chinese Medicine; Beijing Key Laboratory for Animal Models of Emerging and Remerging Infectious Diseases, Beijing 100021, China
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LU Jiahan
LU Jiahan
Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences (CAMS);Comparative Medicine Center, Peking Union Medical College(PUMC);NHC Key Laboratory of Human Disease Comparative Medicine; Key Laboratory of Human Diseases Animal Models, State Administration of Traditional Chinese Medicine; Beijing Key Laboratory for Animal Models of Emerging and Remerging Infectious Diseases, Beijing 100021, China
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XUE Jing
XUE Jing
Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences (CAMS);Comparative Medicine Center, Peking Union Medical College(PUMC);NHC Key Laboratory of Human Disease Comparative Medicine; Key Laboratory of Human Diseases Animal Models, State Administration of Traditional Chinese Medicine; Beijing Key Laboratory for Animal Models of Emerging and Remerging Infectious Diseases, Beijing 100021, China
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CONG Zhe
CONG Zhe
Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences (CAMS);Comparative Medicine Center, Peking Union Medical College(PUMC);NHC Key Laboratory of Human Disease Comparative Medicine; Key Laboratory of Human Diseases Animal Models, State Administration of Traditional Chinese Medicine; Beijing Key Laboratory for Animal Models of Emerging and Remerging Infectious Diseases, Beijing 100021, China
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WEI Qiang
WEI Qiang
Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences (CAMS);Comparative Medicine Center, Peking Union Medical College(PUMC);NHC Key Laboratory of Human Disease Comparative Medicine; Key Laboratory of Human Diseases Animal Models, State Administration of Traditional Chinese Medicine; Beijing Key Laboratory for Animal Models of Emerging and Remerging Infectious Diseases, Beijing 100021, China
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Affiliation:

Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences (CAMS);Comparative Medicine Center, Peking Union Medical College(PUMC);NHC Key Laboratory of Human Disease Comparative Medicine; Key Laboratory of Human Diseases Animal Models, State Administration of Traditional Chinese Medicine; Beijing Key Laboratory for Animal Models of Emerging and Remerging Infectious Diseases, Beijing 100021, China

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摘要:

目的建立微滴式数字 PCR 技术(droplet digital PCR,ddPCR)作为关键指标,实现精准检测艾滋病动物模型中细胞及组织中 SIV DNA 载量,用于储存库隐匿病毒实时动态评估。方法根据成熟的 qPCR 方法,对微滴式 dPCR 的反应条件进行了优化,确定最适退火温度;通过对 10 倍系列稀释 pGEM-SIVgag477 质粒标准品的检测,确定 ddPCR 检测范围。并通过对若干 DNA 样品的多次检测,判断该检测方法的稳定性。结果 ddPCR 的退火温度为 60℃ ;检测范围是 0. 3~ 3. 96×10⁴ copies/ μL;微滴式 dPCR 与 qPCR 有良好的相关性,相关系数 r = 0. 9981。结论本研究建立了基于微滴式 dPCR 的 SIV 病毒 DNA 的绝对定量方法,可有效的对细胞和组织中病毒储存库样本中病毒 DNA 载量进行动态绝对定量,极大提高了模型应用评价的准确度。

关键词:微滴式数字 PCR;猴免疫缺陷病毒;DNA;绝对定量

Abstract:

Objective To establish a sensitive droplet digital PCR ( ddPCR) assay for measuring the proviral DNA load in PBMCs (peripheral blood mononuclear cell) and tissues from SIV-infected animals. Methods According to the standard qPCR method , we first optimized the annealing temperature of the droplet dPCR, and then determined the detection range of the ddPCR. This was followed by correlation analysis of ddPCR and qPCR via detection of the 10-fold serially diluted pGEM-SIVgag477. We repeatedly tested 7 DNA samples extracted from PBMCs of SIV-infected monkeys to study the repeatability of the method . Results The annealing temperature of the ddPCR was determined to be 60°C and the detection range was 0. 3~ 3. 96 × 10⁴ copies/ μL. The correlation coefficient of ddPCR and qPCR was in the range of 0. 9981. Conclusions We established an absolute quantitative method for SIV DNA analysis based on droplet dPCR. This method can be used to accurately quantify the proviral DNA load and represents an effective technique for the measurement of viral reservoirs in SIV research.

Key words:droplet digital PCR; SIV; DNA; absolute quantification

引用本文

张丽丽,陆佳涵,薛婧,丛喆,魏强.艾滋病模型中关键指标 SIV DNA 绝对定量微滴式数字 PCR 技术的创新应用[J].中国比较医学杂志,2020,30(5):27~30,53.

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收稿日期:2019-11-21
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在线发布日期: 2020-06-19
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