The establishment and application of an absolute quantitative method for analyzing SIV DNA via droplet digital PCR

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

Home > Archive>Volume 30, Issue 5, 2020 >27-30,53. DOI:10. 3969 / j.issn.1671-7856. 2020. 05. 005

The establishment and application of an absolute quantitative method for analyzing SIV DNA via droplet digital PCR
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                        10. 3969 / j.issn.1671-7856. 2020. 05. 005
                    
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                        ZHANG LiliZHANG 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 JiahanLU 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 JingXUE 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 ZheCONG 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 QiangWEI 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
Clc Number:R-33
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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

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Received:November 21,2019
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Online: June 19,2020
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