Research on AAV9 Infectious Titer Detection Method Based on TCID<sub>50</sub>

doi:10.13523/j.cb.2106006

China Biotechnology

2021, Vol. 41

Issue (10): 28-32 DOI: 10.13523/j.cb.2106006

Research on AAV9 Infectious Titer Detection Method Based on TCID₅₀

WANG Cong^1,²,LI Xiu²,NIU Miao²,DAI Yang-guang²,DONG Zhe-yue²,DONG Xiao-yan²,YU Shuang-qing^2,^*(

),YANG Yi-shu^1,^*(

)

1 Beijing University of Technology, Beijing 100124,China
2 Beijing FivePlus Gene Technology Co., Ltd., Beijing 102629, China

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Abstract

Objective: To establish a method for detecting the infectious titers of adeno-associated virus type 9 (AAV9) vector products based on the median tissue culture infective dose (TCID₅₀). Methods: HEK-293 cells were co-infected with HSV1 containing rep and cap genes of AAV2 and gradient diluted AAV9 vector products. After 48 hours of culture, the AAV-specific inverted terminal repeats (ITR) were amplified by quantitative real-time PCR(qPCR), and the infectivity titer of the sample was calculated by the Kärber method based on the number of positive and negative infected wells. Results: The AAV9 vector product carrying the enhanced green fluorescent protein reporter gene was used to determine the optimal multiplicity of infection (MOI) of the helper virus HSV1-rc as 5. The infectious titer of AAV9-101 is 1.6×10⁹ TCID₅₀/mL. Conclusion: This method can detect the infectious titer of AAV9 vector products with repeatability.

Key words： Adeno-associated virus type 9 vector Infectious titer Medina tissue culture infective dose

Received: 03 June 2021 Published: 08 November 2021

ZTFLH:

Q819

Corresponding Authors: Shuang-qing YU,Yi-shu YANG E-mail: yushuangqing@five-plus.com.cn;yishu-y@bjut.edu.cn

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	Cong WANG
	Xiu LI
	Miao NIU
	Yang-guang DAI
	Zhe-yue DONG
	Xiao-yan DONG
	Shuang-qing YU
	Yi-shu YANG

Cite this article:

WANG Cong,LI Xiu,NIU Miao,DAI Yang-guang,DONG Zhe-yue,DONG Xiao-yan,YU Shuang-qing,YANG Yi-shu. Research on AAV9 Infectious Titer Detection Method Based on TCID₅₀. China Biotechnology, 2021, 41(10): 28-32.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2106006 OR https://manu60.magtech.com.cn/biotech/Y2021/V41/I10/28

Fig.1 PCR products amplified with rep or cap primers M: The molecular weight standard;1 and 3: HSV1-rc DNA used as template;2 and 4: Water used as template

Fig.2 Expression of AAV9-EGFP at different MOI of HSV1-rc

Table 1 C_T value of AAV9-101 detected by qPCR


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