Highly Sensitive Droplet Digital PCR for Detection of Low-level <i>JAK2V617F</i> Mutation

doi:10.13523/j.cb.2310057

China Biotechnology

2023, Vol. 43

Issue (12): 139-144 DOI: 10.13523/j.cb.2310057

Highly Sensitive Droplet Digital PCR for Detection of Low-level JAK2V617F Mutation

LONG Ling-yu¹,ZHOU Ya-lan^1,^**(

),JIAO Yao¹,YE Lin-hai²,LI Xiu-chun²,LIU Yan-rong¹,HUANG Xiao-jun^1,³,RUAN Guo-rui^1,^**(

)

1 Peking University People’s Hospital, Peking University Institute of Hematology, National Clinical Research Center for Hematologic Disease, Beijing Key Laboratory of Hematopoietic Stem Cell Transplantation, Peking University, Beijing 100044,China
2 Sniper Medical Technology Co., Ltd., Suzhou 215000,China
3 Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100084,China

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Abstract

Objective: To establish a droplet digital PCR (ddPCR) method for detecting the low-level JAK2V617F mutation and to explore its application value in the diagnosis of myeloproliferative neoplasm (MPN). Methods: Site-specific TaqMan-MGB probes were used to establish the ddPCR method for detecting the JAK2V617F mutation in genomic DNA. JAK2V617F mutations were detected by ddPCR, real-time quantitative PCR and next-generation sequencing (NGS). The consistency of the detection results of different methods was compared. Results: The ddPCR method for detecting the JAK2V617F mutation based on specific TaqMan-MGB probes was successfully established, and the detection sensitivity was at least 0.05%. The ddPCR method and qPCR method were used to detect the JAK2V617F mutation in 82 subjects with MPN and 8 healthy controls. The consistency rate was 96.7%. For the 52 samples with positive results by both methods, the correlation coefficient of quantitative data was 0.971 4 (P<0.000 1). Eleven samples were verified by NGS and the correlation coefficient between the two quantitative results was 0.983 9 (P<0.000 1). Conclusions: The ddPCR with the specific TaqMan-MGB probe method can detect the JAK2V617F mutation conveniently and accurately, which may have potential application value in the screening and dynamic monitoring of the JAK2V617F mutation.

Key words： Myeloproliferative neoplasms (MPN) JAK2V617F mutation Droplet digital PCR (ddPCR)

Received: 10 October 2023 Published: 16 January 2024

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	Ling-yu LONG
	Ya-lan ZHOU
	Yao JIAO
	Lin-hai YE
	Xiu-chun LI
	Yan-rong LIU
	Xiao-jun HUANG
	Guo-rui RUAN

Cite this article:

Ling-yu LONG, Ya-lan ZHOU, Yao JIAO, Lin-hai YE, Xiu-chun LI, Yan-rong LIU, Xiao-jun HUANG, Guo-rui RUAN. Highly Sensitive Droplet Digital PCR for Detection of Low-level JAK2V617F Mutation. China Biotechnology, 2023, 43(12): 139-144.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2310057 OR https://manu60.magtech.com.cn/biotech/Y2023/V43/I12/139

Table 1 The results of ddPCR for detecting positive standards and negative controls

Fig.1 Correlation test and sensitivity determination of ddPCR method for detecting JAK2V617F mutation

Fig.2 Reprehensive results of JAK2V617F mutation detected by ddPCR A.Blank control B.Negative control ( K562 cell line DNA ) C and D were the results of 1 JAK2V617F mutation negative clinical sample and 1 JAK2V617F mutation positive clinical sample, respectively

Fig.3 Correlation analysis of quantitative results of JAK2V617F mutation detected by ddPCR and real-time quantitative PCR

Fig.4 Correlation analysis of quantitative results of JAK2V617F mutation detected by ddPCR and NGS


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