Establishment and Optimization of Droplet Digital PCR Method for African Swine Fever Virus

doi:10.11924/j.issn.1000-6850.casb2025-0088

Abstract

Abstract:

The objective of this study is to establish a droplet digital PCR (ddPCR) method for the detection of African swine fever virus. In this study, the specific primers and probes of African swine fever virus were synthesized, and the annealing temperature, primer and probe concentration and reaction conditions were optimized. The specificity, sensitivity and repeatability of the method were comprehensively evaluated, and the preliminary clinical application was carried out according to the optimized conditions. The optimal annealing temperature of the established ddPCR method was 61.4℃. The optimal final primer and probe concentrations were 700 nmol/L and 150 nmol/L, respectively; the results of this method showed that the specific amplification of ASFV was positive, and the amplification of other common epidemic viruses was negative without amplification, and there was no cross-reactivity. The lowest limit of detection of this ddPCR method was 1.37 copies/μL, which was 10-fold more sensitive than that of real-time PCR, the fit of the concentration gradient linear analysis was R²=0.9898, and the coefficient of variation of the repeatability test was less than 10%. The ddPCR method established by the application was used to detect 80 suspected ASFV clinical samples from different sources, and the results were negative, which was consistent with the results of real-time polymerase chain reaction (qPCR). The ddPCR method established in this study has high sensitivity, strong specificity and good reproducibility, and can be used for accurate quantitative analysis of ASFV laboratory detection.

Key words: droplet digital PCR (ddPCR), African swine fever, quantitative detection, number of copies

WANG Yonghao, LIU Pengxiang, SONG Shuai, CHEN Yuting, WEN Xiaohui, XIANG Guoqing, CHEN Meixia, LI Wenchao, LUO Shengjun. Establishment and Optimization of Droplet Digital PCR Method for African Swine Fever Virus[J]. Chinese Agricultural Science Bulletin, 2025, 41(30): 156-164.

Figures/Tables 10

References 34

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组别	引物和探针用量/μL			体系总体积/μL	引物终浓度/(nmol/L)	探针终浓度/(nmol/L)
组别	ASFV-F	ASFV-R	ASFV-P	体系总体积/μL	引物终浓度/(nmol/L)	探针终浓度/(nmol/L)
1	1.4	1.4	0.3	20	700	150
2	1.8	1.8	0.5		900	250
3	2.2	2.2	0.7		1100	350
4	1.4	1.4	0.5		700	250
5	2.2	2.2	0.5		1100	250

组别	引物和探针用量/μL			体系总体积/μL	引物终浓度/(nmol/L)	探针终浓度/(nmol/L)
组别	ASFV-F	ASFV-R	ASFV-P	体系总体积/μL	引物终浓度/(nmol/L)	探针终浓度/(nmol/L)
1	1.4	1.4	0.3	20	700	150
2	1.8	1.8	0.5		900	250
3	2.2	2.2	0.7		1100	350
4	1.4	1.4	0.5		700	250
5	2.2	2.2	0.5		1100	250

检测方法	稀释倍数
检测方法	10¹	10²	10³	10⁴	10⁵	10⁶	10⁷	10⁸
ddPCR	＋	＋	＋	＋	＋	＋	＋	－
qPCR	＋	＋	＋	＋	＋	＋	－	－

检测方法	稀释倍数
检测方法	10¹	10²	10³	10⁴	10⁵	10⁶	10⁷	10⁸
ddPCR	＋	＋	＋	＋	＋	＋	＋	－
qPCR	＋	＋	＋	＋	＋	＋	－	－

样品稀释倍数	批内重复试验			批间重复试验
样品稀释倍数	平均数	标准偏差	CV/%	平均数	标准偏差	CV/%
10²	579.38	20.11	3.47	558.88	22.14	3.96
10³	71.63	4.35	6.07	71.25	4.17	5.85
10⁵	1.51	0.12	7.83	1.57	0.15	9.83