Establishment and Application of Multiplex Droplet Digital PCR for SARS-CoV-2 Omicron Variant

doi:10.13560/j.cnki.biotech.bull.1985.2023-0758

Abstract

Abstract:

【Objective】 This research is aimed to establish an accurate and efficient multiple droplet digital PCR（ddPCR）quantitative analysis detection method, and to develop a detection system that can simultaneously identify severe acute respiratory syndrome coronavirus（SARS-CoV-2）ORF1ab gene, N gene, E gene, and Omicron variant S gene, so as to improve the diagnostic efficiency of viral infectious diseases and the ability to monitor the risk of transmission. 【Method】 Based on it, conserved gene sequences were screened, specific primers and probes were designed, reaction systems and amplification procedures were optimized, and the specificity, sensitivity and stability of the method were evaluated. With clinical samples as experimental materials, the established ddPCR method was applied for testing and verification to determine the positive detection rate.【Result】 Each pair of primers effectively amplified the target fragment in the multiple ddPCR reaction system. The lower detection limits for SARS-CoV-2 ORF1ab gene, N gene, E gene, and Omicron variant S gene were 0.59, 0.68, 1.44 and 1.03 copies/µL, respectively. In the nucleic acid tests of 20 clinical samples, a total of 16 positive samples were detected, with a positive rate of 80%（16/20）. The coincidence rate was consistent with that of retesting by fluorescence quantitative PCR method. 【Conclusion】 The multiple ddPCR method established in this study is highly specific and sensitive, and can be used to accurately quantify trace amounts of SARS-CoV-2 in clinical samples.

Key words: droplet digital PCR, SARS-CoV-2, omicron variant, quantitative

ZHENG Qiao, LIN Hua, XU Hao, AN Wei, XUE Chang-hua, ZHANG Jing, HAN Guo-quan. Establishment and Application of Multiplex Droplet Digital PCR for SARS-CoV-2 Omicron Variant[J]. Biotechnology Bulletin, 2024, 40(2): 80-89.

Figures/Tables 9

References 25

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引物与探针Primer and probe	序列Sequence（5'-3'）	产物长度Product length/bp
ORF1ab-F	TGTGCTAATGACCCTGT	138
ORF1ab-R	GTTTAAAAACGATTGTGCATCAG
ORF1ab-P	FAM-AACTCCGCGAACCCATGCTT-BHQ1
N-F	GTCAAGCCTCTTCTCGTT	101
N-R	ATTGCCAGCCATTCTAGCAG
N-P	HEX-CCTCATCACGTAGTCGCAACAGT-BHQ1
E-F	TTTCTTGCTTTCGTGGT	132
E-R	TTTAACACGAGAGTAAACGTA
E-P	ROX-CTTCGATTGTGTGCGTACTGCT-BHQ2
S-F	GTTGCTGATTATTCTGTCCTA	103
S-R	AGACATTAGTAAAGCAGAGAT
S-P	CY5-ACACTTAAAAGCGAAAAATGGT-MGB

引物与探针Primer and probe	序列Sequence（5'-3'）	产物长度Product length/bp
ORF1ab-F	TGTGCTAATGACCCTGT	138
ORF1ab-R	GTTTAAAAACGATTGTGCATCAG
ORF1ab-P	FAM-AACTCCGCGAACCCATGCTT-BHQ1
N-F	GTCAAGCCTCTTCTCGTT	101
N-R	ATTGCCAGCCATTCTAGCAG
N-P	HEX-CCTCATCACGTAGTCGCAACAGT-BHQ1
E-F	TTTCTTGCTTTCGTGGT	132
E-R	TTTAACACGAGAGTAAACGTA
E-P	ROX-CTTCGATTGTGTGCGTACTGCT-BHQ2
S-F	GTTGCTGATTATTCTGTCCTA	103
S-R	AGACATTAGTAAAGCAGAGAT
S-P	CY5-ACACTTAAAAGCGAAAAATGGT-MGB

基因名称 Gene name	稀释度 Dilution	ddPCR 检测结果Results of ddPCR/（Cp·μL^-1）			平均值AVG	标准差SD	相对标准偏差RSD/%
基因名称 Gene name	稀释度 Dilution	Repeat 1	Repeat 2	Repeat 3	平均值AVG	标准差SD	相对标准偏差RSD/%
ORF1ab	10^-6	11230.63	11200.05	11315.73	11248.80	48.94	0.44
	10^-7	1163.21	1149.84	1172.26	1161.77	9.21	0.79
	10^-8	96.43	88.66	89.98	91.69	3.39	3.70
	10^-9	8.83	6.57	7.33	7.58	0.94	12.39
	10^-10	0.74	0.44	0.58	0.59	0.12	20.89
	10^-11	0	0	0	0	0	/
N	10^-6	6133.75	6226.00	6081.97	6147.21	59.57	0.97
	10^-7	617.10	621.28	598.72	612.37	9.80	1.60
	10^-8	51.33	57.58	52.90	53.94	2.62	4.92
	10^-9	5.33	5.69	4.40	5.14	0.54	10.57
	10^-10	0.82	0.78	0.44	0.68	0.17	25.07
	10^-11	0	0	0	0	0	/
E	10^-6	4350.43	4378.88	4334.00	4387.77	47.95	1.09
	10^-7	461.63	443.83	441.59	449.02	8.97	2.00
	10^-8	45.01	50.60	43.13	46.25	3.17	6.86
	10^-9	4.38	4.87	4.03	4.43	0.34	7.78
	10^-10	1.32	1.69	1.32	1.44	0.17	12.08
	10^-11	0	0.44	0	0.15	0.21	141.42
S	10^-6	5891.09	5839.83	5942.93	5891.28	42.09	0.71
	10^-7	591.21	558.87	593.71	581.26	15.87	2.73
	10^-8	56.59	55.57	60.52	57.56	2.13	3.71
	10^-9	5.06	5.87	5.92	5.62	0.39	7.02
	10^-10	0.88	0.88	1.32	1.03	0.21	20.20
	10^-11	0	0	0	0	0	/

基因名称 Gene name	稀释度 Dilution	ddPCR 检测结果Results of ddPCR/（Cp·μL^-1）			平均值AVG	标准差SD	相对标准偏差RSD/%
基因名称 Gene name	稀释度 Dilution	Repeat 1	Repeat 2	Repeat 3	平均值AVG	标准差SD	相对标准偏差RSD/%
ORF1ab	10^-6	11230.63	11200.05	11315.73	11248.80	48.94	0.44
	10^-7	1163.21	1149.84	1172.26	1161.77	9.21	0.79
	10^-8	96.43	88.66	89.98	91.69	3.39	3.70
	10^-9	8.83	6.57	7.33	7.58	0.94	12.39
	10^-10	0.74	0.44	0.58	0.59	0.12	20.89
	10^-11	0	0	0	0	0	/
N	10^-6	6133.75	6226.00	6081.97	6147.21	59.57	0.97
	10^-7	617.10	621.28	598.72	612.37	9.80	1.60
	10^-8	51.33	57.58	52.90	53.94	2.62	4.92
	10^-9	5.33	5.69	4.40	5.14	0.54	10.57
	10^-10	0.82	0.78	0.44	0.68	0.17	25.07
	10^-11	0	0	0	0	0	/
E	10^-6	4350.43	4378.88	4334.00	4387.77	47.95	1.09
	10^-7	461.63	443.83	441.59	449.02	8.97	2.00
	10^-8	45.01	50.60	43.13	46.25	3.17	6.86
	10^-9	4.38	4.87	4.03	4.43	0.34	7.78
	10^-10	1.32	1.69	1.32	1.44	0.17	12.08
	10^-11	0	0.44	0	0.15	0.21	141.42
S	10^-6	5891.09	5839.83	5942.93	5891.28	42.09	0.71
	10^-7	591.21	558.87	593.71	581.26	15.87	2.73
	10^-8	56.59	55.57	60.52	57.56	2.13	3.71
	10^-9	5.06	5.87	5.92	5.62	0.39	7.02
	10^-10	0.88	0.88	1.32	1.03	0.21	20.20
	10^-11	0	0	0	0	0	/

样品名称No. of samples	总微滴数Total number of droplets	ORF1ab/（Cp·μL^-1）	N/（Cp·μL^-1）	E/（Cp·μL^-1）	S/（Cp·μL^-1）
样本1	20891	47.96	7480.95	335.57	99.29
样本2	21153	9.97	887.71	17.38	4.33
样本3	21372	4.70	219.61	9.71	6.45
样本4	20833	0.00	0.00	0.00	0.00
样本5	20536	28.03	6458.03	276.39	138.53
样本6	21393	14.59	912.05	21.85	11.59
样本7	21106	9.09	234.45	13.01	2.64
样本8	20712	248.53	7949.99	399.37	305.53
样本9	20793	0.00	0.00	0.00	0.00
样本10	21397	0.00	0.00	0.00	0.00
样本11	21368	0.00	0.00	0.00	0.00
样本12	21090	8.73	143.22	24.77	6.09
样本13	20675	5.57	112.42	19.40	2.20
样本14	20874	3.96	24.64	2.24	1.49
样本15	21704	93.39	6887.53	378.12	84.85
样本16	20380	38.92	780.27	357.50	21.19
样本17	21228	26.01	226.01	285.21	9.09
样本18	21104	29.11	269.19	14.39	24.35
样本19	20930	4.08	62.85	5.20	1.76
样本20	20678	118.65	10636.56	342.67	113.30