SARS-CoV-2 Omicron 变异株多重微滴式数字 PCR 定量方法的建立及应用

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

摘要/Abstract

摘要：

【目的】为建立精准、高效的多重微滴式数字 PCR（droplet digital PCR，ddPCR）定量分析检测方法，开发可同时鉴别新型冠状病毒（severe acute respiratory syndrome coronavirus 2，SARS-CoV-2）ORF1ab 基因、N 基因、E 基因以及 Omicron 变异株 S 基因的检测体系，提高病毒性传染疾病的诊断效率及传播风险监测能力。【方法】通过筛选保守基因序列并设计特异性引物与探针，优化反应体系和扩增程序，对该方法的特异性、灵敏度及稳定性进行评价。以临床样本为实验材料，利用建立的 ddPCR 方法进行检测和验证，确定阳性检出率。【结果】多重 ddPCR 反应体系中，各对引物探针对目的片段均能有效扩增，SARS-CoV-2 ORF1ab 基因、N 基因、E 基因以及 Omicron 变异株 S 基因灵敏度检测下限分别为0.59、0.68、1.44、1.03 copies/µL。在20份临床样本的核酸检测中，共检出16份阳性样本，阳性率达80%（16/20），经荧光定量 PCR 方法复测符合率一致。【结论】研究建立的多重 ddPCR 方法特异性强、灵敏度高，可实现对临床样本中微量新冠病毒的精确定量检测。

关键词: 微滴式数字 PCR, 新型冠状病毒, Omicron 变异株, 定量

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

郑巧, 林华, 徐浩, 安微, 薛昌华, 张婧, 韩国全. SARS-CoV-2 Omicron 变异株多重微滴式数字 PCR 定量方法的建立及应用[J]. 生物技术通报, 2024, 40(2): 80-89.

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.

图/表 9

表1 引物与探针序列

Table 1 Primer and probe sequences

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

图1 SARS-CoV-2 ORF1ab 基因、N 基因、E 基因、S 基因扩增产物电泳图 M：DL2000；1：ORF1ab 基因扩增产物；2：N 基因扩增产物；3：E 基因扩增产物；4：S 基因扩增产物

Fig. 1 Electrophoresis map of SARS-CoV-2 ORF1ab, N, E, and S gene amplification products M: DL2000; 1: ORF1ab gene amplified product; 2: N gene amplified products; 3: E gene amplified products; 4: S gene amplified product

图2 SARS-CoV-2 ORF1ab 基因、N 基因、E 基因、S 基因单重ddPCR 检测一维图 A：ORF1ab 阳性质粒标准品，蓝色液滴；B：N 基因阳性质粒标准品，红色液滴；C：E 基因阳性质粒标准品，绿色液滴；D：S 基因阳性质粒标准品，黄色液滴；空白对照，灰色液滴

Fig. 2 One-dimensional map of SARS-CoV-2 ORF1ab gene, N gene, E gene and S gene by single ddPCR A: ORF1ab positive plasmid standard, blue droplets; B: N gene positive plasmid standard, red droplet; C: E gene positive plasmid standard, green droplet; D: S gene positive plasmid standard, yellow droplet; blank control, grey droplets

图3 四重ddPCR 特异性检测一维图 1F：Beta株；1G：Gamma株；1H：Delta株；2A：SARS-CoV-2 阳性质粒标准品；2B：猪流行性腹泻病毒；2C：猪传染性胃肠炎病毒；2D：甲型流感病毒；2E：诺如病毒；2F：腺病毒；2G：轮状病毒；2H：空白对照

Fig. 3 One-dimensional map of specificity detected by quadruple ddPCR 1F: Beta strain; 1G: Gamma strain; 1H: Delta strain; 2A: SARS-CoV-2 positive plasmid standard; 2B: porcine epidemic diarrhea virus; 2C: transmissible gastroenteritis of swine virus; 2D: influenza A virus; 2E: norovirus; 2F: adenovirus; 2G: rotavirus; 2H: blank control

表2 四重 ddPCR 灵敏度与稳定性检测结果

Table 2 Sensitivity and stability detection results by quadruple ddPCR

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	/

图4 四重 ddPCR 灵敏度检测一维图 A：FAM 通道，ORF1ab 基因检测一维图；B：HEX 通道，N 基因检测一维图；C：ROX 通道，E 基因检测一维图；D：CY5 通道，S 基因检测一维图

Fig. 4 One-dimensional map of sensitivity detected by quadruple ddPCR A: FAM channel, one-dimensional map of ORF1ab gene detection; B: HEX channel, one-dimensional map of N-gene detection; C: ROX channel, one-dimensional map of E gene detection; D: CY5 channel, one-dimensional map of S gene detection

图5 四重 ddPCR 灵敏度检测线性关系拟合曲线图 A：ORF1ab 基因灵敏度检测线性关系拟合曲线图；B：N 基因灵敏度检测线性关系拟合曲线图；C：E 基因灵敏度检测线性关系拟合曲线图；D：S 基因灵敏度检测线性关系拟合曲线图

Fig. 5 Fitting curve of linear relationship for quadruple ddPCR sensitivity detection A: The fitting curve of the linear relationship of the sensitivity detection of ORF1ab gene. B: The fitting curve of the linear relationship of the sensitivity detection of N gene.C: The fitting curve of the linear relationship of the sensitivity detection of E gene. D: The fitting curve of the linear relationship of the sensitivity detection of S gene

表3 20份样本多重微滴数字 PCR 拷贝数检测结果

Table 3 Copy number detection results for 20 samples by quadruple ddPCR

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

表4 20份样本荧光定量 PCR 检测结果

Table 4 Results of 20 samples via fluorescence quantitative PCR

样品名称 No. of samples	qPCR 检测结果（ct值）qPCR results
样品名称 No. of samples	FAM（ORF1ab）	HEX（N）	ROX（E）
样本1	29.63	25.62	28.91
样本2	32.06	28.29	31.70
样本3	33.06	29.55	32.73
样本4	NA	NA	NA
样本5	30.63	26.95	29.49
样本6	31.61	28.17	30.55
样本7	32.75	29.41	31.78
样本8	27.36	25.29	25.46
样本9	NA	NA	NA
样本10	NA	NA	NA
样本11	NA	NA	NA
样本12	31.45	30.18	30.23
样本13	32.18	30.89	30.75
样本14	34.35	33.91	35.34
样本15	28.47	26.79	26.82
样本16	29.86	28.24	28.2
样本17	30.83	29.40	29.42
样本18	30.83	29.23	31.17
样本19	33.00	31.68	33.53
样本20	28.29	24.97	28.78

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