Biotechnology Bulletin ›› 2024, Vol. 40 ›› Issue (2): 80-89.doi: 10.13560/j.cnki.biotech.bull.1985.2023-0758
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ZHENG Qiao1,2(), LIN Hua1, XU Hao3, AN Wei1, XUE Chang-hua1, ZHANG Jing1(), HAN Guo-quan2()
Received:
2023-08-09
Online:
2024-02-26
Published:
2024-03-13
Contact:
ZHANG Jing, HAN Guo-quan
E-mail:zq950222@163.com;zjeane@163.com;hans_980306@sicau.edu.cn
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.
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 |
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 |
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
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
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
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/% | ||
---|---|---|---|---|---|---|---|
Repeat 1 | Repeat 2 | Repeat 3 | |||||
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 | / |
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/% | ||
---|---|---|---|---|---|---|---|
Repeat 1 | Repeat 2 | Repeat 3 | |||||
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 | / |
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
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
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 |
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 |
样品名称 No. of samples | qPCR 检测结果(ct值)qPCR results | ||
---|---|---|---|
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 |
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 | ||
---|---|---|---|
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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