Biotechnology Bulletin ›› 2021, Vol. 37 ›› Issue (6): 286-294.doi: 10.13560/j.cnki.biotech.bull.1985.2020-1587
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PAN Zhi-wen(), CHEN Wei-ting, GAO Jie-er, ZHOU Feng, YAO Juan, WANG Sheng-bin, JIANG Da-gang()
Received:
2020-12-30
Online:
2021-06-26
Published:
2021-07-08
Contact:
JIANG Da-gang
E-mail:panzhiwen@scau.edu.cn;dagangj@scau.edu.cn
PAN Zhi-wen, CHEN Wei-ting, GAO Jie-er, ZHOU Feng, YAO Juan, WANG Sheng-bin, JIANG Da-gang. Genomic DNA Rapid Preparation and PCR Detection Methods for Genetically Modified Papaya[J]. Biotechnology Bulletin, 2021, 37(6): 286-294.
检测对象 Detection targets | 引物/探针 Primers/Probes | 引物/探针序列 Primers/probes sequence(5'-3') | 引物出处 Reference |
---|---|---|---|
Papain | Papain-QF | TGCTTGACTGCGACAGAC | [12] |
Papain-QR | CTTTCTCCCTTGAGCGAC | ||
Papain-QP | FAM-AGCTACGGGTGCAATGGAGGTTACC-TAMRA | ||
NPT Ⅱ | NPT-QF | AGGATCTCGTCGTGACCCAT | [13] |
NPT-QR | GCACGAGGAAGCGGTCA | ||
NPT-QP | FAM-CACCCAGCCGGCCACAGTCGAT-TAMRA | ||
CaMV35S | P35S-F | GCTCCTACAAATGCCATCA | [14] |
P35S-R | GATAGTGGGATTGTGCGTCA | ||
P35S-QF | CGACAGTGGTCCCAAAGA | ||
P35S-QR | AAGACGTGGTTGGAACGTCTTC | ||
P35S-QP | FAM-TGGACCCCCACCCACGAGGAGCATC-TAMRA | ||
NOS | TNOS-F | GAATCCTGTTGCCGGTCTTG | [14] |
TNOS-R | TTATCCTAGTTTGCGCGCTA | ||
TNOS-QF | ATCGTTCAAACATTTGGCA | ||
TNOS-QR | ATTGCGGGACTCTAATCATA | ||
TNOS-QP | FAM-CATCGCAAGACCGGCAACAGG-TAMRA |
Table 1 Primers and probes sequence information used in transgenic papaya real-time PCR detection
检测对象 Detection targets | 引物/探针 Primers/Probes | 引物/探针序列 Primers/probes sequence(5'-3') | 引物出处 Reference |
---|---|---|---|
Papain | Papain-QF | TGCTTGACTGCGACAGAC | [12] |
Papain-QR | CTTTCTCCCTTGAGCGAC | ||
Papain-QP | FAM-AGCTACGGGTGCAATGGAGGTTACC-TAMRA | ||
NPT Ⅱ | NPT-QF | AGGATCTCGTCGTGACCCAT | [13] |
NPT-QR | GCACGAGGAAGCGGTCA | ||
NPT-QP | FAM-CACCCAGCCGGCCACAGTCGAT-TAMRA | ||
CaMV35S | P35S-F | GCTCCTACAAATGCCATCA | [14] |
P35S-R | GATAGTGGGATTGTGCGTCA | ||
P35S-QF | CGACAGTGGTCCCAAAGA | ||
P35S-QR | AAGACGTGGTTGGAACGTCTTC | ||
P35S-QP | FAM-TGGACCCCCACCCACGAGGAGCATC-TAMRA | ||
NOS | TNOS-F | GAATCCTGTTGCCGGTCTTG | [14] |
TNOS-R | TTATCCTAGTTTGCGCGCTA | ||
TNOS-QF | ATCGTTCAAACATTTGGCA | ||
TNOS-QR | ATTGCGGGACTCTAATCATA | ||
TNOS-QP | FAM-CATCGCAAGACCGGCAACAGG-TAMRA |
Fig. 2 PCR amplification of endogenous gene Papain A: Fresh leaf. B: Wilted leaf. C: Fruit. M: DL2000 marker. 1: Positive control. 2 and 3: Undiluted solution. 4 and 5: 5 times dilution. 6 and 7: 25 times dilution. 8 and 9: 125 times dilution
样品名称 Sample name | DNA浓度/稀释倍数 DNA concentration/ Dilution time | 平行1 Repeat 1 | 平行2 Repeat 2 | 平行3 Repeat 3 | Mean Ct | SD | RSD/% | 扩增效率 Amplification efficiency/% |
---|---|---|---|---|---|---|---|---|
DNA-Std1 | 25 ng/μL | 21.41 | 21.35 | 21.34 | 21.37 | 0.038 | 0.177 | 90.73 |
DNA-Std2 | 5 ng/μL | 24.47 | 24.48 | 24.38 | 24.44 | 0.055 | 0.225 | |
DNA-Std3 | 1 ng/μL | 26.72 | 26.70 | 26.72 | 26.71 | 0.012 | 0.043 | |
DNA-Std4 | 0.2 ng/μL | 29.29 | 29.34 | 29.29 | 29.31 | 0.029 | 0.099 | |
DNA-Std5 | 0.04 ng/μL | 31.70 | 31.63 | 31.57 | 31.63 | 0.065 | 0.206 | |
DNA-Std6 | 0.008 ng/μL | 34.04 | 34.18 | 34.38 | 34.20 | 0.171 | 0.500 | |
DNA-Std7 | 0.0016 ng/μL | 36.68 | 36.14 | 36.64 | 36.49 | 0.301 | 0.825 | |
鲜叶片样品 Fresh leaf sample | 原液 | 29.42 | 29.26 | 29.41 | 29.36 | 0.090 | 0.305 | 95.83 |
5倍稀释 | 31.98 | 31.75 | 31.72 | 31.82 | 0.142 | 0.447 | ||
25倍稀释 | 33.77 | 33.59 | 33.66 | 33.67 | 0.091 | 0.270 | ||
125倍稀释 | 36.63 | 37.35 | 36.20 | 36.73 | 0.581 | 1.582 | ||
干叶片样品 Wilted leaf sample | 原液 | 27.43 | 27.39 | 27.48 | 27.43 | 0.045 | 0.164 | 102.09 |
5倍稀释 | 29.49 | 29.75 | 29.62 | 29.62 | 0.130 | 0.439 | ||
25倍稀释 | 31.67 | 31.97 | 31.99 | 31.88 | 0.179 | 0.562 | ||
125倍稀释 | 34.21 | 34.60 | 34.11 | 34.31 | 0.259 | 0.755 | ||
果肉样品 Fruit sample | 原液 | 33.01 | 32.86 | 33.00 | 32.96 | 0.084 | 0.255 | 145.32 |
5倍稀释 | 34.15 | 34.24 | 34.03 | 34.14 | 0.105 | 0.309 | ||
25倍稀释 | 35.99 | 36.68 | 36.23 | 36.30 | 0.350 | 0.965 | ||
125倍稀释 | 37.22 | 39.21 | N/A | 38.22 | 1.407 | 3.682 |
Table 2 Real-time fluorescence PCR of papaya endogenous Papain gene
样品名称 Sample name | DNA浓度/稀释倍数 DNA concentration/ Dilution time | 平行1 Repeat 1 | 平行2 Repeat 2 | 平行3 Repeat 3 | Mean Ct | SD | RSD/% | 扩增效率 Amplification efficiency/% |
---|---|---|---|---|---|---|---|---|
DNA-Std1 | 25 ng/μL | 21.41 | 21.35 | 21.34 | 21.37 | 0.038 | 0.177 | 90.73 |
DNA-Std2 | 5 ng/μL | 24.47 | 24.48 | 24.38 | 24.44 | 0.055 | 0.225 | |
DNA-Std3 | 1 ng/μL | 26.72 | 26.70 | 26.72 | 26.71 | 0.012 | 0.043 | |
DNA-Std4 | 0.2 ng/μL | 29.29 | 29.34 | 29.29 | 29.31 | 0.029 | 0.099 | |
DNA-Std5 | 0.04 ng/μL | 31.70 | 31.63 | 31.57 | 31.63 | 0.065 | 0.206 | |
DNA-Std6 | 0.008 ng/μL | 34.04 | 34.18 | 34.38 | 34.20 | 0.171 | 0.500 | |
DNA-Std7 | 0.0016 ng/μL | 36.68 | 36.14 | 36.64 | 36.49 | 0.301 | 0.825 | |
鲜叶片样品 Fresh leaf sample | 原液 | 29.42 | 29.26 | 29.41 | 29.36 | 0.090 | 0.305 | 95.83 |
5倍稀释 | 31.98 | 31.75 | 31.72 | 31.82 | 0.142 | 0.447 | ||
25倍稀释 | 33.77 | 33.59 | 33.66 | 33.67 | 0.091 | 0.270 | ||
125倍稀释 | 36.63 | 37.35 | 36.20 | 36.73 | 0.581 | 1.582 | ||
干叶片样品 Wilted leaf sample | 原液 | 27.43 | 27.39 | 27.48 | 27.43 | 0.045 | 0.164 | 102.09 |
5倍稀释 | 29.49 | 29.75 | 29.62 | 29.62 | 0.130 | 0.439 | ||
25倍稀释 | 31.67 | 31.97 | 31.99 | 31.88 | 0.179 | 0.562 | ||
125倍稀释 | 34.21 | 34.60 | 34.11 | 34.31 | 0.259 | 0.755 | ||
果肉样品 Fruit sample | 原液 | 33.01 | 32.86 | 33.00 | 32.96 | 0.084 | 0.255 | 145.32 |
5倍稀释 | 34.15 | 34.24 | 34.03 | 34.14 | 0.105 | 0.309 | ||
25倍稀释 | 35.99 | 36.68 | 36.23 | 36.30 | 0.350 | 0.965 | ||
125倍稀释 | 37.22 | 39.21 | N/A | 38.22 | 1.407 | 3.682 |
Fig. 3 Real-time fluorescence PCR standard curve of Papain gene A: Purified papaya genomic DNA. B: Fresh leaf sample. C: Wilted leaf sample. D: Fruit sample
Fig. 4 PCR electrophoresis results of transgenic papaya A: Papain gene. B: CaMV35S promoter. C: NOS terminator. D: NPT II gene. M: DL2000 marker, 1: blank, 2: negative control, 3 and 4: 0.1% fresh leaf, 5 and 6: 0.1% wilted leaf, 7 and 8: 0.1% fruit, 9 and 10: 1% fresh leaf, 11 and 12: 1% wilted leaf, 13 and 14: 1% fruit, 15 and 16: 5% fresh leaf, 17 and 18: 5% wilted leaf, 19 and 20: 5% fruit, 21: 0.1% positive control, 22: 1% positive control
样品名称 Sample name | 检测参数 Detection item | 平行1 Repeat 1 | 平行2 Repeat 2 | 平行3 Repeat 3 | Mean Ct | SD | RSD |
---|---|---|---|---|---|---|---|
阴性对照 Negative control | Papain | 22.94 | 22.75 | 22.80 | 22.83 | 0.099 | 0.431% |
CaMV35S启动子 | N/A | N/A | N/A | N/A | N/A | N/A | |
NOS终止子 | N/A | N/A | N/A | N/A | N/A | N/A | |
NPT Ⅱ | N/A | N/A | N/A | N/A | N/A | N/A | |
1%阳性对照 1% positive control | Papain | 23.26 | 23.15 | 23.20 | 23.20 | 0.055 | 0.237% |
CaMV35S启动子 | 26.43 | 26.49 | 26.55 | 26.49 | 0.060 | 0.227% | |
NOS终止子 | 27.22 | 27.17 | 27.18 | 27.19 | 0.027 | 0.097% | |
NPT Ⅱ | 26.25 | 26.39 | 26.28 | 26.31 | 0.074 | 0.280% | |
5%鲜叶片样品 5% fresh leaf sample | Papain | 27.40 | 27.47 | 27.46 | 27.44 | 0.038 | 0.138% |
CaMV35S启动子 | 29.05 | 29.07 | 28.80 | 28.97 | 0.150 | 0.519% | |
NOS终止子 | 29.85 | 29.82 | 29.99 | 29.89 | 0.091 | 0.304% | |
NPT Ⅱ | 29.17 | 29.30 | 29.33 | 29.27 | 0.085 | 0.291% | |
5%干叶片样品 5% wilted leaf sample | Papain | 27.96 | 28.07 | 28.02 | 28.02 | 0.055 | 0.197% |
CaMV35S启动子 | 30.33 | 30.14 | 30.22 | 30.23 | 0.095 | 0.316% | |
NOS终止子 | 30.55 | 30.57 | 30.31 | 30.48 | 0.145 | 0.475% | |
NPT Ⅱ | 29.91 | 29.90 | 29.85 | 29.89 | 0.032 | 0.108% | |
5%果肉样品 5% fruit sample | Papain | 29.27 | 29.29 | 29.28 | 29.28 | 0.010 | 0.034% |
CaMV35S启动子 | 30.45 | 30.49 | 30.59 | 30.51 | 0.072 | 0.236% | |
NOS终止子 | 31.35 | 31.45 | 31.53 | 31.44 | 0.090 | 0.287% | |
NPT Ⅱ | 30.62 | 30.45 | 30.47 | 30.51 | 0.093 | 0.305% | |
1%鲜叶片样品 1% fresh leaf sample | Papain | 27.09 | 27.12 | 27.16 | 27.12 | 0.035 | 0.130% |
CaMV35S启动子 | 30.68 | 30.86 | 30.57 | 30.70 | 0.146 | 0.477% | |
NOS终止子 | 31.96 | 32.04 | 32.01 | 32.00 | 0.040 | 0.126% | |
NPT Ⅱ | 31.23 | 31.15 | 31.11 | 31.16 | 0.061 | 0.196% | |
1%干叶片样品 1% wilted leaf sample | Papain | 28.28 | 28.27 | 28.26 | 28.27 | 0.010 | 0.035% |
CaMV35S启动子 | 31.54 | 31.73 | 32.31 | 31.86 | 0.401 | 1.259% | |
NOS终止子 | 32.49 | 32.52 | 32.10 | 32.37 | 0.234 | 0.724% | |
NPT Ⅱ | 31.36 | 32.12 | 31.75 | 31.74 | 0.380 | 1.197% | |
1%果肉样品 1% fruit sample | Papain | 29.30 | 29.27 | 29.33 | 29.30 | 0.030 | 0.102% |
CaMV35S启动子 | 33.72 | 33.39 | 32.73 | 33.28 | 0.504 | 1.515% | |
NOS终止子 | 33.55 | 33.73 | 33.30 | 33.53 | 0.216 | 0.644% | |
NPT Ⅱ | 32.71 | 32.52 | 32.46 | 32.56 | 0.131 | 0.401% | |
0.1%鲜叶片样品 0.1% fresh leaf sample | Papain | 27.38 | 27.48 | 27.29 | 27.38 | 0.095 | 0.347% |
CaMV35S启动子 | 33.76 | 33.91 | 33.90 | 33.86 | 0.084 | 0.248% | |
NOS终止子 | 34.82 | 33.88 | 34.59 | 34.43 | 0.490 | 1.423% | |
NPT Ⅱ | 33.79 | 33.61 | 34.04 | 33.81 | 0.216 | 0.639% | |
0.1%干叶片样品 0.1% wilted leaf sample | Papain | 27.74 | 27.72 | 27.67 | 27.71 | 0.036 | 0.130% |
CaMV35S启动子 | 34.05 | 33.97 | 33.51 | 33.84 | 0.291 | 0.861% | |
NOS终止子 | 34.83 | 35.79 | 34.84 | 35.15 | 0.551 | 1.569% | |
NPT Ⅱ | 34.39 | 34.27 | 34.56 | 34.41 | 0.146 | 0.424% | |
0.1%果肉样品 0.1% fruit sample | Papain | 29.38 | 29.22 | 29.06 | 29.22 | 0.160 | 0.548% |
CaMV35S启动子 | 36.35 | 36.36 | 39.23 | 37.31 | 1.660 | 4.449% | |
NOS终止子 | 36.50 | 36.11 | 37.35 | 36.65 | 0.634 | 1.730% | |
NPT Ⅱ | 35.46 | 34.58 | 37.35 | 35.80 | 1.415 | 3.954% |
Table 3 Data and analysis of real-time fluorescence PCR of transgenic papaya
样品名称 Sample name | 检测参数 Detection item | 平行1 Repeat 1 | 平行2 Repeat 2 | 平行3 Repeat 3 | Mean Ct | SD | RSD |
---|---|---|---|---|---|---|---|
阴性对照 Negative control | Papain | 22.94 | 22.75 | 22.80 | 22.83 | 0.099 | 0.431% |
CaMV35S启动子 | N/A | N/A | N/A | N/A | N/A | N/A | |
NOS终止子 | N/A | N/A | N/A | N/A | N/A | N/A | |
NPT Ⅱ | N/A | N/A | N/A | N/A | N/A | N/A | |
1%阳性对照 1% positive control | Papain | 23.26 | 23.15 | 23.20 | 23.20 | 0.055 | 0.237% |
CaMV35S启动子 | 26.43 | 26.49 | 26.55 | 26.49 | 0.060 | 0.227% | |
NOS终止子 | 27.22 | 27.17 | 27.18 | 27.19 | 0.027 | 0.097% | |
NPT Ⅱ | 26.25 | 26.39 | 26.28 | 26.31 | 0.074 | 0.280% | |
5%鲜叶片样品 5% fresh leaf sample | Papain | 27.40 | 27.47 | 27.46 | 27.44 | 0.038 | 0.138% |
CaMV35S启动子 | 29.05 | 29.07 | 28.80 | 28.97 | 0.150 | 0.519% | |
NOS终止子 | 29.85 | 29.82 | 29.99 | 29.89 | 0.091 | 0.304% | |
NPT Ⅱ | 29.17 | 29.30 | 29.33 | 29.27 | 0.085 | 0.291% | |
5%干叶片样品 5% wilted leaf sample | Papain | 27.96 | 28.07 | 28.02 | 28.02 | 0.055 | 0.197% |
CaMV35S启动子 | 30.33 | 30.14 | 30.22 | 30.23 | 0.095 | 0.316% | |
NOS终止子 | 30.55 | 30.57 | 30.31 | 30.48 | 0.145 | 0.475% | |
NPT Ⅱ | 29.91 | 29.90 | 29.85 | 29.89 | 0.032 | 0.108% | |
5%果肉样品 5% fruit sample | Papain | 29.27 | 29.29 | 29.28 | 29.28 | 0.010 | 0.034% |
CaMV35S启动子 | 30.45 | 30.49 | 30.59 | 30.51 | 0.072 | 0.236% | |
NOS终止子 | 31.35 | 31.45 | 31.53 | 31.44 | 0.090 | 0.287% | |
NPT Ⅱ | 30.62 | 30.45 | 30.47 | 30.51 | 0.093 | 0.305% | |
1%鲜叶片样品 1% fresh leaf sample | Papain | 27.09 | 27.12 | 27.16 | 27.12 | 0.035 | 0.130% |
CaMV35S启动子 | 30.68 | 30.86 | 30.57 | 30.70 | 0.146 | 0.477% | |
NOS终止子 | 31.96 | 32.04 | 32.01 | 32.00 | 0.040 | 0.126% | |
NPT Ⅱ | 31.23 | 31.15 | 31.11 | 31.16 | 0.061 | 0.196% | |
1%干叶片样品 1% wilted leaf sample | Papain | 28.28 | 28.27 | 28.26 | 28.27 | 0.010 | 0.035% |
CaMV35S启动子 | 31.54 | 31.73 | 32.31 | 31.86 | 0.401 | 1.259% | |
NOS终止子 | 32.49 | 32.52 | 32.10 | 32.37 | 0.234 | 0.724% | |
NPT Ⅱ | 31.36 | 32.12 | 31.75 | 31.74 | 0.380 | 1.197% | |
1%果肉样品 1% fruit sample | Papain | 29.30 | 29.27 | 29.33 | 29.30 | 0.030 | 0.102% |
CaMV35S启动子 | 33.72 | 33.39 | 32.73 | 33.28 | 0.504 | 1.515% | |
NOS终止子 | 33.55 | 33.73 | 33.30 | 33.53 | 0.216 | 0.644% | |
NPT Ⅱ | 32.71 | 32.52 | 32.46 | 32.56 | 0.131 | 0.401% | |
0.1%鲜叶片样品 0.1% fresh leaf sample | Papain | 27.38 | 27.48 | 27.29 | 27.38 | 0.095 | 0.347% |
CaMV35S启动子 | 33.76 | 33.91 | 33.90 | 33.86 | 0.084 | 0.248% | |
NOS终止子 | 34.82 | 33.88 | 34.59 | 34.43 | 0.490 | 1.423% | |
NPT Ⅱ | 33.79 | 33.61 | 34.04 | 33.81 | 0.216 | 0.639% | |
0.1%干叶片样品 0.1% wilted leaf sample | Papain | 27.74 | 27.72 | 27.67 | 27.71 | 0.036 | 0.130% |
CaMV35S启动子 | 34.05 | 33.97 | 33.51 | 33.84 | 0.291 | 0.861% | |
NOS终止子 | 34.83 | 35.79 | 34.84 | 35.15 | 0.551 | 1.569% | |
NPT Ⅱ | 34.39 | 34.27 | 34.56 | 34.41 | 0.146 | 0.424% | |
0.1%果肉样品 0.1% fruit sample | Papain | 29.38 | 29.22 | 29.06 | 29.22 | 0.160 | 0.548% |
CaMV35S启动子 | 36.35 | 36.36 | 39.23 | 37.31 | 1.660 | 4.449% | |
NOS终止子 | 36.50 | 36.11 | 37.35 | 36.65 | 0.634 | 1.730% | |
NPT Ⅱ | 35.46 | 34.58 | 37.35 | 35.80 | 1.415 | 3.954% |
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