转基因番木瓜基因组DNA的快速制备和PCR检测方法

doi:10.13560/j.cnki.biotech.bull.1985.2020-1587

摘要/Abstract

摘要：

转基因番木瓜检测中,模板DNA的制备是关键一步。为了提高检测效率,建立快速制备番木瓜样品基因组DNA和PCR检测方法十分重要。建立了番木瓜基因组DNA的快速制备方法,包括样品准备、制备缓冲液匀浆和稀释上清。在完成不同番木瓜样品的基因组DNA快速制备后,对获得的DNA进行PCR检测验证。普通PCR验证结果显示,本方法制备的叶片和果肉基因组DNA溶液稀释5倍时,PCR扩增条带清晰,更高稀释倍数时,叶片基因组DNA扩增条带强于果肉基因组DNA;实时荧光PCR验证结果显示,叶片基因组DNA的PCR扩增效率位于合理区间。灵敏度测试结果表明,含量低至0.1%的叶片和果肉样品,内标准基因和外源基因特异性片段普通PCR和实时荧光PCR均能得到预期扩增,且重复性较好。本研究建立的番木瓜基因组DNA快速制备与PCR方法效果良好,体系稳定。

关键词: 转基因番木瓜, DNA制备, PCR, 检测方法

Abstract:

The preparation of genomic DNA template is the key step in the detection of genetically modified papaya. In order to improve detection efficiency,it is very important to establish rapid genomic DNA preparation and PCR detection methods for papaya samples. In this study,a rapid preparation method of papaya genomic DNA was developed,including sample preparation,homogenisation with preparation buffer and supernatant dilution. After the rapid preparation of genomic DNA solutions from different papaya samples,the obtained genomic DNA solutions were verified by PCR method. The results of qualitative PCR showed that the amplified bands were clear when the genomic DNA solutions of the leaves and fruits were diluted for 5 times. The PCR amplification results of leaf genomic DNA were better than that of the fruits when the DNA solution were diluted for 25 times. The PCR amplification efficiencies of leaf genomic DNA were reasonable in the real-time fluorescence PCR reaction. Sensitivity testing results indicated the specific fragments of endogenous reference and exogenous genes were amplified as expected by both qualitative and real-time fluorescence PCR methods when the contents in the leaves and fruits samples was 0.1%. The rapid preparation and PCR detection methods of papaya genomic DNA established here is efficient and stable.

Key words: genetically modified papaya, DNA preparation, PCR, detection method

潘志文, 陈伟庭, 高洁儿, 周峰, 姚涓, 王声斌, 姜大刚. 转基因番木瓜基因组DNA的快速制备和PCR检测方法[J]. 生物技术通报, 2021, 37(6): 286-294.

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.

图/表 7

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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

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

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

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

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%