不同玉米转化体通用PCR检测体系建立

doi:10.13560/j.cnki.biotech.bull.1985.2024-0361

摘要/Abstract

摘要：

【目的】为解决不同转化体扩增体系和反应条件不统一问题，建立通用PCR检测体系，进而提升转化体鉴定效率。【方法】通过收集国内外转基因材料的转化体特异性PCR鉴定方法，比较其扩增体系和反应条件的差异，选择其中使用频率最高的参数作为通用参数。利用不同玉米转基因材料，验证转化体特异性通用PCR定性检测方法。【结果】建立通用普通PCR扩增体系：总体积25.0 µL、25 mmol/L MgCl₂溶液1.5 µL、2.5 mmol/L dNTPs混合溶液2.0 µL、靶标和内参上下游引物终浓度0.4 µmol/L、Taq DNA聚合酶0.025 U/µL、25 mg/L DNA模板2.0 µL，LOD 0.1%；反应条件：94℃预变性5 min、94℃变性30 s、58℃退火30 s，72℃延伸30 s、共进行35次循环、72℃终延伸7 min。通用实时荧光PCR定性鉴定扩增体系：总体积20.0 µL、25 mmol/L MgCl₂溶液2.0 µL、dNTPs混合溶液（各2.5 mmol/L）1.6 µL、靶标和内参上下游引物和探针终浓度0.4 µmol/L、Taq DNA聚合酶0.04 U/µL、25 mg/L DNA模板2.5 µL，LOD 0.1%；反应条件为95℃预变性5 min、95℃起始变性15 s、60℃退火延伸60 s、40个循环。【结论】不同转化体材料可以利用本研究建立的通用PCR扩增体系和反应条件进行检测。

关键词: 转化体, 聚合酶链式反应, 扩增体系, 反应条件, 玉米

Abstract:

【Objective】Establishing a universal PCR detection system would address the issue of inconsistent amplification systems and reaction conditions of different events, thereby enhancing the efficiency of event detection. 【Method】 In this study, we analyzed and compared the differences in amplification systems and reaction conditions of qualitative and quantitative PCR detection methods for the specificity of transgenic material events. The most widely used PCR systems and conditions parameters were selected as unified parameters to validate the PCR qualitative detection methods for different maize events in the laboratory. 【Result】 General PCR amplification system: Total volume 25.0 μL, 25 mmol/L MgCl₂ solution 1.5 μL, 2.5 mmol/L dNTPs mixed solution 2.0 μL, final concentration of upstream and downstream primer 0.4 μmol/L, Taq DNA polymerase 0.025 U/μL, 25 mg/L DNA template 2.0 μL, LOD 0.1%. Reaction conditions: Denaturation at 94℃ for 5 min, 35 cycles of denaturation at 94℃ for 30 s, annealing at 58℃ for 30 s, extension at 72℃ for 30 s, and final extension at 72℃ for 7 min. Universal real-time fluorescent qualitative PCR amplification system: Total volume 20.0 μL, 25 mmol/L MgCl₂ solution 2.0 μL, dNTPs mixed solution（2.5 mmol/L each）1.6 μL, final concentration of upstream and downstream primers and probe 0.4 μmol/L, Taq DNA polymerase 0.04 U/μL, 25 mg/L DNA template 2.5 μL, LOD 0.1%. Reaction conditions: denaturation at 95℃ for 5 min; 95℃ denaturation 15 s, 60℃ annealing extension 60 s, 40 cycles.【Conclusion】This system and its associated amplification system and reaction conditions can be used to detect different event materials.

Key words: event, polymerase chain reaction（PCR）, amplification system, reaction conditions, Zea mays L.

王晶, 张晓磊, 白玉, 盛宇欣, 关海涛, 温洪涛. 不同玉米转化体通用PCR检测体系建立[J]. 生物技术通报, 2024, 40(12): 34-44.

WANG Jing, ZHANG Xiao-lei, BAI Yu, SHENG Yu-xin, GUAN Hai-tao, WEN Hong-tao. Establishment of a Universal PCR Detection System for Different Maize Events[J]. Biotechnology Bulletin, 2024, 40(12): 34-44.

图/表 7

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物种 Species	转化体 Events	检出限 LOD	25 mmol/L氯化镁25 mmol/L MgCl₂/µL	10 µmol/L脱氧核糖核苷三磷酸 10 μmol/L dNTP /µL	引物终浓度 Final concentration of primers /（µmol·L^-1）	DNA聚合酶 DNA polymerase /（U·µL^-1）	25 mg/L模板 25 mg/L template /µL	终体积 Final volume（ddH₂O）/µL
玉米Maize（Zea mays L.）	IE09S034, 12-5	0.10%	1.5	0.2	0.2	0.025	4	25
	5307, C0010.3.7, GH5112E-117C, Bt506	0.10%	1.5	0.2	0.2	0.025	2	25
	DBN9858, MON87427, 4114, DBN9936, VCO-Ø1981-5	0.10%	1.5	0.2	0.4	0.025	2	25
	C0010.1.3	0.10%	1.5	0.2	0.6	0.025	2	25
	MON88017	-	2.5	0.2	0.2	0.025	2	25
	59122, BVLA430101, BVLA430101, MON89034	-	1.5	0.2	0.4	0.025	2	25
	TC1507, MON863, GA21, NK603, T25, BT10, CBH351	-	2.5	0.2	0.5	0.025	1	50（31.7）
	MIR604	-	2.5	0.2	0.5	0.025	2	25
	MON87460, DAS-40278-9, BT11	1 g/kg	1.5	0.2	0.2	0.025	2	25
	3272, BT176, MON810	1 g/kg	1.5	0.2	0.4	0.025	2	25
	MIR162	1 g/kg	1.5	0.2	0.5	0.025	2	25
	CC-2	20拷贝	1.5	0.2	0.2	0.025	2	25
	G1105E-823C	20拷贝	1.5	0.2	0.4	0.025	2	25
	C0010.3.1	39拷贝	1.5	0.2	0.6	0.025	4	25
	C0030.2.4, C0030.2.5	40拷贝	1.5	0.25	0.3	0.025	4	25
大豆Soybean（Glycine max L.）	MON89788, A2704-12	-	1.5	0.2	0.2	0.025	2	25
	SHZD32-1	0.10%	1.5	0.2	0.2	0.025	2	25
	MON87701, SYHT0H2	0.10%	1.5	0.2	0.4	0.025	2	25
	MON87708	0.10%	1.5	0.2	0.5	0.025	2	25
	DAS68416-4	0.10%	1.5	0.2	0.8	0.025	2	25
	DAS-444Ø6-6	-	1.5	0.2	0.2	0.050	2	25
	A5547-127	-	2.5	0.2	0.4	0.050	2	25
	DP356043, DP305423, FG72, CV127	-	1.5	0.2	0.4	0.025	2	25
	GTS40-3-2	1 g/kg	2.0	0.2	0.4	0.025	1	25
	MON87705, MON87769	1 g/kg	1.5	0.2	0.4	0.025	2	25
棉花Cotton（Gossypium hirsutum L.）	LLcotton25, COT102	-	1.5	0.2	0.4	0.025	2	25
	GHB614	-	1.5	0.2	0.2	0.025	2	25
	Bt, MON88913, MON1598, MON1445	-	2.5	0.2	0.4	0.05	2	25
油菜Rape（Brassica napus L.）	MON88302	-	1.5	0.2	0.2	0.025	2	50
	73496	0.05%	1.5	0.2	0.6	0.025	4	25
	Topas19/2	-	2.5	0.2	0.25	0.05	1	50
	RF1, T45, Oxy-235	-	2.5	0.2	0.5	0.025	1	50
	RF3, RF2, GT73, MS1	-	2.5	0.2	0.5	0.05	1	50
使用频率最高的扩增体系 The most frequently used amplification system			1.5	0.2	0.4	0.025	2	25
使用频率最高的扩增体系占比 The percentage of amplification systems with the highest frequency of use			47/70	68/70	29/70	59/70	48/70	54/70

物种 Species	转化体 Events	检出限 LOD	25 mmol/L氯化镁25 mmol/L MgCl₂/µL	10 µmol/L脱氧核糖核苷三磷酸 10 μmol/L dNTP /µL	引物终浓度 Final concentration of primers /（µmol·L^-1）	DNA聚合酶 DNA polymerase /（U·µL^-1）	25 mg/L模板 25 mg/L template /µL	终体积 Final volume（ddH₂O）/µL
玉米Maize（Zea mays L.）	IE09S034, 12-5	0.10%	1.5	0.2	0.2	0.025	4	25
	5307, C0010.3.7, GH5112E-117C, Bt506	0.10%	1.5	0.2	0.2	0.025	2	25
	DBN9858, MON87427, 4114, DBN9936, VCO-Ø1981-5	0.10%	1.5	0.2	0.4	0.025	2	25
	C0010.1.3	0.10%	1.5	0.2	0.6	0.025	2	25
	MON88017	-	2.5	0.2	0.2	0.025	2	25
	59122, BVLA430101, BVLA430101, MON89034	-	1.5	0.2	0.4	0.025	2	25
	TC1507, MON863, GA21, NK603, T25, BT10, CBH351	-	2.5	0.2	0.5	0.025	1	50（31.7）
	MIR604	-	2.5	0.2	0.5	0.025	2	25
	MON87460, DAS-40278-9, BT11	1 g/kg	1.5	0.2	0.2	0.025	2	25
	3272, BT176, MON810	1 g/kg	1.5	0.2	0.4	0.025	2	25
	MIR162	1 g/kg	1.5	0.2	0.5	0.025	2	25
	CC-2	20拷贝	1.5	0.2	0.2	0.025	2	25
	G1105E-823C	20拷贝	1.5	0.2	0.4	0.025	2	25
	C0010.3.1	39拷贝	1.5	0.2	0.6	0.025	4	25
	C0030.2.4, C0030.2.5	40拷贝	1.5	0.25	0.3	0.025	4	25
大豆Soybean（Glycine max L.）	MON89788, A2704-12	-	1.5	0.2	0.2	0.025	2	25
	SHZD32-1	0.10%	1.5	0.2	0.2	0.025	2	25
	MON87701, SYHT0H2	0.10%	1.5	0.2	0.4	0.025	2	25
	MON87708	0.10%	1.5	0.2	0.5	0.025	2	25
	DAS68416-4	0.10%	1.5	0.2	0.8	0.025	2	25
	DAS-444Ø6-6	-	1.5	0.2	0.2	0.050	2	25
	A5547-127	-	2.5	0.2	0.4	0.050	2	25
	DP356043, DP305423, FG72, CV127	-	1.5	0.2	0.4	0.025	2	25
	GTS40-3-2	1 g/kg	2.0	0.2	0.4	0.025	1	25
	MON87705, MON87769	1 g/kg	1.5	0.2	0.4	0.025	2	25
棉花Cotton（Gossypium hirsutum L.）	LLcotton25, COT102	-	1.5	0.2	0.4	0.025	2	25
	GHB614	-	1.5	0.2	0.2	0.025	2	25
	Bt, MON88913, MON1598, MON1445	-	2.5	0.2	0.4	0.05	2	25
油菜Rape（Brassica napus L.）	MON88302	-	1.5	0.2	0.2	0.025	2	50
	73496	0.05%	1.5	0.2	0.6	0.025	4	25
	Topas19/2	-	2.5	0.2	0.25	0.05	1	50
	RF1, T45, Oxy-235	-	2.5	0.2	0.5	0.025	1	50
	RF3, RF2, GT73, MS1	-	2.5	0.2	0.5	0.05	1	50
使用频率最高的扩增体系 The most frequently used amplification system			1.5	0.2	0.4	0.025	2	25
使用频率最高的扩增体系占比 The percentage of amplification systems with the highest frequency of use			47/70	68/70	29/70	59/70	48/70	54/70

物种 Species	转化体 Events	预变性 Pre-denaturation	变性 Denaturation	退火 Annealing	72℃延伸 Extension at 72℃/s	72℃终延伸 Final extension at 72℃/s
玉米 Maize（Zea mays L.）	5307, MON87427, 4114, C0010.3.7, GH5112E-117C, Bt506, C0010.1.3, CC-2, DBN9858	94℃, 300 s	94℃, 30 s	58℃, 30 s	30	300
	MON89034, C0010.3.1, MON87460, BT176, MON810, 12-5, VCO-Ø1981-5, G1105E-823C	94℃, 300 s	94℃, 30 s	58℃, 30 s	30	420
	BVLA430101, BVLA430101	94℃, 300 s	94℃, 30 s	60℃, 30 s	30	420
	TC1507, MON863, GA21, NK603, T25, BT10, CBH351, 59122, MON88017, MIR604, IE09S034, DBN9936, C0030.2.4, C0030.2.5	95℃, 300 s	94℃, 30 s	58℃, 30 s	30	420
	MIR162, 3272	95℃, 300 s	94℃, 30 s	58℃, 30 s	30	420
	DAS-40278-9, BT11	94℃, 300 s	94℃, 30 s	56℃, 30 s	30	420
大豆 Soybean（Glycine max L.）	MON89788	94℃, 180 s	94℃, 30 s	58℃, 30 s	30	300
	A2704-12, A5547-127	94℃, 300 s	94℃, 30 s	56℃, 30 s	30	420
	DP356043	94℃, 300 s	94℃, 30 s	58℃, 30 s	30	420
	DP305423, GTS40-3-2, MON87705, MON87769, MON87708	94℃, 300 s	94℃, 30 s	60℃, 30 s	30	420
	MON87701	95℃, 180 s	94℃, 30 s	58℃, 30 s	30	420
	FG72	95℃, 300 s	94℃, 30 s	58℃, 30 s	30	420
	SHZD32-1	95℃, 300 s	95℃, 30 s	58℃, 30 s	30	300
	CV127, DAS68416-4	95℃, 300 s	95℃, 30 s	58℃, 30 s	30	420
	SYHT0H2	95℃, 300 s	95℃, 30 s	58℃, 45 s	45	420
	DAS-444Ø6-6	95℃, 420 s	94℃, 30 s	58℃, 30 s	30	420
棉花 Cotton（Gossypium hirsutum L.）	MON1445	94℃, 300 s	94℃, 30 s	55℃, 30 s	30	420
	LLcotton25	94℃, 300 s	94℃, 30 s	58℃, 30 s	30	120
	Bt	94℃, 300 s	94℃, 30 s	60℃, 30 s	30	120
	MON88913	95℃, 300 s	95℃, 30 s	58℃, 30 s	30	420
	MON1598	95℃, 300 s	95℃, 30 s	56℃, 30 s	30	420
	GHB614, COT102	95℃, 300 s	95℃, 30 s	58℃, 30 s	30	600
油菜 Rape（Brassica napus L.）	MS1, RF1	94℃, 300 s	94℃, 30 s	58℃, 30 s	45	420
	RF3	94℃, 300 s	94℃, 30 s	58℃, 30 s	45	600
	RF2	94℃, 300 s	94℃, 30 s	58℃, 30 s	30	120
	GT73	94℃, 300 s	94℃, 30 s	59℃, 30 s	40	600
	T45	94℃, 300 s	94℃, 30 s	60℃, 30 s	30	600
	Oxy-235	94℃, 300 s	94℃, 30 s	61℃, 30 s	45	420
	Topas19/2	94℃, 300 s	95℃, 30 s	56℃, 30 s	30	120
	MON88302	95℃, 300 s	94℃, 30 s	58℃, 30 s	30	120
	73496	95℃, 300 s	95℃, 30 s	60℃, 30 s	30	120
使用频率最高的反应条件		94℃, 300 s	94℃, 30 s	58℃, 30 s	30	420
使用频率最高的反应条件占比		40/70	59/70	49/70	63/70	48/70

物种 Species	转化体 Events	预变性 Pre-denaturation	变性 Denaturation	退火 Annealing	72℃延伸 Extension at 72℃/s	72℃终延伸 Final extension at 72℃/s
玉米 Maize（Zea mays L.）	5307, MON87427, 4114, C0010.3.7, GH5112E-117C, Bt506, C0010.1.3, CC-2, DBN9858	94℃, 300 s	94℃, 30 s	58℃, 30 s	30	300
	MON89034, C0010.3.1, MON87460, BT176, MON810, 12-5, VCO-Ø1981-5, G1105E-823C	94℃, 300 s	94℃, 30 s	58℃, 30 s	30	420
	BVLA430101, BVLA430101	94℃, 300 s	94℃, 30 s	60℃, 30 s	30	420
	TC1507, MON863, GA21, NK603, T25, BT10, CBH351, 59122, MON88017, MIR604, IE09S034, DBN9936, C0030.2.4, C0030.2.5	95℃, 300 s	94℃, 30 s	58℃, 30 s	30	420
	MIR162, 3272	95℃, 300 s	94℃, 30 s	58℃, 30 s	30	420
	DAS-40278-9, BT11	94℃, 300 s	94℃, 30 s	56℃, 30 s	30	420
大豆 Soybean（Glycine max L.）	MON89788	94℃, 180 s	94℃, 30 s	58℃, 30 s	30	300
	A2704-12, A5547-127	94℃, 300 s	94℃, 30 s	56℃, 30 s	30	420
	DP356043	94℃, 300 s	94℃, 30 s	58℃, 30 s	30	420
	DP305423, GTS40-3-2, MON87705, MON87769, MON87708	94℃, 300 s	94℃, 30 s	60℃, 30 s	30	420
	MON87701	95℃, 180 s	94℃, 30 s	58℃, 30 s	30	420
	FG72	95℃, 300 s	94℃, 30 s	58℃, 30 s	30	420
	SHZD32-1	95℃, 300 s	95℃, 30 s	58℃, 30 s	30	300
	CV127, DAS68416-4	95℃, 300 s	95℃, 30 s	58℃, 30 s	30	420
	SYHT0H2	95℃, 300 s	95℃, 30 s	58℃, 45 s	45	420
	DAS-444Ø6-6	95℃, 420 s	94℃, 30 s	58℃, 30 s	30	420
棉花 Cotton（Gossypium hirsutum L.）	MON1445	94℃, 300 s	94℃, 30 s	55℃, 30 s	30	420
	LLcotton25	94℃, 300 s	94℃, 30 s	58℃, 30 s	30	120
	Bt	94℃, 300 s	94℃, 30 s	60℃, 30 s	30	120
	MON88913	95℃, 300 s	95℃, 30 s	58℃, 30 s	30	420
	MON1598	95℃, 300 s	95℃, 30 s	56℃, 30 s	30	420
	GHB614, COT102	95℃, 300 s	95℃, 30 s	58℃, 30 s	30	600
油菜 Rape（Brassica napus L.）	MS1, RF1	94℃, 300 s	94℃, 30 s	58℃, 30 s	45	420
	RF3	94℃, 300 s	94℃, 30 s	58℃, 30 s	45	600
	RF2	94℃, 300 s	94℃, 30 s	58℃, 30 s	30	120
	GT73	94℃, 300 s	94℃, 30 s	59℃, 30 s	40	600
	T45	94℃, 300 s	94℃, 30 s	60℃, 30 s	30	600
	Oxy-235	94℃, 300 s	94℃, 30 s	61℃, 30 s	45	420
	Topas19/2	94℃, 300 s	95℃, 30 s	56℃, 30 s	30	120
	MON88302	95℃, 300 s	94℃, 30 s	58℃, 30 s	30	120
	73496	95℃, 300 s	95℃, 30 s	60℃, 30 s	30	120
使用频率最高的反应条件		94℃, 300 s	94℃, 30 s	58℃, 30 s	30	420
使用频率最高的反应条件占比		40/70	59/70	49/70	63/70	48/70

物种 Species	转化体 Events	检出限 LOD	25 mmol/L MgCl₂/µL	10 mmol/L dNTP/µL	引物终浓度 Final concentration of primers/（µmol·L^-1）	探针终浓度 Final concentration of probes /（µmol·L^-1）	DNA聚合酶 DNA polymerase /（U·µL^-1）	25 mg/L模板 25 mg/L template/µL	终体积 Final volume /µL
玉米 Maize（Zea mays L.）	BVLA430101	-	2.5	0.2	0.4	0.2	0.04	2	50
	12-5	0.05%	1.2	0.6	0.8	0.4	0.04	5	20
	4114	0.05%	2.5	0.2	0.4	0.4	0.04	2.5	20
	C0030.2.4, C0030.2.5	20拷贝	1.5	0.25	0.4	0.2	0.025	4	25
	C0010.3.1	9.8拷贝	2.5	0.2	1	0.5	0.04	5	20
	CC-2	10拷贝	2.5	0.2	1	0.5	0.04	5	20
	G1105E-823C	10拷贝	2.5	0.2	0.4	0.4	0.04	2.5	20
大豆 Soybean（Glycine max L.）	SHZD32-1	0.05%	1.5	0.2	0.4	0.3	0.025	2.5	25
	MON87708	0.05%	1.5	0.3	0.3	0.15	0.04	2.5	20
	SYHT0H2	0.05%, Ct≤36	2.5	0.2	0.4	0.4	0.04	2.5	20
	MON87701	0.05%	2.5	0.3	0.6	0.4	0.04	2.5	20
棉花 Cotton（Gossypium hirsutum L.）	COT102	-	1.5	0.2	0.4	0.2	0.04	2	25
油菜 Rape（Brassica napus L.）	73496	0.03%	2.5	0.2	1.0	0.5	0.04	5	20
使用频率最高的扩增体系 The most frequently used amplification system			2.5	0.2	0.4	0.4	0.04	2.5	20
使用频率最高的扩增体系占比 The percentage of amplification systems with the highest frequency of use			8/14	9/14	8/14	5/14	11/14	6/14	9/14