基于荧光自淬灭引物的沙门氏菌新型荧光定量PCR方法的开发

doi:10.13560/j.cnki.biotech.bull.1985.2021-0101

摘要/Abstract

摘要：

沙门氏菌是一种常见的食源性致病菌,由沙门氏菌引起的食源性疾病位居榜首。荧光定量聚合酶链式反应（fluorescence quantitative polymerase chain reaction,FQ-PCR）是一种准确、可靠的用于核酸定量的扩增技术,常用的荧光探针法使得扩增体系复杂、引物合成成本高。为了简化FQ-PCR的扩增体系,减少标记基团的修饰,拟通过单标记的荧光自淬灭引物实现新型荧光定量聚合酶链式反应（innovative FQ-PCR,IFQ-PCR）用于模板DNA的定量检测。根据沙门氏菌特异性基因设计单标记发卡型荧光自淬灭引物,并将其应用于FQ-PCR实现了沙门氏菌的检测。设计的自淬灭引物探针一体化,只需单标记,无需额外探针或染料的加入,简化了扩增体系,降低了检测成本;同时引物的发卡结构提高了检测特异性。在最优的引物浓度（0.4 μmol/L）下,沙门氏菌在10¹-10⁵ CFU/mL的浓度范围内其浓度的对数值与循环阈值（cycle threshold,CT）值之间呈现良好的线性关系,R²高达0.99,检测限低至2 CFU/mL,并且在1.5 h内即可完成扩增检测,方法的稳定性符合要求。因此,一种基于荧光自淬灭引物的IFQ-PCR方法被开发出来并实现了沙门氏菌的简便、快速、灵敏、特异、低成本的检测。

关键词: 荧光定量PCR, 荧光自淬灭引物, 沙门氏菌, 定量检测

Abstract:

Salmonella is a common food-borne pathogen,and food-borne disease caused by Salmonella is in the top of a list. Fluorescence quantitative polymerase chain reaction（FQ-PCR）is an accurate and reliable amplification technique for nucleic acid quantification. The commonly used fluorescent-labeled probe methods make the amplification systems complicated and the cost of primer synthesis high. In order to simplify the FQ-PCR amplification system and reduce the modification of the labeling group,it is planned to conduct an innovative FQ-PCR（IFQ-PCR）for the quantitative detection of template DNA through single-labeled fluorescent self-quenching primers. The single-labeled hairpin type fluorescent self-quenching primer was designed according to Salmonella-specific genes,and applied to FQ-PCR to conduct the detection of Salmonella. The designed self-quenching primer was integrated with probe,only a single label was required,and the amplification system did not require the additional probes or dyes,thus the amplification system was simplified and detection cost was reduced. Meanwhile,the hairpin structure of the primers improved the specificity of the system. At the optimal primer concentration（0.4μmol/L）,the logarithm of the Salmonella concentration showed a good linear relationship with cycle threshold value in the concentration range of 10¹-10⁵ CFU/mL,with R² as high as 0.99 and detection limit as low as 2 CFU/ mL,the amplification detection was completed within 1.5 h,and the stability of the method met the requirements. Therefore,an IFQ-PCR based on fluorescent self-quenching primers was developed to have the simple,rapid,sensitive,specific and low-cost detection of Salmonella.

Key words: fluorescence quantitative PCR, fluorescence self-quenching primers, Salmonella, quantitative detection

丰敏, 李舒婷, 张洋子, 粟元, 朱龙佼, 曹际娟, 刘海燕, 许文涛. 基于荧光自淬灭引物的沙门氏菌新型荧光定量PCR方法的开发[J]. 生物技术通报, 2021, 37(11): 285-292.

FENG Min, LI Shu-ting, ZHANG Yang-zi, SU Yuan, ZHU Long-jiao, CAO Ji-juan, LIU Hai-yan, XU Wen-tao. Development of a Innovative Fluorescent Quantitative PCR Method for Salmonella Based on Fluorescent Self-quenching Primers[J]. Biotechnology Bulletin, 2021, 37(11): 285-292.

图/表 10

表1 引物及靶基因序列表

Table 1 Primer and target gene sequence

名称Name	序列Sequence（5'-3'）	碱基数Number of bases/nt
上游引物Forward primer（FP）	CGGGTCAAGGCTGAGGAA	18
原始下游引物Original reverse primer（ORP）	TGCTGAAGTTGAGGATGTTATTCG	24
下游引物Reverse primer（RP）靶基因Target gene	CGAATATGCTGAAGTTGAGGATGTTATT（FAM）CG TGCTGAAGTTGAGGATGTTATTCGCAAAGGGATCCGTCA GACCTCTGGCAGTACCTTCCTCAGCCTTGACCCG	30 85

图1 原理图

Fig.1 Schematic diagram

图2 引物浓度优化 A:CT值对比图;B:扩增曲线;C:琼脂糖凝胶电泳图,泳道1-5分别为引物浓度为500 nmol/L、400 nmol/L、300 nmol/L、200 nmol/L、100 nmol/L得到的扩增产物

Fig.2 Primer concentration optimization A: CT value comparison chart. B: Amplification curve. C: Agarose gel electrophoresis chart. Lanes 1-5 are amplified product at primer concentrations of 500 nmol/L, 400 nmol/L, 300 nmol/L, 200 nmol/L, 100 nmol/L, respectively

图3 灵敏度检测 A:扩增曲线:大红色线、黑色线、绿色线、橙色线、紫色线、粉红色线对应的沙门氏菌浓度依次为105 CFU/mL、104 CFU/mL、103 CFU/mL、102 CFU/mL、101 CFU/mL、100 CFU/mL（附图为扩增曲线对应的CT值与lgCsalmonella之间的关系图）;B:琼脂糖凝胶电泳图:泳道1-6分别对应的沙门氏菌的浓度为105 CFU/mL、104 CFU/mL、103 CFU/mL、102 CFU/mL、101 CFU/mL、100 CFU/mL

Fig.3 Sensitivity detection A: Amplification curve: the Salmonella concentrations corresponding to the big red line, black line, green line, orange line, purple line and pink line are 105 CFU/mL, 104 CFU/mL、103 CFU/mL, 102 CFU/mL, 101 CFU/mL and 100 CFU/mL (the attached shows the relationship between the CT value corresponding to the amplification curve and lgCsalmonella); B: agarose gel electrophoresis diagram: the concentration of Salmonella corresponding to lanes 1-6 is 105 CFU/mL, 104 CFU/mL, 103 CFU/mL, 102 CFU/mL, 101 CFU/mL and 100 CFU/mL

图4 标准曲线

Fig.4 Standard curve

图5 特异性检测 A:扩增曲线:大红色线对应的食源性致病菌为沙门氏菌,其它颜色线分别代表金黄色葡萄球菌、蜡样芽胞杆菌、大肠杆菌、单增李斯特菌、副溶血弧菌;B:琼脂糖凝胶电泳图:泳道1-6分别为沙门氏菌、金黄色葡萄球菌、蜡样芽胞杆菌、大肠杆菌、单增李斯特菌、副溶血弧菌的DNA扩增得到的产物

Fig.5 Specific detection A: Amplification curve: The food-borne pathogen corresponding to the big red line is Salmonella, and the other color lines represent Staphylococcus aureus, Bacillus cereus, Escherichia coli, Listeria monocytogenes, and Vibrio parahaemolyticus. B :Agarose gel electrophoresis diagram: lanes 1-6 are the products of DNA amplification of Salmonella, Staphylococcus aureus, Bacillus cereus, Escherichia coli, Listeria monocytogenes, and Vibrio parahaemolyticus, respectively

图6 实际样品检测结果

Fig. 6 Test results of actual samples

表2 IFQ-PCR方法对牛奶样品中不同浓度的沙门氏菌的检测结果

Table 2 IFQ-PCR method for the detection results of different concentrations of Salmonella in milk samples

沙门氏菌的浓度 Concentration of Salmonella/（CFU·mL^-1）	CT值 CT value			±S	RSD
10⁵	16.9	16.98	16.89	16.9±0.05	0.29%
10⁴	20.26	20.29	20.18	20.2±0.05	0.28%
10³	23.66	23.63	23.55	23.6±0.06	0.24%
10²	26.49	26.43	26.34	26.4±0.08	0.29%

图7 基于染料法的FQ-PCR的灵敏度检测 A:扩增曲线:蓝色线、橙色线、紫色线、黄色线、灰色线、绿色线对应的沙门氏菌浓度依次为105 CFU/mL、104 CFU/mL、103 CFU/mL、102 CFU/mL、101 CFU/mL、100 CFU/mL（附图为扩增曲线对应的CT值与lgCsalmonella之间的关系图）;B:FQ-PCR法标准曲线图

Fig.7 Sensitivity detection of FQ-PCR based on dye method A: Amplification curve: The Salmonella concentrations corresponding to the blue line, orange line, purple line, yellow line, gray line, and green line are 105 CFU/mL, 104 CFU/mL, 103 CFU/mL, 102 CFU/mL, 101 CFU/mL and 100 CFU/mL (The attached shows the relationship between the CT value corresponding to the amplification curve and lgCsalmonella). B: FQ-PCR method standard curve

表3 IFQ-PCR方法与FQ-PCR方法的比较[13-16,19-23]

Table 3 Comparison of IFQ-PCR method and FQ-PCR method [13-16, 19-23]

方法Method	优点Advantage	缺点Disadvantage
IFQ-PCR法 IFQ-PCR method	引物的修饰简单、合成成本相对较低、扩增体系简单、可减少引物二聚体和碱基错配的发生	引物的设计要求高
FQ-PCR染料法 FQ-PCR dye method	无需标记、灵敏度高、成本低、可通过熔解曲线分析结果	特异性差、扩增体系需额外添加染料
探针法 Probe method	特异性最强、不受非特异性扩增与引物二聚体的影响	探针的设计复杂、标记成本高、扩增体系复杂

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