基于大肠埃希菌 O157∶H7的荧光定量冻干检测试剂盒的研制

doi:10.13560/j.cnki.biotech.bull.1985.2021-0768

摘要/Abstract

摘要：

大肠埃希菌O157∶H7（Escherichia coli O157∶H7,E. coli O157∶H7）作为一种食源性致病菌,其分布广泛、危害性大。为了研制一种基于E. coli O157∶H7的既能实现快速、简单和灵敏检测,又能够实现常温储存及运输的试剂盒,本研究利用实时荧光定量聚合酶链式反应（real-time fluorescence quantitative polymerase chain reaction,qPCR）技术,结合真空冷冻干燥技术,研制了保留核酸检测性能、易于常温储存及运输、减少气溶胶污染的E. coli O157∶H7荧光定量冻干检测试剂盒。研制的试剂盒采用冻干技术保留了核酸扩增试剂的检测性能,复水后,在Taq DNA聚合酶的作用下通过循环扩增实时监测荧光信号的积累,实现荧光定量检测,所提出的方法在40 min内可检测到2.1 copies/μL的eaeA基因。该技术为E. coli O157∶H7的检测提供良好的研究基础和技术参考,填补了市场灵敏度高、便于储存的E. coli O157∶H7检测试剂盒的缺乏。

关键词: E. coli O157∶H7, 核酸, 荧光定量PCR, 冻干保护剂, 试剂盒

Abstract:

Escherichia coli O157∶H7,as a food-borne pathogen,is widely distributed and causes great harms. To develop a kit based on E. coli O157∶H7 that can not only achieve rapid,simple and sensitive detection,but also achieve room temperature storage and transportation,this study utilized real-time fluorescence quantitative polymerase chain reaction（qPCR）,combinig with vacuum lyophilizing technology,and developed an E. coli O157∶H7 fluorescence quantitative lyophilizing detection kit. This kit retains nucleic acid detection performance,and is easy to store and transport at room temperature,and reduces aerosol pollution. The developed kit adopts lyophilizing technology to retain the detection performance of nucleic acid amplification reagents. After rehydration,the accumulation of fluorescent signals is monitored in real time through cyclic amplification under the action of Taq DNA polymerase to achieve quantitative fluorescence detection. By the proposed method 2.1 copies/μL of eaeA gene can be detected within 40 min. This technology provides a fine research foundation and technical reference for the detection of E. coli O157∶H7,and fills the lack of detection kits with high sensitivity and easy storage in the market.

Key words: E. coli O157∶H7, nucleic acid, fluorescent quantitative PCR, lyophilized protective agent, kits

粟元, 朱龙佼, 曹继娟, 刘建龙, 许文涛. 基于大肠埃希菌 O157∶H7的荧光定量冻干检测试剂盒的研制[J]. 生物技术通报, 2022, 38(3): 264-275.

SU Yuan, ZHU Long-jiao, CAO Ji-juan, LIU Jian-long, XU Wen-tao. Development of Fluorescence Quantitative Lyophilized Detection Kit Based on Escherichia coli O157∶H7[J]. Biotechnology Bulletin, 2022, 38(3): 264-275.

图/表 14

表1 冻干保护剂的分类

Table 1 Classification of the lyoprotectant[16]

分类 Classification	冻干保护剂名称 Name of lyoprotectant	功能 Function
糖类	海藻糖、乳糖、蔗糖、果糖、麦芽糖	稳定剂
氨基酸类	精氨酸、甘氨酸、色氨酸	缓冲剂
盐类	磷酸盐（PBS）、醋酸盐、柠檬酸盐和琥珀酸盐	缓冲剂
聚合物类	聚乙烯吡咯烷酮（PVP）、聚乙二醇	增强效果
糖脂类	牛血清白蛋白（BSA）	增强效果
多元醇类	甘露醇、甘油、山梨醇	填充剂

表2 qPCR引物序列

Table 2 Primers' sequences of qPCR

引物名称 Primer name	引物序列 Primer sequence（5'-3'）
16S rDNA-F	CAACGCGAAGAACCTTACC
16S rDNA-R	CGACAGCCATGCANCACCT
E. coli O157:H7-F	CGTCATGGTACGGGTAATGAA
E. coli O157:H7-R	TAACGGCTGCCCGATAATG
E. coli O157:H7-T	FAM-TGGTCTCAGCAAATCGAGCCACA-TAMRA

图1 琼脂糖凝胶电泳图 A：E. coli O157:H7基因组;M：2 000 bp DNA ladder;1-3：大肠埃希菌O157:H7模板;B：16S rDNA PCR 产物,M：2 000 bp DNA ladder;1-3：大肠埃希菌O157:H7 90 bp 目的片段

Fig. 1 Agarose gel electrophoresis image A：E. coli O157:H7 genome. M：2 000 bp DNA ladder. 1-3：E. coli O157:H7 template. B：16S rDNA PCR product. M：2 000 bp DNA ladder. 1-3：90 bp target fragment of E. coli O157:H7

图2 大肠埃希菌O157:H7引物 F、R 的浓度优化 A：定量图;B：电泳图;C：柱形图。M：DNA marker Ι;1-3：0.2 μmol/L F 和 R、阴性对照、空白对照;4-6：0.3 μmol/L F 和 R、阴性对照、空白对照;7-9：0.4 μmol/L F 和 R、阴性对照、空白对照;10-12：0.5 μmol/L F 和 R、阴性对照、空白对照;13-15：0.6 μmol/L F 和 R、阴性对照、空白对照

Fig. 2 Concentration optimization of F and R of E. coli O157：H7 A：Quantitative graph. B：Electrophoresis graph. C：Bar graph. M：DNA marker Ι;1-3：0.2 μmol/L F and R,negative control,and control check;4-6：0.3 μmol/L F and R,negative control,and control check;7-9：0.4 μmol/L F and R,negative control,and control check;10-12：0.5 μmol/L F and R,negative control,and control check;13-15：0.6 μmol/L F and R,negative control,and control check

图3 大肠埃希菌O157:H7的引物与探针的比例优化 A：定量图;B：电泳图;C：柱形图。M：DNA marker I;1-3：引物:探针为 3:1、阴性对照、空白对照;4-6：引物:探针为 2:1、阴性对照、空白对照;7-9：引物:探针为 1:1、阴性对照、空白对照;10-12：引物:探针为 1:2、阴性对照、空白对照;13-15：引物:探针为 1:3、阴性对照、空白对照

Fig. 3 Optimization of the ratios of primers to probes of E. coli O157:H7 A：Quantitative graph. B：Electrophoresis graph. C：Bar graph. M：DNA marker I;1-3：primer:probe is 3:1,negative control,and control check;4-6：primer:probe is 2:1,negative control,and control check;7-9：primer:probe is 1:1,negative control,and control check;10-12：primer:probe is 1:2,negative control,and control check;13-15：primer:probe is 1:3,negative control,and control check

图4 大肠埃希菌O157:H7的退火温度优化 A：定量图;B：电泳图;C：柱形图;M：DNA marker Ι;1-3：50℃、阴性对照、空白对照;4-6：53℃、阴性对照、空白对照;7-9：56℃、空白对照;10-12：59℃、阴性对照、空白对照;13-15：62℃、阴性对照、空白对照;16-18：65℃、阴性对照、空白对照

Fig. 4 Optimization of annealing temperature for E. coli O157:H7 A：Quantitative graph. B：Electrophoresis graph. C：Bar graph. M：DNA marker Ι;1-3：50℃,negative control,and control check;4-6：53℃,negative control,control check;7-9：56℃,control check;10-12：59℃,negative control,and control check;13-15：62℃,negative control,and control check;16-18：65℃,negative control,and control check

图5 建立的大肠埃希菌O157：H7检测方法的灵敏度 A：检测方法灵敏度的定量图;B：Ct值与E. coli O157：H7拷贝数的线性关系

Fig. 5 Sensitivity of the established E. coli O157：H7 detection method A：Quantitative graph of the sensitivity of the detection method. B：The linear relationship between the Ct and the copies of E. coli O157：H7

图6 建立的大肠埃希菌O157:H7检测方法的特异性

Fig. 6 Specificity of the established E. coli O157:H7 detection method

图7 冻干保护剂对 qPCR 扩增的影响

Fig. 7 Effect of lyoprotectant on qPCR amplification

图8 冻干保护剂对 qPCR 体系的冻干保护作用

Fig. 8 Freeze-drying protective effect of lyoprotectant on qPCR system

图9 荧光定量冻干检测试剂盒的建立 A：检测效果;B：冻干的样品的形态

Fig. 9 Establishment of the fluorescent quantitative lyoph-ilized detection kit A：Detection effect. B：Morphology of lyophilized samples

图10 荧光定量冻干检测试剂盒的重复性 A：批内试剂盒的扩增曲线;B：批间试剂盒的扩增曲线

Fig. 10 Reproducibility of the fluorescence quantitative lyophilized detection kit A：Amplification curve of the intra-batch kit. B：Amplification curve of the inter-batch kit

图11 荧光定量冻干检测试剂盒的检测效果

Fig. 11 Detection effect of the fluorescence quantitative lyophilized detection kit

图12 实际样品的检测 A：荧光定量冻干检测试剂盒;B：国标法

Fig. 12 Detection of actual samples A：Fluorescence quantitative lyophilized detection kit. B：National standard method

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