生物技术通报 ›› 2022, Vol. 38 ›› Issue (3): 264-275.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0768
粟元1(), 朱龙佼1, 曹继娟2, 刘建龙3, 许文涛1()
收稿日期:
2021-06-17
出版日期:
2022-03-26
发布日期:
2022-04-06
作者简介:
粟元,女,硕士研究生,研究方向:功能核酸生物传感器;E-mail: 基金资助:
SU Yuan1(), ZHU Long-jiao1, CAO Ji-juan2, LIU Jian-long3, XU Wen-tao1()
Received:
2021-06-17
Published:
2022-03-26
Online:
2022-04-06
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。
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.
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 |
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 |
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
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