灌溉水源中沙门氏菌的SYBR Green I实时荧光定量PCR检测方法的建立

吕新; 刘兰英; 李玥仁

doi:10.19303/j.issn.1008-0384.2023.08.013

灌溉水源中沙门氏菌的SYBR Green I实时荧光定量PCR检测方法的建立

doi: 10.19303/j.issn.1008-0384.2023.08.013

吕新^{1, 2,},
刘兰英^{1, 2},
李玥仁^{1, 2, ,}

1.
福建省农业科学院农业质量标准与检测技术研究所，福建　福州　350003
2.
福建省农产品质量安全重点实验室，福建　福州　350003

基金项目: 福建省科技计划公益类专项（2020R1022005）；福建省农业科学院科技创新团队建设项目（CXTD2021011-3）；福建省高质量发展超越“5511”协同创新工程项目（XTCXGC2021020）

详细信息

作者简介:
吕新（1980 —），男，硕士，助理研究员，主要从事农产品质量安全研究，E-mail：lux_ing@126.com

通讯作者:
李玥仁（1966 —），男，博士，研究员，主要从事农产品质量安全研究，E-mail：yuerenli@yeah.net

中图分类号: S19
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出版历程
- 收稿日期: 2023-02-10
- 修回日期: 2023-05-22
- 网络出版日期: 2023-08-16
- 刊出日期: 2023-08-28

SYBR Green I RT-PCR Assay for Quantitative Detection of Salmonella in Irrigation Water

LV Xin^{1, 2
,},
LIU Lanying^{1, 2},
LI Yueren^{1, 2
, ,}

1.
Institute of Quality Standards & Testing Technology for Agro-Products, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian　350003, China
2.
Fujian Key Laboratory of Agro-products Quality & Safety, Fuzhou, Fujian　350003, China

摘要

摘要: 目的建立基于SYBR Green I嵌合染料法的灌溉水源中沙门氏菌实时荧光定量PCR（Quantitative Real-time PCR, qPCR）检测方法。方法以沙门氏菌侵袭蛋白A （invA）基因为靶基因，设计1对特异性qPCR检测引物，在对qPCR反应体系和反应条件进行优化后，通过特异性实验和灵敏度实验对qPCR方法进行验证，并制作灌溉水源中沙门氏菌qPCR定量标准曲线，最后与国标方法对比检测灌溉水源中沙门氏菌污染来验证该方法的准确性。结果经过优化的qPCR检测方法灵敏度检测结果显示最低检测限量为1×10⁻¹ pg·μL⁻¹，特异性检测结果显示具有良好的特异性，干扰菌株对本qPCR检测方法无影响，所制作的qPCR扩增标准曲线在2×10⁰～2×10⁵ cfu·mL⁻¹有较好的线性关系，相关系数为0.999 6，能对沙门氏菌进行准确的定量分析。该方法在与国标方法对比检测灌溉水源中沙门氏菌污染时具有同等准确性。结论本研究建立的基于SYBR Green I嵌合荧光法的实时荧光定量PCR检测方法，可以满足灌溉水源中沙门氏菌的定量检测要求。
- 实时荧光定量PCR /
- 灌溉水源 /
- 沙门氏菌
Abstract: Objective A SYBR Green I-based quantitative RT-PCR (qPCR) method was developed for detecting salmonella in irrigation water. Method For the methodology development a pair of primers were designed based on the sequences of the invasion protein A gene (invA) of Salmonella. qPCR reaction conditions were optimized, the assay tested for specificity and sensitivity, and a standard curve of amplification constructed. Test results on a specimen of contaminated irrigation water using the qPCR assay were compared for detection accuracy with those obtained from the national standard method. Result The newly developed qPCR assay showed a minimum detection limit of 1×10⁻¹pg·μL⁻¹ and free of interference from genomic nucleic acids of non-target microbes. The constructed linear standard curve between 2×10⁰cfu·mL⁻¹ and 2×10⁵ cfu·mL⁻¹ had a high correlation coefficient of 0.999 6. The assay demonstrated same accuracy as did the national standard method in detecting salmonella in irrigation water. Conclusion The newly established qPCR assay could be adequately applied for salmonella detection in agriculture irrigation water.
- Quantitative RT-PCR /
- Irrigated water /
- Salmonella

HTML全文

图 1 qPCR方法检测沙门氏菌

A：qPCR荧光扩增曲线；B：qPCR溶解曲线。

Figure 1. Salmonella detection by newly developed qPCR assay

A: qPCR amplification curve; B: qPCR melting curve.

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图 2 qPCR方法检测沙门氏菌的特异性

Figure 2. Specificity of qPCR assay in salmonella detection

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图 3 qPCR方法检测沙门氏菌的灵敏度

Figure 3. Sensitivity of qPCR assay in salmonella detection

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图 4 不同浓度沙门氏菌qPCR扩增曲线

Figure 4. qPCR amplification curve of detecting Salmonella at different concentrations

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图 5 沙门氏菌qPCR标准曲线

Figure 5. Standard curves of qPCR for salmonella detection

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图 6 qPCR扩增检测灌溉水源样品

Figure 6. qPCR quantification curves of detecting Salmonella in irrigation water

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表 1 供试菌株

Table 1. Salmonella strains applied for experimentation

序号 No.	菌株名称 Strains name	拉丁学名 Scientific name of strains	菌株编号 Strains number	菌株来源 Source of strains
1	鼠伤寒沙门氏菌	Salmonella typhimurium	1.1174	CGMCC
2	金黄色葡萄球菌	Staphylococcus aureus	25923	ATCC
3	大肠埃希氏菌	Escherichia coli	25922	ATCC
4	阿氏肠杆菌	Enterobacter asburiae	1.876	CGMCC
5	大肠埃希氏菌O157:H7	Escherichia coli O157:H7	10907	CICC
6	单核细胞增生李斯特氏菌	Listeria monocytogenes	1.9136	CGMCC
7	英诺克李斯特氏菌	Listeria innocua	1.2990	CGMCC

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