聚乙二醇沉淀富集和预扩增反转录实时定量聚合酶链式反应高灵敏检测小浆果中低浓度甲型肝炎病毒

引用本文: 王娉, 田卓, 齐欣, 郑秋月, 胡冰, 麻丽丹, 杨莉莉, 陈颖, 曹际娟. 聚乙二醇沉淀富集和预扩增反转录实时定量聚合酶链式反应高灵敏检测小浆果中低浓度甲型肝炎病毒[J]. 分析化学, 2022, 50(8): 1168-1178. doi: 10.19756/j.issn.0253-3820.210775 shu

Citation: WANG Ping, TIAN Zhuo, QI Xin, ZHENG Qiu-Yue, HU Bing, MA Li-Dan, YANG Li-Li, CHEN Ying, CAO Ji-Juan. Highly Sensitive Detection of Low Concentration Hepatitis A Virus in Small Berries Using Polyethylene Glycol Precipitation Enrichment and Pre-Amplification Reverse Transcription- Quantitative Polymerase Chain Reaction[J]. Chinese Journal of Analytical Chemistry, 2022, 50(8): 1168-1178. doi: 10.19756/j.issn.0253-3820.210775 shu

聚乙二醇沉淀富集和预扩增反转录实时定量聚合酶链式反应高灵敏检测小浆果中低浓度甲型肝炎病毒

王娉 ^1, ,
田卓 ^2,3,5, ,
齐欣 ^3, ,
郑秋月 ^2, ,
胡冰 ^2, ,
麻丽丹 ^4, ,
杨莉莉 ^2, ,
陈颖 ^{1,
,} ,
曹际娟 ^{2,
,}

1.
中国检验检疫科学研究院, 北京 322001;
2.
大连民族大学生命科学学院,生物技术与资源利用教育部重点实验室, 大连 116600;
3.
大连海关技术中心, 大连 116001;
4.
丹东海关综合技术服务中心, 丹东 118017;
5.
辽宁师范大学生命科学学院, 大连 116023

通讯作者: 陈颖,E-mail:chenyingcaiq@163.com; 曹际娟,E-mail:caojijuan@dlnu.edu.cn

基金项目:
国家重大研发计划项目(No.2018YFC1603504)和辽宁省"兴辽英才计划"项目(No.XLYC2002106)资助。 # 共同第一作者

摘要: 基于聚乙二醇沉淀富集和改进的预扩增反转录实时定量聚合酶链式反应(RT-qPCR),建立了高灵敏检测小浆果中低剂量感染甲型肝炎病毒(Hepatitis A virus,HAV)的方法。优化了聚乙二醇沉淀富集草莓中HAV的处理时间,探索设置预扩增步骤以加速样本核酸裂解和富集模板,建立了一种高灵敏的预扩增RT-qPCR检测新方法。回收率为15.51%,HAV减毒疫苗检测灵敏度可低至滴度4.49 CCID₅₀/mL,检测HAV质粒的灵敏度低至3.5 copies/μL。与ISO/TS15216-2:2019标准方法相比,本方法具有更高的检测效率,适于对小浆果中微量污染HAV的检测。对78批市售小浆果样品中HAV的阳性检出率为5.13%,高于采用ISO/TS 15216-2:2019标准方法的检出率(1.28%)。本方法对低剂量HAV病毒污染小浆果的检测具有很好的适用性和推广应用前景。

关键词:

English

Highly Sensitive Detection of Low Concentration Hepatitis A Virus in Small Berries Using Polyethylene Glycol Precipitation Enrichment and Pre-Amplification Reverse Transcription- Quantitative Polymerase Chain Reaction

WANG Ping ^1, ,
TIAN Zhuo ^2,3,5, ,
QI Xin ^3, ,
ZHENG Qiu-Yue ^2, ,
HU Bing ^2, ,
MA Li-Dan ^4, ,
YANG Li-Li ^2, ,
CHEN Ying ^{1,
,} ,
CAO Ji-Juan ^{2,
,}

1.
Chinese Academy of Inspection and Quarantine, Beijing 322001, China;
2.
Key Laboratory of Biotechnology and Bioresources Utilization of Ministry of Education, College of Life Science, Dalian Minzu University, Dalian 116600, China;
3.
Dalian Customs Technology Center, Dalian 116001, China;
4.
Dandong Customs Technology Center, Dandong 118017, China;
5.
School of Life Science, Liaoning Normal University, Dalian 116023, China

Corresponding author: CHEN Ying, ; CAO Ji-Juan,

Received Date: 02 October 2021
Revised Date: 25 March 2022
Fund Project:
Supported by the National Key R&D Program of China (No.2018YFC1603504) and the "Rejuvenating Liaoning Talents Plan" of Liaoning Province, China (No.XLYC2002106).

Abstract: On the basis of polyethylene glycol (PEG) precipitation enrichment and improved pre-amplified reverse transcription-quantitative polymerase chain reaction (RT-qPCR), a method was established for highly sensitive detection of low-dose contamination of hepatitis A virus (HAV) in small berries. The treatment time of HAV in strawberry enriched by PEG precipitation was optimized, and the pre-amplification steps were set up to accelerate the lysis of sample nucleic acid and enrichment of templates. By using this method, the recovery was 15.51%, the lowest detection sensitivity could be up to 4.49 CCID₅₀/mL of HAV attenuated vaccine titer, and the sensitivity of HAV plasmid was as low as 3.5 copies/μL. Compared with ISO/TS15216-2:2019 standard method, this method had higher detection efficiency and was suitable for detection of trace contaminated HAV in small berries. The positive rate of HAV in 78 batches of commercially available small berry samples was 5.13%, which was higher than that of ISO/TS 15216-2:2019 standard (1.28%). This method showed good applicability and application prospect for detection of low-dose HAV virus contamination in small berries.

Key words:

Small berries
/ Low-dose hepatitis A virus
/ Polyethylene glycol
/ Pre-amplification reverse transcription-quantitative polymerase chain reaction
/ Detection

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沈阳化工大学材料科学与工程学院沈阳 110142

聚乙二醇沉淀富集和预扩增反转录实时定量聚合酶链式反应高灵敏检测小浆果中低浓度甲型肝炎病毒

通讯作者: 陈颖,E-mail:chenyingcaiq@163.com; 曹际娟,E-mail:caojijuan@dlnu.edu.cn

English

Highly Sensitive Detection of Low Concentration Hepatitis A Virus in Small Berries Using Polyethylene Glycol Precipitation Enrichment and Pre-Amplification Reverse Transcription- Quantitative Polymerase Chain Reaction

Corresponding author: CHEN Ying, ; CAO Ji-Juan,

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中国化学会秘书处

聚乙二醇沉淀富集和预扩增反转录实时定量聚合酶链式反应高灵敏检测小浆果中低浓度甲型肝炎病毒

通讯作者: 陈颖,E-mail:chenyingcaiq@163.com; 曹际娟,E-mail:caojijuan@dlnu.edu.cn

English

Highly Sensitive Detection of Low Concentration Hepatitis A Virus in Small Berries Using Polyethylene Glycol Precipitation Enrichment and Pre-Amplification Reverse Transcription- Quantitative Polymerase Chain Reaction

Corresponding author: CHEN Ying, ; CAO Ji-Juan,

CCS Chemistry：嵌段共聚物自组装成 “三明治”二维有机材料，实现纳米级压力传感功能

CCS Chemistry：颜徐州、鲍哲南： 你的皮肤可能是一片SPMs

CCS Chemistry：工作高效不疲劳，只须让催化剂动起来

CCS Chemistry：静电组装导向聚合- 聚电解质纳米凝胶量化制备新策略

CCS Chemistry：金黄色葡萄球菌醛基脱氢酶是如何识别特异性底物的？

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