NIU Pei Hua; ZHANG Chen; WANG Ji; TAN Wen Jie; MA Xue Jun

doi:10.3967/bes2014.112

Detection and Identification of Six Foodborne Bacteria by Two-tube Multiplex Real Time PCR and Melting Curve Analysis

NIU Pei Hua, ZHANG Chen, WANG Ji, TAN Wen Jie, MA Xue Jun

文章导航 > Biomedical and Environmental Sciences > 2014 > 27(10): 770-778

NIU Pei Hua, ZHANG Chen, WANG Ji, TAN Wen Jie, MA Xue Jun. Detection and Identification of Six Foodborne Bacteria by Two-tube Multiplex Real Time PCR and Melting Curve Analysis[J]. Biomedical and Environmental Sciences, 2014, 27(10): 770-778. doi: 10.3967/bes2014.112

Citation:

NIU Pei Hua, ZHANG Chen, WANG Ji, TAN Wen Jie, MA Xue Jun. Detection and Identification of Six Foodborne Bacteria by Two-tube Multiplex Real Time PCR and Melting Curve Analysis[J]. Biomedical and Environmental Sciences, 2014, 27(10): 770-778. doi: 10.3967/bes2014.112

doi: 10.3967/bes2014.112

基金项目: the China Mega Project for Infectious Disease (2013ZX10004-001,2012ZX10004-215,2013ZX10004-202, and2013ZX10004804-007)

Detection and Identification of Six Foodborne Bacteria by Two-tube Multiplex Real Time PCR and Melting Curve Analysis

Key Laboratory for Medical Virology, Ministry of Health, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China

Funds: the China Mega Project for Infectious Disease (2013ZX10004-001,2012ZX10004-215,2013ZX10004-202, and2013ZX10004804-007)

摘要: ObjectiveThis study is aimed to develop a two-tube melting curve-based multiplex real time PCR assay (MCMRT-PCR) for the simultaneous detection of six common foodborne pathogenic bacteria (diarrhoeagenic Escherichia coli, Salmonella,andShigellain tube 1, Staphylococcus aureus,Vibrio parahaemolyticus, and Listeria monocytogenesin tube 2). MethodsA two-tube MCMRT-PCR assaywas performed on 7900HT Fast Real-Time PCR System (Applied Biosystems, USA). Amplification by PCR was optimized to obtain high efficiency. The sensitivity and specificity of assays were investigated. ResultsThe detection limit of optimized MCMRT-PCR assay was 3.9×102 CFU/mLforS. aureus, 4.4×102 CFU/mL for L. monocytogenes, 3.0×102 CFU/mL forSalmonella, 2.5×102 CFU/mL forShigella, 2.1×102 CFU/mL forV. parahaemolyticus, and 1.2×102 CFU/mL forE.coli. The feasibility of MCMRT-PCR was further evaluated using artificially contaminated milk, the sensitivity was at the level of 105 CFU/mL. ConclusionA two-tube MCMRT-PCR assay using six primer sets was developed for detection of multiple pathogens. Our findings demonstrates that the proposed two-tube assay is reliable, useful and rapid for simultaneous detection of six foodborne pathogenic bacteria with an intended application in provincial Centers for Diseases Control and Prevention (CDC).
- /
- /
- /
Abstract: ObjectiveThis study is aimed to develop a two-tube melting curve-based multiplex real time PCR assay (MCMRT-PCR) for the simultaneous detection of six common foodborne pathogenic bacteria (diarrhoeagenic Escherichia coli, Salmonella,andShigellain tube 1, Staphylococcus aureus,Vibrio parahaemolyticus, and Listeria monocytogenesin tube 2). MethodsA two-tube MCMRT-PCR assaywas performed on 7900HT Fast Real-Time PCR System (Applied Biosystems, USA). Amplification by PCR was optimized to obtain high efficiency. The sensitivity and specificity of assays were investigated. ResultsThe detection limit of optimized MCMRT-PCR assay was 3.9×102 CFU/mLforS. aureus, 4.4×102 CFU/mL for L. monocytogenes, 3.0×102 CFU/mL forSalmonella, 2.5×102 CFU/mL forShigella, 2.1×102 CFU/mL forV. parahaemolyticus, and 1.2×102 CFU/mL forE.coli. The feasibility of MCMRT-PCR was further evaluated using artificially contaminated milk, the sensitivity was at the level of 105 CFU/mL. ConclusionA two-tube MCMRT-PCR assay using six primer sets was developed for detection of multiple pathogens. Our findings demonstrates that the proposed two-tube assay is reliable, useful and rapid for simultaneous detection of six foodborne pathogenic bacteria with an intended application in provincial Centers for Diseases Control and Prevention (CDC).
- Detection /
- Real-time PCR /
- Melting curve /
- Bacteria

[1]	CHE Jie, CHEN Bo Han, XU Li, GAO Yuan, YUE Meng Meng, CHEN Zi Man, ZHANG Mao Jun, SHAO Zhu Jun. Establishment and Modification of Ninety-seven Pneumococcal Serotyping Assays Based on Quantitative Real-time Polymerase Chain Reaction . Biomedical and Environmental Sciences, 2023, 36(9): 787-799. doi: 10.3967/bes2023.078
[2]	YIN Cai Hong, SONG Zhan Yun, LIU Xing Xing, WANG Xiao Mu, WANG Ying, GAO Cheng Cheng, SONG Xiu Ling, FENG Xin. Comparison of Microdroplet Digital PCR Assays with Real-time Fluorescence Quantitative PCR for Clostridioides difficile Detection . Biomedical and Environmental Sciences, 2023, 36(7): 653-657. doi: 10.3967/bes2023.094
[3]	CHEN Hao, HOU Zhi Yuan, CHEN Die, LI Ting, WANG Yi Ming, DE LIMA Marcelo Andrade, YANG Ying, GUO Zhen Zhong. Highly Sensitive Poly-N-isopropylacrylamide Microgel-based Electrochemical Biosensor for the Detection of SARS-COV-2 Spike Protein . Biomedical and Environmental Sciences, 2023, 36(3): 269-278. doi: 10.3967/bes2023.029
[4]	LIAO Lei, SHI Wen, MA Chao, TANG Wen Lian, QIAN Qi Lan, WANG Yu, LI Jiao Jiao, SHEN Jin Yang, JI Jing, MA Jin Ming, GAO Song. Duplex Detection of Vibrio Cholerae and Vibrio Parahaemolyticus by Real-time Recombinase Polymerase Amplification . Biomedical and Environmental Sciences, 2022, 35(12): 1161-1165. doi: 10.3967/bes2022.148
[5]	SHAO Ya Kun, XU Juan, GONG Jie, CHANG Jian Min. Development of a High-Resolution Melting Curve Analysis to Differentiate Candida parapsilosis Complex Species . Biomedical and Environmental Sciences, 2021, 34(6): 478-482. doi: 10.3967/bes2021.064
[6]	WEI Xiao, LI Yan, LU Xin, ZHAO Rong Tao, YUAN Zheng Quan, SHI Hua, ZHAO Xiang Na. Rapid Detection of Yersinia pestis by Real-time Recombinase-aided Amplification . Biomedical and Environmental Sciences, 2021, 34(4): 309-313. doi: 10.3967/bes2021.040
[7]	CHE Jie, LU Jin Xing, LI Wen Ge, ZHANG Yun Fei, ZHAO Xiao Fei, YUAN Min, BAI Xue Mei, CHEN Xia, LI Juan. A New High-throughput Real-time PCR Assay for the Screening of Multiple Antimicrobial Resistance Genes in Broiler Fecal Samples from China . Biomedical and Environmental Sciences, 2019, 32(12): 881-892. doi: 10.3967/bes2019.111
[8]	WANG Qian Ying, LI Fan, SHEN Xin Xin, FU Shi Hong, HE Ying, LEI Wen Wen, LIANG Guo Dong, WANG Huan Yun, MA Xue Jun. A Reverse-transcription Recombinase-aided Amplification Assay for the Rapid Detection of the Far-Eastern Subtype of Tick-borne Encephalitis Virus . Biomedical and Environmental Sciences, 2019, 32(5): 357-362. doi: 10.3967/bes2019.047
[9]	FAN Guo Hao, SHEN Xin Xin, LI Fan, LI Xin Na, BAI Xue Ding, ZHANG Rui Qing, WANG Rui Huan, LEI Wen Wen, WANG Huan Yu, MA Xue Jun, WU Gui Zhen. Development of an Internally Controlled Reverse Transcription Recombinase-aided Amplification Assay for the Rapid and Visual Detection of West Nile Virus . Biomedical and Environmental Sciences, 2019, 32(12): 926-929. doi: 10.3967/bes2019.116
[10]	WANG Hui, ZHANG Jing, HU Shao Hua, TAN Sheng Zhi, ZHANG Bo, ZHOU Hong Mei, PENG Rui Yun. Real-time Microwave Exposure Induces Calcium Efflux in Primary Hippocampal Neurons and Primary Cardiomyocytes . Biomedical and Environmental Sciences, 2018, 31(8): 561-571. doi: 10.3967/bes2018.077
[11]	SHAO Nan, LI Fan, NIE Kai, FU Shi Hong, ZHANG Wei Jia, HE Ying, LEI Wen Wen, WANG Qian Ying, LIANG Guo Dong, CAO Yu Xi, WANG Huan Yu. TaqMan Real-time RT-PCR Assay for Detecting and Differentiating Japanese Encephalitis Virus . Biomedical and Environmental Sciences, 2018, 31(3): 208-214. doi: 10.3967/bes2018.026
[12]	XIE Na, CHEN Zhao Yun, CHEN Tao, ZHU Bing Qing, XU Li, GAO Yuan, ZHANG Ai Yu, ZHAO Pan, LIU Ji Wen, SHAO Zhu Jun. A Cross-sectional Survey Assessing Carriage of Streptococcus pneumoniae in a Healthy Population in Xinjiang Uygur Autonomous Region of China . Biomedical and Environmental Sciences, 2018, 31(3): 233-237. doi: 10.3967/bes2018.029
[13]	CAO Yu Xi, HE Xiao Xia, FU Shi Hong, HE Ying, LI Hao, GAO Xiao Yan, LIANG Guo Dong. Real-time RT-PCR Assay for the Detection of Culex flavivirus . Biomedical and Environmental Sciences, 2015, 28(12): 917-919. doi: 10.3967/bes2015.019
[14]	LI Hao, CAO Yu Xi, HE Xiao Xia, FU Shi Hong, LYU Zhi, HE Ying, GAO Xiao Yan, LIANG Guo Dong, WANG Huan Yu. Real-time RT-PCR Assay for the detection of Tahyna Virus . Biomedical and Environmental Sciences, 2015, 28(5): 374-377. doi: 10.3967/bes2015.052
[15]	CHEN Xue Lan, ZHANG Bin, TANG Li, JIAO Hai Tao, XU Heng Yi, XU Feng, XU Hong, WEI Hua, XIONG Yong Hua. Expression and Characterization of ArgR, An Arginine Regulatory Protein in Corynebacterium crenatum . Biomedical and Environmental Sciences, 2014, 27(6): 436-443. doi: 10.3967/bes2014.072
[16]	LI Qiao, ZHANG Shu Hua, YU Yuan Hua, WANG Li Ping, GUAN Shu Wen, LI Peng Fei. Toxicity of Sodium Fluoride to Caenorhabditis elegans . Biomedical and Environmental Sciences, 2012, 25(2): 216-223. doi: 10.3967/0895-3988.2012.02.014
[17]	ZHAO Fei, CAO Bin, HE Li Hua, YIN Yu Dong, TAO Xiao Xia, SONG Shu Fan, MENG Fan Liang, ZHANG Jian Zhong. Evaluation of a New Real-time PCR Assay for Detection of Mycoplasma Pneumoniae in Clinical Specimens . Biomedical and Environmental Sciences, 2012, 25(1): 77-81. doi: 10.3967/0895-3988.2012.01.011
[18]	ZAHOOR QADIR SAMRA, MARIAM NASEEM, SUMARIA JAVED KHAN, NADIA DAR, MUHAMMAD AMIN ATHAR. PCR Targeting of Antibiotic Resistant Bacteria in Public Drinking Water of Lahore Metropolitan, Pakistan . Biomedical and Environmental Sciences, 2009, 22(6): 458-463.
[19]	ZENG-HUI WU, YONG-LIANG LOU, YI-YU LU, JIE YAN. Development of Quantitative Real-time Polymerase Chain Reaction for the Detection of Vibrio vulnificus Based on Hemolysin (vvhA) Coding System . Biomedical and Environmental Sciences, 2008, 21(4): 296-301.
[20]	Jun-Wen Li, XIU-QUAN SHI, FU-HUAN CHAO, Xin-Wei Wang, Jin-Lai Zheng, NONG Song. A Study on Detecting and Identifying Enteric Pathogens With PCR . Biomedical and Environmental Sciences, 2004, 17(1): 109-120.

点击查看大图

计量

文章访问数: 1427
HTML全文浏览量: 415
PDF下载量: 81
被引次数: 0

Detection and Identification of Six Foodborne Bacteria by Two-tube Multiplex Real Time PCR and Melting Curve Analysis

doi: 10.3967/bes2014.112

基金项目: the China Mega Project for Infectious Disease (2013ZX10004-001,2012ZX10004-215,2013ZX10004-202, and2013ZX10004804-007)

刊出日期: 2014-10-20

关键词:

Detection /

Real-time PCR /

Melting curve /

Bacteria

摘要: ObjectiveThis study is aimed to develop a two-tube melting curve-based multiplex real time PCR assay (MCMRT-PCR) for the simultaneous detection of six common foodborne pathogenic bacteria (diarrhoeagenic Escherichia coli, Salmonella,andShigellain tube 1, Staphylococcus aureus,Vibrio parahaemolyticus, and Listeria monocytogenesin tube 2). MethodsA two-tube MCMRT-PCR assaywas performed on 7900HT Fast Real-Time PCR System (Applied Biosystems, USA). Amplification by PCR was optimized to obtain high efficiency. The sensitivity and specificity of assays were investigated. ResultsThe detection limit of optimized MCMRT-PCR assay was 3.9×102 CFU/mLforS. aureus, 4.4×102 CFU/mL for L. monocytogenes, 3.0×102 CFU/mL forSalmonella, 2.5×102 CFU/mL forShigella, 2.1×102 CFU/mL forV. parahaemolyticus, and 1.2×102 CFU/mL forE.coli. The feasibility of MCMRT-PCR was further evaluated using artificially contaminated milk, the sensitivity was at the level of 105 CFU/mL. ConclusionA two-tube MCMRT-PCR assay using six primer sets was developed for detection of multiple pathogens. Our findings demonstrates that the proposed two-tube assay is reliable, useful and rapid for simultaneous detection of six foodborne pathogenic bacteria with an intended application in provincial Centers for Diseases Control and Prevention (CDC).

English Abstract

Detection and Identification of Six Foodborne Bacteria by Two-tube Multiplex Real Time PCR and Melting Curve Analysis

Key Laboratory for Medical Virology, Ministry of Health, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China

Funds: the China Mega Project for Infectious Disease (2013ZX10004-001,2012ZX10004-215,2013ZX10004-202, and2013ZX10004804-007)

Publish Date: 2014-10-20

Keywords:

Abstract: ObjectiveThis study is aimed to develop a two-tube melting curve-based multiplex real time PCR assay (MCMRT-PCR) for the simultaneous detection of six common foodborne pathogenic bacteria (diarrhoeagenic Escherichia coli, Salmonella,andShigellain tube 1, Staphylococcus aureus,Vibrio parahaemolyticus, and Listeria monocytogenesin tube 2). MethodsA two-tube MCMRT-PCR assaywas performed on 7900HT Fast Real-Time PCR System (Applied Biosystems, USA). Amplification by PCR was optimized to obtain high efficiency. The sensitivity and specificity of assays were investigated. ResultsThe detection limit of optimized MCMRT-PCR assay was 3.9×102 CFU/mLforS. aureus, 4.4×102 CFU/mL for L. monocytogenes, 3.0×102 CFU/mL forSalmonella, 2.5×102 CFU/mL forShigella, 2.1×102 CFU/mL forV. parahaemolyticus, and 1.2×102 CFU/mL forE.coli. The feasibility of MCMRT-PCR was further evaluated using artificially contaminated milk, the sensitivity was at the level of 105 CFU/mL. ConclusionA two-tube MCMRT-PCR assay using six primer sets was developed for detection of multiple pathogens. Our findings demonstrates that the proposed two-tube assay is reliable, useful and rapid for simultaneous detection of six foodborne pathogenic bacteria with an intended application in provincial Centers for Diseases Control and Prevention (CDC).

Citation:

NIU Pei Hua, ZHANG Chen, WANG Ji, TAN Wen Jie, MA Xue Jun. Detection and Identification of Six Foodborne Bacteria by Two-tube Multiplex Real Time PCR and Melting Curve Analysis[J]. Biomedical and Environmental Sciences, 2014, 27(10): 770-778. doi: 10.3967/bes2014.112

全文HTML

下载: 全尺寸图片幻灯片

返回文章

用微信扫码二维码

分享至好友和朋友圈

Biomedical and Environmental Sciences ISSN 0895-3988 CN 11-2816 / Q

Publish Under the Auspices of Chinese Center for Disease Control and Prevention

Distributed by Chinese Center for Disease Control and Prevention

Quick Links

Supported by Beijing Renhe Information Technology Co. Ltd E-mail: info@rhhz.net

doi: 10.3967/bes2014.112

Detection and Identification of Six Foodborne Bacteria by Two-tube Multiplex Real Time PCR and Melting Curve Analysis

计量

出版历程

Detection and Identification of Six Foodborne Bacteria by Two-tube Multiplex Real Time PCR and Melting Curve Analysis

doi: 10.3967/bes2014.112

English Abstract

Detection and Identification of Six Foodborne Bacteria by Two-tube Multiplex Real Time PCR and Melting Curve Analysis

Key Laboratory for Medical Virology, Ministry of Health, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China

全文HTML

目录