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Development of a Two Triplex Real-Time Polymerase Chain Reaction for Rapid Detection of Six Carbapenemase Genes in Enterobacteriaceae
Ji Ae Choi, Song Mee Bae, Jung Wook Kim, Kwang Jun Lee
Osong Public Health Res Perspect. 2020;11(1):53-59.   Published online February 28, 2020
DOI: https://doi.org/10.24171/j.phrp.2020.11.1.08
  • 5,119 View
  • 114 Download
  • 1 Web of Science
  • 1 Crossref
AbstractAbstract PDFSupplementary Material
Objectives

Carbapenem resistance is a serious clinical and public health threat. Carbapenemase can confer carbapenem resistance, and most carbapenemase genes are plasmid encoded so resistance can easily spread. In this study, we aimed to develop a novel system based on the TaqMan platform for the rapid detection of 6 clinically prevalent carbapenemase genes: Klebsiella pneumoniae carbapenemase, New Delhi metallo-β-lactamase, oxacillinase, imipenem-hydrolyzing, Verona integron-encoded metallo-β-lactamase, and Guiana extended-spectrum β-lactamase.

Methods

The triplex assay was verified by testing genomic DNA of 6 carbapenemase-producing Klebsiella pneumoniae. It was validated with a blinded panel of 310 Enterobacteriaceae isolates, including 225 carbapenemase-producers and 85 non-producers, by direct colony triplex real-time polymerase chain reaction (PCR). The real-time PCR was performed using the ABI 7500 fast instrument (Applied Biosystems, CA, USA) and specific primers for each carbapenemase target were designed to include modified peptide-nucleic acid oligonucleotides.

Results

No amplification was detected among the negative samples. The result showed 100% concordance with the genotypes previously identified. The entire assay, including DNA extraction and real-time PCR, was completed within 2 hours.

Conclusion

The newly developed triplex real-time PCR assay was useful for the rapid, accurate and simultaneous detection of 6 carbapenemase genes in Enterobacteriaceae, suggesting its potential to allow an early decision on the appropriate treatment, management, and prevention of the spread of resistant infections in hospitals.

Citations

Citations to this article as recorded by  
  • Fast Track Diagnostic Tools for Clinical Management of Sepsis: Paradigm Shift from Conventional to Advanced Methods
    Ena Gupta, Juhi Saxena, Sanni Kumar, Umang Sharma, Saundarya Rastogi, Vijay Kumar Srivastava, Sanket Kaushik, Anupam Jyoti
    Diagnostics.2023; 13(2): 277.     CrossRef
Multiplex Real-time Polymerase Chain Reaction Assays for Simultaneous Detection of Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus
Jie Yeun Park, Semi Jeon, Jun Young Kim, Misun Park, Seonghan Kim
Osong Public Health Res Perspect. 2013;4(3):133-139.   Published online June 30, 2013
DOI: https://doi.org/10.1016/j.phrp.2013.04.004
  • 3,123 View
  • 31 Download
  • 19 Crossref
AbstractAbstract PDF
Objectives
A multiplex real-time polymerase chain reaction (RT-PCR) method was developed for the identification of three Vibrio species: Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus.
Methods
Specific primers and probes targeting the hlyA, tlh, and vvhA genes were selected and used for multiplex real-time PCR to confirm the identification of V. cholerae, V. parahaemolyticus, and V. vulnificus, respectively. This method was applied to screen Vibrio species from environmental samples and combining it with a culture-based method, its effectiveness was evaluated in comparison with culture-based methods alone.
Results
Specific PCR fragments were obtained from isolates belonging to the target species, indicating a high specificity of this multiplex real-time PCR. No cross-reactivity with the assay was observed between the tested bacteria. The sensitivity of the multiplex real-time PCR was found to have a lower limit of 104 colony-forming units/reaction for all three Vibrio species. The combination strategy raised the isolation ratio of all three Vibrio species 1.26- to 2.75-fold.
Conclusion
This assay provides a rapid, sensitive, and specific technique to detect these three Vibrio species in the environment.

Citations

Citations to this article as recorded by  
  • Vibrio cholerae and Salmonella Typhi culture-based wastewater or non-sewered sanitation surveillance in a resource-limited region
    Petros Chigwechokha, Ruth Lusungu Nyirenda, Davie Dalitsani, Ranken Lorvin Namaumbo, Yohanny Kazembe, Ted Smith, Rochelle H. Holm
    Journal of Exposure Science & Environmental Epidem.2024;[Epub]     CrossRef
  • Incidence, virulence genes and antimicrobial resistance of Vibrio parahaemolyticus isolated from seafood
    Deyan Stratev, Rumyana Fasulkova, Gergana Krumova-Valcheva
    Microbial Pathogenesis.2023; 177: 106050.     CrossRef
  • Exploring the Effect of Functional Diets Containing Phytobiotic Compounds in Whiteleg Shrimp Health: Resistance to Acute Hepatopancreatic Necrotic Disease Caused by Vibrio parahaemolyticus
    Carla Hernández-Cabanyero, Esther Carrascosa, Silvia Jiménez, Belén Fouz
    Animals.2023; 13(8): 1354.     CrossRef
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    Jirakrit Saetang, Phutthipong Sukkapat, Suriya Palamae, Prashant Singh, Deep Nithun Senathipathi, Jirayu Buatong, Soottawat Benjakul
    Biosensors.2023; 13(7): 698.     CrossRef
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    Sharmin Quazi Bonny, M. A. Motalib Hossain, Syed Muhammad Kamal Uddin, Thiruchelvi Pulingam, Suresh Sagadevan, Mohd Rafie Johan
    Critical Reviews in Food Science and Nutrition.2022; 62(5): 1317.     CrossRef
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    Feng Yang, Limei Xu, Wanzhen Huang, Fang Li
    Aquaculture.2022; 548: 737605.     CrossRef
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    Duanduan Chen, Leifeng Guo, Cao Yi, Shouquan Wang, Yuanyuan Ru, Hui Wang
    Ecotoxicology and Environmental Safety.2021; 217: 112266.     CrossRef
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    Lingzhi Li, Hongmei Meng, Dan Gu, Yang Li, Mengdie Jia
    Microbiological Research.2019; 222: 43.     CrossRef
  • Application of digital PCR and next generation sequencing in the etiology investigation of a foodborne disease outbreak caused by Vibrio parahaemolyticus
    Ying Li, Shuang Zhang, Jie Li, Meiling Chen, Mu He, Yuanyuan Wang, Yanchun Zhang, Hongbo Jing, Hongmei Ma, Yindong Li, Lin Zhao, Hongqun Zhao, Biao Kan, Bo Pang
    Food Microbiology.2019; 84: 103233.     CrossRef
  • Cholera Outbreak due to Raw Seafood Consumption in South Korea, 2016
    Jeong Hyun Kim, Jin Lee, Sahyun Hong, Sangwon Lee, Hae-young Na, Young-Il Jeong, Eun Jin Choi, Junyoung Kim, Hyo Sun Kawk, Enhi Cho
    The American Journal of Tropical Medicine and Hygi.2018; 99(1): 168.     CrossRef
  • Detection and differentiation of Vibrio parahaemolyticus by multiplexed real-time PCR
    Deshun Xu, Lei Ji, Xiaofang Wu, Wei Yan, Liping Chen
    Canadian Journal of Microbiology.2018; 64(11): 809.     CrossRef
  • Development of a rapid PCR protocol to detect Vibrio parahaemolyticus in clams
    Sara Federici, Diana I. Serrazanetti, M. Elisabetta Guerzoni, Raffaella Campana, Eleonora Ciandrini, Wally Baffone, Andrea Gianotti
    Journal of Food Science and Technology.2018; 55(2): 749.     CrossRef
  • First Case of Necrotizing Fasciitis Caused by Skermanella aerolata Infection Mimicking Vibrio Sepsis
    Sang Taek Heo, Ki Tae Kwon, Jeong Rae Yoo, Ji Young Choi, Keun Hwa Lee, Kwan Soo Ko
    Annals of Laboratory Medicine.2018; 38(6): 604.     CrossRef
  • Vibrio cholerae O1 with Reduced Susceptibility to Ciprofloxacin and Azithromycin Isolated from a Rural Coastal Area of Bangladesh
    Shah M. Rashed, Nur A. Hasan, Munirul Alam, Abdus Sadique, Marzia Sultana, Md. Mozammel Hoq, R. Bradley Sack, Rita R. Colwell, Anwar Huq
    Frontiers in Microbiology.2017;[Epub]     CrossRef
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    Talanta.2016; 147: 220.     CrossRef
  • Toxigenic Vibrio cholerae O1 in vegetables and fish raised in wastewater irrigated fields and stabilization ponds during a non-cholera outbreak period in Morogoro, Tanzania: an environmental health study
    Yaovi M. G. Hounmanou, Robinson H. Mdegela, Tamègnon V. Dougnon, Ofred J. Mhongole, Edward S. Mayila, Joseph Malakalinga, George Makingi, Anders Dalsgaard
    BMC Research Notes.2016;[Epub]     CrossRef
  • Isolation of Vibrio vulnificus Biotype I from Disease Outbreaks on Cultured Tiger Grouper Epinephelus fuscoguttatus Forsskal, 1775
    Jumroensri Thawonsuwan, Jiraporn Kasornchandra, Patcharee Soonsan, Chantana Keawtapee
    Fish Pathology.2016; 51(Special-is): S39.     CrossRef
  • Detection of Vibrio cholerae by isothermal cross-priming amplification combined with nucleic acid detection strip analysis
    Xia Zhang, Xin-Jun Du, Chun Guan, Ping Li, Wen-Jie Zheng, Shuo Wang
    Molecular and Cellular Probes.2015; 29(4): 208.     CrossRef
  • lolB gene, a valid alternative for qPCR detection of Vibrio cholerae in food and environmental samples
    Alejandro Garrido-Maestu, María-José Chapela, Juan M. Vieites, Ana G. Cabado
    Food Microbiology.2015; 46: 535.     CrossRef
Article
Multiplex Real-Time Polymerase Chain Reaction-Based Method for the Rapid Detection of gyrA and parC Mutations in Quinolone-Resistant Escherichia coli and Shigella spp.
Junyoung Kim, Semi Jeon, Hyungjun Kim, Misun Park, Soobok Kim, Seonghan Kim
Osong Public Health Res Perspect. 2012;3(2):113-117.   Published online June 30, 2012
DOI: https://doi.org/10.1016/j.phrp.2012.04.004
  • 2,938 View
  • 14 Download
  • 6 Crossref
AbstractAbstract PDF
Two real-time polymerase chain reaction assays were developed to detect mutations in codons 83 and 87 in gyrA and in codons 80 and 91 in parC, the main sites that causes quinolone resistance in pathogenic Escherichia coli and Shigella spp. isolates. These assays can be employed as a useful method for controlling infections caused by quinolone-resistant E coli and Shigella isolates.

Citations

Citations to this article as recorded by  
  • Various Techniques for Molecular and Rapid Detection of Infectious and Epidemic Diseases
    Mohamad Hesam Shahrajabian, Wenli Sun
    Letters in Organic Chemistry.2023; 20(9): 779.     CrossRef
  • Molecular detection and Frequency of Fluoroquinolone-Resistant Escherichia coli by Multiplex Allele Specific Polymerase Chain Reaction (MAS-PCR)
    Noha Tharwat Abou El-Khier, Maysaa El Sayed Zaki
    Egyptian Journal of Basic and Applied Sciences.2020; 7(1): 1.     CrossRef
  • Identification of new DNA gyrase inhibitors based on bioactive compounds from streptomyces: structure-based virtual screening and molecular dynamics simulations approaches
    Hourieh Kalhor, Solmaz Sadeghi, Mahya Marashiyan, Reyhaneh Kalhor, Sanaz Aghaei Gharehbolagh, Mohammad Reza Akbari Eidgahi, Hamzeh Rahimi
    Journal of Biomolecular Structure and Dynamics.2020; 38(3): 791.     CrossRef
  • Soft sweep development of resistance in Escherichia coli under fluoroquinolone stress
    Xianxing Xie, Ruichen Lv, Chao Yang, Yajun Song, Yanfeng Yan, Yujun Cui, Ruifu Yang
    Journal of Microbiology.2019; 57(12): 1056.     CrossRef
  • Rapid Detection of Genomic Mutations in gyrA and parC Genes of Escherichia coli by Multiplex Allele Specific Polymerase Chain Reaction
    Sukanlayanee Onseedaeng, Panan Ratthawongjirakul
    Journal of Clinical Laboratory Analysis.2016; 30(6): 947.     CrossRef
  • New concepts in diagnostics for infectious diarrhea
    J A Platts-Mills, J Liu, E R Houpt
    Mucosal Immunology.2013; 6(5): 876.     CrossRef

PHRP : Osong Public Health and Research Perspectives