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A Duplex Digital PCR Assay for Simultaneous Quantification of the Enterococcus spp. and the Human Fecal-associated HF183 Marker in Waters
Published on: March 9, 2016
Multiplex real-time SYBR Green I PCR assays for simultaneous detection of 15 common enteric pathogens in stool
Yike Zhong1, Yongxia Wang1, Tong Zhao1
1College of Life Sciences and Food Engineering, Hebei University of Engineering, Handan, 056038, China.
Abstract:
Diarrheal diseases account for more than 50% of foodborne diseases worldwide, the majority of which occur in infants and young children. The traditional bacterial detection method is complex and time-consuming; therefore, it is necessary to establish a rapid and convenient detection method that can detect multiple pathogens simultaneously. In this study, we developed a set of five multiplex real-time SYBR Green I PCR assays to simultaneously detect 15 common enteric pathogens based on the Homo-Tag Assisted Non-Dimer system. These assays effectively reduced primer-dimer formation and improved the stability, uniformity, and amplification efficiency of multiplex PCR. The detection limit of the multiplex SYBR Green I PCR system was approximately 104-106 CFU/mL for stool specimens. Furthermore, we vitrified heat-unstable components on the cap of a reaction tube, showing that Taq DNA polymerase, dNTPs, primers, and SYBR Green I remained stable at 25 °C. In summary, we developed multiplex SYBR Green I PCR assays that can simultaneously detect 15 enteric pathogens. This method is comprehensive, rapid, inexpensive, accurate, and simple and displays high specificity.
Insights
A new multiplex PCR method rapidly detects 15 enteric pathogens, crucial for diagnosing diarrheal diseases in children. This technique offers a stable, efficient, and cost-effective diagnostic tool for foodborne illnesses.
Area of Science:
- Molecular Biology
- Microbiology
- Public Health
Background:
- Diarrheal diseases are a major global health concern, particularly in infants and young children, often stemming from foodborne pathogens.
- Traditional methods for detecting bacterial pathogens are complex and time-consuming, hindering rapid diagnosis and treatment.
Purpose of the Study:
- To develop a rapid, convenient, and simultaneous detection method for multiple enteric pathogens.
- To establish multiplex real-time PCR assays for efficient pathogen identification.
Main Methods:
- Developed five multiplex real-time SYBR Green I PCR assays utilizing the Homo-Tag Assisted Non-Dimer system.
- Optimized assays to minimize primer-dimer formation and enhance multiplex PCR performance.
- Assessed the stability of heat-unstable reaction components at 25°C.
Main Results:
- Successfully developed multiplex PCR assays capable of simultaneously detecting 15 common enteric pathogens.
- Achieved a detection limit of approximately 10^4-10^6 CFU/mL in stool specimens.
- Demonstrated the stability of key reagents (Taq DNA polymerase, dNTPs, primers, SYBR Green I) at room temperature (25°C).
Conclusions:
- The developed multiplex SYBR Green I PCR assays provide a comprehensive, rapid, inexpensive, accurate, and simple method for simultaneous detection of 15 enteric pathogens.
- This innovative method offers high specificity and improved stability, addressing the limitations of traditional diagnostic approaches.
- The room-temperature stability of reagents facilitates broader application and accessibility of this diagnostic technology.
