Related Experiment Video
Updated: Aug 29, 2025
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
Qualitative and Quantitative Real-Time PCR Methods for Assessing False-Positive Rates in Genetically Modified
Yunjing Li1, Fang Xiao1, Chao Zhai2
1Key Laboratory of Agricultural Genetically Modified Organisms Traceability, Ministry of Agriculture and Rural Affairs, Oil Crops Research Institute of Chinese Academy of Agricultural Science/Supervision and Test Center (Wuhan) for Plant Ecological Environment Safety, Ministry of Agriculture and Rural Affairs, Wuhan 430062, China.
Abstract:
The hygromycin phosphotransferase (HPT) gene as a selective marker is normally used in screening tests as a first step in detecting and quantifying genetically modified organisms (GMOs) in seeds, food, and feed materials. Nevertheless, if researchers only focus on the HPT gene, it is difficult to distinguish genetically modified (GM) crops from microbial infection, leading to miscalculation of the rate of GM materials in a given sample set. Here, we cloned the 7259 bp sequence carrying the HPT gene from soybean sprouts using the genome walking strategy. BLAST analysis revealed that this sequence was derived from plasmids naturally occurring in microorganisms, such as Escherichia coli, Klebsiella pneumoniae or Salmonella sp. Using the reconstructed plasmid pFP-hpt, qualitative PCR and quantitative real-time PCR (qPCR) methods were established, and 261 bp and 156 bp products were produced. The specificity of these assays was assessed against related pFP-hpt plasmids, plant species with important agronomic traits, and GM crops containing the HPT gene. No unexpected results were observed between samples using these qualitative PCR and qPCR methods. The sensitivity of this qualitative PCR assay was determined at 20 copies, while the limit of detection (LOD) and limit of quantification (LOQ) of qPCR were both 5 copies per reaction. Our in-house validation indicated that the amplification efficiency, linearity, and repeatability of this qPCR assay were in line with performance requirements. Furthermore, a qualitative and quantitative duplex PCR showed high reliability for the simultaneous detection of the HPT gene in a plant sample and environmental micro-organisms harboring the HPT gene in one PCR reaction. These qualitative PCR and qPCR assays were able to differentiate between plants infected with E. coli harboring the HPT gene from GM plants, indicating that these two methods are broadly applicable for routine GMO testing.
Insights
New PCR methods accurately detect genetically modified (GM) crops by distinguishing the hygromycin phosphotransferase (HPT) gene from microbial infections. This improves GMO testing accuracy in food and feed.
Area of Science:
- Molecular Biology
- Biotechnology
- Food Safety
Background:
- The hygromycin phosphotransferase (HPT) gene is a common marker for genetically modified organisms (GMOs).
- Distinguishing HPT in GM crops from microbial contamination is challenging, potentially leading to miscalculations in GMO detection.
- Existing methods may not reliably differentiate between plant-derived and microbial HPT genes.
Purpose of the Study:
- To develop and validate sensitive and specific PCR-based methods for detecting the HPT gene.
- To differentiate HPT in genetically modified (GM) plants from HPT in microorganisms.
- To enhance the accuracy of GMO testing in food and feed materials.
Main Methods:
- Genome walking was used to clone the HPT gene sequence from soybean sprouts.
- Qualitative and quantitative real-time PCR (qPCR) assays were established using a reconstructed plasmid (pFP-hpt).
- Duplex PCR was developed for simultaneous detection of HPT in plant and microbial samples.
Main Results:
- Developed qualitative PCR with 20 copies sensitivity and qPCR with LOD/LOQ of 5 copies per reaction.
- Validated assays showed high specificity, amplification efficiency, linearity, and repeatability.
- Duplex PCR demonstrated reliable simultaneous detection of HPT in plant and microbial sources.
- Assays successfully differentiated GM plants from plants infected with HPT-harboring E. coli.
Conclusions:
- The developed qualitative PCR and qPCR assays are highly sensitive and specific for HPT detection.
- These methods effectively distinguish GM plants from microbial contamination, improving GMO testing reliability.
- The duplex PCR offers a robust solution for simultaneous detection, broadly applicable for routine GMO analysis.
