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Establishment of Specific Multiplex PCR Detection Methods for the Predominant tet(X)-Positive Acinetobacter Species
Chong Chen1,2, Jing Liu1,2, Jie Gao1,2
1Joint International Research Laboratory of Agriculture and Agri-Product Safety, Ministry of Education of China, Institutes of Agricultural Science and Technology Development, Yangzhou University, Yangzhou 225009, China.
Abstract:
The increasing prevalence of the mobile tigecycline resistance gene tet(X) poses a severe global health threat, and the genus Acinetobacter is a major reservoir. This study aimed to develop a rapid and specific multiplex PCR assay for detecting the predominant tet(X)-positive Acinetobacter species. Through pan-genome analyses of 390 tet(X)-positive Acinetobacter genomes, a total of 20 tet(X) variants were identified in 24 Acinetobacter species, including 17 published lineages and seven taxonomically unresolved Taxa. Acinetobacter indicus (30.8%), Acinetobacter amyesii (17.2%), and Acinetobacter towneri (16.1%) were the top three hosts of diverse tet(X) variants. Species-specific signature genes were identified and used for primer design, yielding amplicons of 267 bp (tet(X)), 424 bp (A. indicus), 690 bp (A. amyesii), and 990 bp (A. towneri). The assay was rigorously adjusted for an optimal annealing temperature of 52.8 °C and a primer ratio of 1:1:1:1, demonstrating high sensitivity with a detection limit of 0.3 ng/μL DNA and excellent stability under -20 °C, 4 °C, 20 °C storage conditions. Validation experiments on 151 bacterial strains showed high accuracy for DNA templates (≥97.8%) and bacterial suspensions (≥93.5%) within two hours. This cost-effective and highly accurate multiplex PCR provides a powerful tool for proactive surveillance and control of the critical Acinetobacter sp. pathogens.
Insights
A new multiplex PCR assay rapidly detects tigecycline resistance gene tet(X) in Acinetobacter species. This tool aids in controlling the spread of critical Acinetobacter pathogens.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- The mobile tigecycline resistance gene tet(X) is a growing global health concern.
- Acinetobacter species are significant reservoirs for tet(X) genes, necessitating effective detection methods.
Purpose of the Study:
- To develop a rapid, specific multiplex PCR assay for identifying tet(X)-positive Acinetobacter species.
- To enable proactive surveillance and control of Acinetobacter pathogens carrying tet(X).
Main Methods:
- Pan-genome analysis of 390 tet(X)-positive Acinetobacter genomes to identify tet(X) variants and species-specific genes.
- Design and optimization of a multiplex PCR assay targeting tet(X), Acinetobacter indicus, Acinetobacter amyesii, and Acinetobacter towneri.
- Validation of the assay's sensitivity, stability, and accuracy using DNA templates and bacterial suspensions.
Main Results:
- Identified 20 tet(X) variants across 24 Acinetobacter species, with A. indicus, A. amyesii, and A. towneri being major hosts.
- Developed a multiplex PCR assay yielding specific amplicons for tet(X) and key Acinetobacter species.
- Achieved high sensitivity (0.3 ng/μL DNA detection limit), excellent stability, and high accuracy (≥97.8% for DNA, ≥93.5% for suspensions) within two hours.
Conclusions:
- The developed multiplex PCR assay is a cost-effective, accurate, and rapid tool for detecting tet(X)-positive Acinetobacter.
- This assay supports enhanced surveillance and control strategies against critical Acinetobacter pathogens contributing to tigecycline resistance.
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