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Subtype Screening of blaIMP Genes Using Bipartite Primers for DNA Sequencing
Ryuji Kawahara1, Masanori Watahiki2, Yuko Matsumoto3
1Division of Microbiology, Osaka Institute of Public Health, Japan.
Abstract:
Genes conferring carbapenem resistance have spread worldwide among gram-negative bacteria. Subtyping of these genes has epidemiological value due to the global cross-border movement of people. Subtyping of blaIMP genes that frequently detected in Japan appears to be important in public health settings; however, there are few useful tools for this purpose. We developed a subtyping screening tool based on PCR direct sequencing, which targets the internal sequences of almost all blaIMP genes. The tool used bipartite multiplex primers with M13 universal sequences at the 5'-end. According to in silico analysis, among the 78 known IMP-type genes, except for blaIMP-81, 77 detected genes were estimated to be differentiated. In vitro evaluation indicated that sequences of amplicons of IMP-1, IMP-6, IMP-7, and IMP-20 templates were identical to their respective subtypes. Even if the amplicons were small or undetectable through the first PCR, sufficient amplicons for DNA sequencing were obtained through a second PCR using the M13 universal primers. In conclusion, our tool can be possibly used for subtype screening of blaIMP, which is useful for the surveillance of bacteria with blaIMP in clinical and public health settings or environmental fields.
Insights
A new PCR-based tool effectively subtypes blaIMP genes, crucial for tracking carbapenem resistance in gram-negative bacteria. This method aids public health surveillance by differentiating common IMP types found globally and in Japan.
Area of Science:
- Microbiology
- Genetics
- Epidemiology
Background:
- Carbapenem resistance genes are spreading globally in gram-negative bacteria.
- Subtyping carbapenemase genes like blaIMP is vital for epidemiological tracking.
- Existing tools for subtyping blaIMP genes, particularly prevalent in Japan, are limited.
Purpose of the Study:
- To develop and evaluate a novel PCR-based tool for subtyping blaIMP genes.
- To enhance the surveillance of carbapenem-resistant gram-negative bacteria.
Main Methods:
- Development of a PCR screening tool utilizing bipartite multiplex primers with M13 universal sequences.
- In silico analysis of 78 known IMP-type genes.
- In vitro evaluation using various blaIMP gene templates and a two-step PCR approach for sequencing.
Main Results:
- The tool is estimated to differentiate 77 out of 78 known IMP-type genes in silico.
- In vitro tests confirmed accurate subtyping for IMP-1, IMP-6, IMP-7, and IMP-20.
- A secondary PCR with M13 primers successfully generated sequencing amplicons even from initially undetectable or small products.
Conclusions:
- The developed PCR direct sequencing tool is effective for blaIMP gene subtyping.
- This tool can significantly aid in the surveillance of blaIMP-carrying bacteria in clinical, public health, and environmental settings.
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