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. 2024 May 14;14(1):11055.
doi: 10.1038/s41598-024-60987-7.

Melting temperature mapping method using imperfect-match linear long probes

Shinya Ootsuki #  1 , Hideki Niimi #  2 , Tomohiro Ueno  3 , Masashi Mori  4 , Homare Tabata  5 , Hiroshi Minami  5 , Isao Kitajima  6
Affiliations

Melting temperature mapping method using imperfect-match linear long probes

Shinya Ootsuki et al. Sci Rep. .

Abstract

Identifying pathogenic microorganisms as early as possible is critical for selecting the appropriate antimicrobial therapy in infected patients. We previously reported the development of the Tm mapping method for identifying a broad range of pathogenic bacteria within 3 h of blood collection. However, the Tm mapping identification requires an analytical instrument with a tube-to-tube variation of no more than 0.1 °C, so we can only use a few instruments that have such high thermal accuracy. To address the problem, we developed the improved Tm mapping method using imperfect-match linear long quenching probes (IMLL Q-probes). Using IMLL Q-probes, almost all commercially available analytical instruments can be used for the Tm mapping method. Some bacterial species cannot be narrowed down to one species, but they can at least be narrowed down to the genus level. The Tm mapping method using IMLL Q-probes is useful for deciding on antimicrobial therapy in infected patients.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Workflow of the melting temperature mapping method using imperfect-match linear long quenching probes (IMLL Q-probes). (a) Workflow of the Tm mapping method using IMLL Q-probes for identifying unknown pathogenic bacteria within three hours of whole blood collection. (b) The strategy for the primer designs is shown. Nested PCR is performed using five bacterial universal primer sets, seven IMLL Q-probes are additionally used for PCR amplicons respectively, and then seven Tm values are obtained. (c) Binding sites of each IMLL Q-probes.
Figure 2
Figure 2
The Tm mapping shapes (IMLL Q-probes) of the 68 species of bacteria registered in the database. The horizontal line drawn in each Tm mapping shape is the average of the seven or fewer Tm values using IMLL Q-probes.
Figure 2
Figure 2
The Tm mapping shapes (IMLL Q-probes) of the 68 species of bacteria registered in the database. The horizontal line drawn in each Tm mapping shape is the average of the seven or fewer Tm values using IMLL Q-probes.
See this image and copyright information in PMC

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