283. The Tm Mapping Method: A Novel Method That Enables Rapid Identification of Unknown Pathogenic Microorganisms within Three Hours of Patient Sample Collection
Session: Poster Abstract Session: Diagnostic Microbiology; Novel Molecular Methods
Thursday, October 3, 2013
Room: The Moscone Center: Poster Hall C
Background: As the current pathogen-identification methods require several days, the use of inappropriate antimicrobial agents often leads to life-threating conditions in patients with severe infection, and the overuse of broad-spectrum antimicrobial agents has led to the emergence of drug-resistant bacteria. To solve the problems above, we developed the Tm mapping method. 

Methods: The Tm mapping method consists of three major steps: First, bacterial DNA is extracted directly from a clinical sample. Then, nested PCR is performed using the seven bacterial universal primer sets. To achieve accuracy in this PCR step, we have developed a eukaryote-made Taq polymerase which is free from bacterial DNA contamination. Using this polymerase, sensitive and reliable detection of bacteria without any false-positive results becomes feasible, thereby making PCR directly from patient samples to identify bacterial pathogens possible. Second, seven melting temperature (Tm) values are obtained by high resolution melting (HRM) analysis of the amplicons. These seven Tm values, when mapped on two dimensions, create a unique shape of a specific bacteria, like a constellation. This unique shape reflects the different DNA base sequences present among bacterial species. Finally, by comparing this Tm mapping shape to the shapes in the database, the dominant bacteria in a patient sample can be rapidly identified.

Results: We first performed blind tests using the 107 kinds of bacterial DNA registered in the database, and all results matched the pre-sequenced bacterial DNA. Next, to judge the credibility of the Tm mapping results, we set judgment criteria for identification based on the Difference Value. Using 130 bacterial colonies suitable for Tm mapping method, 97% (126/130) of the results matched the sequencing results. Finally, using 34 patient samples suitable for Tm mapping method, 97% (33/34) of the results matched the sequencing results. In our evaluation study, the Tm mapping method was more accurate than conventional culture-based identification.

Conclusion: The Tm mapping method would be especially useful for infectious diseases that require prompt treatment, and would contribute to the rescue of patients with severe infections, as well as a decrease in the development of antibiotic resistance.

Hideki Niimi, MD. PhD.1, Tomohiro Ueno2, Masashi Mori, PhD.3, Homare Tabata, PhD4, Hiroshi Minami, PhD4, Michihiko Goto, MD5, Sugeru Saito, MD. PhD.2 and Isao Kitajima, MD. PhD.2, (1)Department of Molecular Genetic Pathology, Toyama University Hospital, Toyama, Japan, (2)Toyama University Hospital, Toyama, Japan, (3)Ishikawa Prefectural University, Ishikawa, Japan, (4)Hokkaido Mitsui Chemicals, Inc., Sunagawa, Japan, (5)Division of Infectious Diseases, Department of Internal Medicine, University of Iowa Carver College of Medicine, Iowa City, IA

Disclosures:

H. Niimi, None

T. Ueno, None

M. Mori, None

H. Tabata, None

H. Minami, None

M. Goto, None

S. Saito, None

I. Kitajima, None

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