Melting Temperature Mapping Method: A Novel Method for Rapid Identification of Unknown Pathogenic Microorganisms within Three Hours of Sample Collection

Abstract

Acquiring the earliest possible identification of pathogenic microorganisms is critical for selecting the appropriate antimicrobial therapy in infected patients. We herein report the novel "melting temperature (Tm) mapping method" for rapidly identifying the dominant bacteria in a clinical sample from sterile sites. Employing only seven primer sets, more than 100 bacterial species can be identified. In particular, using the Difference Value, it is possible to identify samples suitable for Tm mapping identification. Moreover, this method can be used to rapidly diagnose the absence of bacteria in clinical samples. We tested the Tm mapping method using 200 whole blood samples obtained from patients with suspected sepsis, 85% (171/200) of which matched the culture results based on the detection level. A total of 130 samples were negative according to the Tm mapping method, 98% (128/130) of which were also negative based on the culture method. Meanwhile, 70 samples were positive according to the Tm mapping method, and of the 59 suitable for identification, 100% (59/59) exhibited a "match" or "broad match" with the culture or sequencing results. These findings were obtained within three hours of whole blood collection. The Tm mapping method is therefore useful for identifying infectious diseases requiring prompt treatment.

Figure 1. Workflow of the novel rapid method for identifying unknown pathogenic bacteria within three hours of whole blood collection.

Figure 2. Concept of the Tm mapping method.

(A) The strategy for the primer designs is shown. Nested PCR is performed using seven bacterial universal primer sets, and then the seven Tm values are obtained. (B) Mapping the seven Tm values on two dimensions leads to the identification of the unique bacterial species-specific shape. The average of all seven Tm values includes the measurement error among trials; however, the Tm mapping shape is not affected by this type of error. (C) Using an analytical instrument with a high degree of thermal accuracy among PCR tubes and Tm value analysis with EvaGreen dye in 36 samples of the same bacterial DNA in the same trial, the tube-to-tube variation is within ±0.1 °C. (D) In order to analyze the Tm mapping “shape”, we developed a method to measure the distance of each individual Tm value from the average value. Tm values above the average receive a “+” designation, while those below the average receive a “−” designation. The Tm mapping shape is identified by comparing the seven distances obtained from the unknown bacteria to those in the database. (E) In order to identify a bacterial isolate, the identification software program calculates the Difference Values using the indicated formula. The closer the Difference Value is to zero, the more similar the Tm mapping shape is to the shape of a given species of pathogenic bacteria in the database.