The University of Toyama

National University Corporation University of Toyama

Hokkaido Mitsui Chemicals Inc.

Hitachi, Ltd.

Mitsui Chemicals, Inc.

Isao Kitajima

Hideki Niimi

Tomohiro Ueno

Homare Tabata

Shiroh Hayashi

Hiroshi Minami

Hideyuki Noda

Koh Amano

Hisaaki Yanai

Masashi Mori

Hiroko Tada

Atsushi Matsui

Yoshitsugu Higashi

Tomomi Sakai

Shinya Otsuki

Kazushige Sugie

Akio Miyakoshi

A method of pretreating a blood sample for measuring ATP of a pathogenic microorganism in blood, comprising: preparing a pellet of platelets and the pathogenic microorganism from the blood sample; and subjecting the pellet of platelets and the pathogenic microorganism to the following steps (A) to (C) in any order (including multiple simultaneous steps). A method of pretreating a cultured blood sample, wherein the method is a simplified method, does not comprise the following step (A), and comprises the steps (B) and (C) only simultaneously performed. (A) digesting cell membrane proteins of the platelets with a protease; (B) swelling the platelets in a hypotonic solution; and (C) disrupting cell membranes of the platelets with a detergent solution under a condition of suppressing an effect on the pathogenic microorganism.

Method of pretreating blood sample

A method for enabling rapid and accurate determination of the number of bacterial cells in a specimen using a PCR method includes the following steps: (1) a first PCR step of carrying out a PCR method using a nucleic acid derived from a specimen as a template and a universal primer pair for amplifying a bacterial 16S rRNA gene to obtain a first amplification product; (2) a second PCR step of carrying out a nested PCR method using a primer pair(s) for amplifying an internal sequence(s) of the sequence of the first amplification product obtained by the first PCR step to obtain a second amplification product; and (3) a bacterial number determination step of obtaining the number of bacterial cells in the specimen based on the amount of the second amplification product obtained in the second PCR step and using calibration data.

Method for determining bacterial number in specimen

The improved Tm mapping method using imperfect-match linear long quenching probes can accurately distinguish among and identify microorganisms at least at the genus level and often at the species level even in a real-time PCR instrument having measurement errors of Tm values between PCR tubes within &#xb1;0.5&#xb0; C. Therefore, the Tm mapping method can be performed in almost all real-time PCR instruments and can identify unspecified infection-causing pathogenic microorganisms in about 4 hours after sample collection.

Tm mapping method

When bacteria growth is determined in the related art using an ATP method, the bacteria growth is determined, based on an increase or a decrease in live bacteria ATP with the lapse of a culture time. Accordingly, it is necessary to measure the live bacteria ATP multiple times while antimicrobial susceptibility culture is carried out. It takes time and labor in sample preparation for each measurement, and it is difficult to quickly obtain an antimicrobial susceptibility result. Therefore, according to the present invention, the presence or absence of antimicrobial susceptibility of bacteria is determined, based on an ATP luminescence amount derived from dead bacteria in a culture liquid.

Method, device and system for testing drug sensitivity

Disclosed is a thermostable DNA polymerase preparation which can illimitably reduce the risk of false positivity in the detection of a subject microorganism utilizing a gene amplification reaction and therefore enables the selective amplification of DNA for detecting the subject microorganism even when the amount of the subject microorganism is small and therefore the amount of DNA collected therefrom is extremely small, and can be produced at a reduced cost. Also disclosed is a method for quantifying or quantifying/identifying a subject organism to be detected rapidly, conveniently and with high sensitivity using the preparation of the present invention.

Enzyme preparation containing thermostable DNA polymerase, method for producing same, and method for detecting subject organism to be detected

A system rapidly detects and identifies pathogenic bacteria responsible for infection (particularly septicemia), and selects an appropriate antimicrobial drug. A method according to the present invention for detecting and identifying pathogenic bacteria includes performing gene amplification such as real-time PCR, and analyzing the combination of the melting temperatures (Tm values) determined by gene amplification product melting curve analysis or the difference between the Tm values. Specifically, real-time PCR is performed using 4 to 16 primer sets including 1 to 7 primer sets for the 16S ribosomal RNA of bacteria, 1 to 6 primer sets for the 18S ribosomal RNA of fungi, and one primer set respectively for the spa gene and the mecA gene specific to MRSA, and the combination of the Tm values of the amplification product or the combination of the differences between the Tm values is compared with a database to identify pathogenic bacteria responsible for septicemia. Pathogenic bacteria responsible for infection (particularly septicemia) can be rapidly detected and identified using the method according to the invention so that a rapid septicemia diagnosis method and evidence-based medicine in septicemia treatment are implemented.

Method for identifying pathogenic microorganisms responsible for infection by extracting the DNA of a microorganism

Growing community of inventors

Toyama, Japan

Isao Kitajima