US 12,312,643 B2
Method for determining bacterial number in specimen
Hideki Niimi, Toyama (JP); Isao Kitajima, Toyama (JP); Akio Miyakoshi, Toyama (JP); and Yoshitsugu Higashi, Toyama (JP)
Assigned to MITSUI CHEMICALS, INC., Tokyo (JP); and NATIONAL UNIVERSITY CORPORATION UNIVERSITY OF TOYAMA, Toyama (JP)
Appl. No. 15/733,166
Filed by MITSUI CHEMICALS, INC., Tokyo (JP); and NATIONAL UNIVERSITY CORPORATION UNIVERSITY OF TOYAMA, Toyama (JP)
PCT Filed Jun. 21, 2018, PCT No. PCT/JP2018/023597
§ 371(c)(1), (2) Date Jun. 3, 2020,
PCT Pub. No. WO2019/123692, PCT Pub. Date Jun. 27, 2019.
Claims priority of application No. 2017-246333 (JP), filed on Dec. 22, 2017; and application No. 2017-246724 (JP), filed on Dec. 22, 2017.
Prior Publication US 2021/0102239 A1, Apr. 8, 2021
Int. Cl. C12Q 1/68 (2018.01); C12Q 1/686 (2018.01); C12Q 1/689 (2018.01)
CPC C12Q 1/689 (2013.01) [C12Q 1/686 (2013.01)] 23 Claims
 
1. A method for determining the number of test bacterial cells in a test specimen, comprising:
(1) a first PCR step of carrying out a universal PCR method using a test nucleic acid derived from the test bacterial cells in the test specimen as a template and a universal primer pair for amplifying a bacterial 16S rRNA gene to obtain a universal test amplification product, wherein the universal test amplification product has a sequence comprising an internal sequence, wherein the universal primer pair comprises two forward primers in first equivalent amounts and two reverse primers in second equivalent amounts, wherein the two forward primers differ from each other in a first single base and/or the two reverse primers differ from each other in a second single base;
(2) a second PCR step of carrying out a nested PCR method using a nested primer pair for amplifying the internal sequence to obtain a nested test amplification product; and
(3) a bacterial cell number determination step of obtaining the number of the test bacterial cells in the test specimen based on the amount of the nested test amplification product and using calibration data showing a relationship between an amount of at least one nested control amplification product derived from at least one control sample of control bacterial cells of a known species and a cell number of the control bacterial cells in at least one control sample.