US 11,834,695 B2
Marker and method for determination of Parkinson's disease
Hirokazu Tsuji, Minato-ku (JP); Takashi Asahara, Minato-ku (JP); Koji Nomoto, Minato-ku (JP); Masaaki Hirayama, Nagoya (JP); and Kinji Ohno, Nagoya (JP)
Assigned to KABUSHIKI KAISHA YAKULT HONSHA, Minato-ku (JP); and NATIONAL UNIVERSITY CORPORATION NAGOYA UNIVERSITY, Nagoya (JP)
Appl. No. 16/612,968
Filed by KABUSHIKI KAISHA YAKULT HONSHA, Minato-ku (JP); and NATIONAL UNIVERSITY CORPORATION NAGOYA UNIVERSITY, Nagoya (JP)
PCT Filed May 17, 2018, PCT No. PCT/JP2018/019148
§ 371(c)(1), (2) Date Nov. 12, 2019,
PCT Pub. No. WO2018/212288, PCT Pub. Date Nov. 22, 2018.
Claims priority of application No. 2017-098973 (JP), filed on May 18, 2017.
Prior Publication US 2021/0010052 A1, Jan. 14, 2021
Int. Cl. C12Q 1/06 (2006.01); C12Q 1/6851 (2018.01); C12Q 1/689 (2018.01); C12N 1/20 (2006.01); G01N 33/92 (2006.01); C12R 1/24 (2006.01); C12R 1/25 (2006.01)
CPC C12Q 1/06 (2013.01) [C12N 1/20 (2013.01); C12Q 1/689 (2013.01); G01N 33/92 (2013.01); C12Q 1/6851 (2013.01); C12R 2001/24 (2021.05); C12R 2001/25 (2021.05)] 7 Claims
 
1. A method for detecting deterioration of a disease condition of a patient having a Parkinson's disease, the method comprising:
obtaining samples from the patient comprising at least one intestinal bacterium selected from the group consisting of Bifidobacterium, Bacteroides fragilis group, Lactobacillus brevis, and Lactobacillus plantarum subgroup at two or more different time points,
measuring the number of the at least one intestinal bacterium or the total numbers of the intestinal bacteria in the samples of the patient and comparing the numbers between the samples,
wherein the measuring comprises culturing the at least one intestinal bacterium or the intestinal bacteria in a medium and counting the number of the at least one intestinal bacterium or the intestinal bacteria; culturing the at least one intestinal bacterium or the intestinal bacteria in a medium and measuring turbidity or absorbance of the culture; using a FISH method; using a quantitative RT-PCR method; or a combination thereof, and
determining that the disease condition of the patient is getting severe when the number of the at least one intestinal bacterium or the intestinal bacteria is decreased over time or determining that the disease condition of the patient is getting mild when the number of the at least one intestinal bacterium or the intestinal bacteria is increased over time; or determining that the disease condition of the patient is getting severe when an amount of change in the number of the at least one intestinal bacterium or the intestinal bacteria over time is smaller than a reference or determining that the disease condition of the patient is getting mild when the amount of change in the number of the at least one intestinal bacterium or the intestinal bacteria over time is greater than the reference, wherein the reference is a value Q of a vertical axis at the time point of 0 in a horizontal axis in an approximate line equation created in advance for a correlation between the amount of change in the number of the at least one intestinal bacterium or the number of the intestinal bacteria measured at two or more different time points in the vertical axis and the deterioration of the disease condition in the horizontal axis of Parkinson's disease patients,
wherein the measuring is conducted by using a quantitative RT-PCR method comprising:
extracting RNA from the samples,
synthesizing cDNA by a reverse transcription (RT) reaction using a nucleic acid fragment or primer that hybridizes to extracted RNA and subsequently performing a PCR using the cDNA as a template, and
detecting a DNA fragment amplified by the PCR,
wherein the PCR is performed using at least one primer set selected from the group consisting of:
a primer set comprising a primer comprising the nucleotide sequence of SEQ ID NO: 5 and a primer comprising the nucleotide sequence of SEQ ID NO: 6;
a primer set comprising a primer comprising the nucleotide sequence of SEQ ID NO: 7 and a primer comprising the nucleotide sequence of SEO ID NO: 8;
a primer set comprising a primer comprising the nucleotide sequence of SEQ ID NO: 21 and a primer comprising the nucleotide sequence of SEQ ID NO: 22;
a primer set comprising a primer comprising the nucleotide sequence of SEQ ID NO: 29 and a primer comprising the nucleotide sequence of SEQ ID NO: 30; and
a primer set comprising primers comprising the nucleotide sequences of SEO ID NOs: 1 to 38.