JP2024053821A - Method for quantifying Faecalibacterium prausnitzii and Faecalibacterium prausnitzii-related bacteria, primer set, and Faecalibacterium prausnitzii and Faecalibacterium prausnitzii-related bacteria quantitatively - Google Patents

Abstract

[Problem] To provide a quantitative determination method, primer set, and quantitative determination kit for Faecalibacterium prausnitzii and Faecalibacterium prausnitzii-related bacteria that can more accurately quantify Faecalibacterium prausnitzii and Faecalibacterium prausnitzii-related bacteria. [Solution] A quantitative determination method for Faecalibacterium prausnitzii and Faecalibacterium prausnitzii-related bacteria, comprising: carrying out a DNA amplification reaction using a sample containing nucleic acid and a primer set for amplifying at least a part of a gene encoding an RNA polymerase of Faecalibacterium prausnitzii and Faecalibacterium prausnitzii-related bacteria as a template; and quantifying Faecalibacterium prausnitzii and Faecalibacterium prausnitzii-related bacteria contained in the sample from the results of the DNA amplification reaction. [Selected Figures] None

Description

The present invention relates to a method for quantifying Faecalibacterium prausnitzii and bacteria related to Faecalibacterium prausnitzii, a primer set, and a kit for quantifying Faecalibacterium prausnitzii and bacteria related to Faecalibacterium prausnitzii.

The human intestine is home to as many as 100 trillion bacteria, and it is known that these intestinal bacteria are deeply involved in the health of the host. The intestinal bacteria are made up of over 1,000 diverse bacterial species, and disruption of the balance of these intestinal bacteria is thought to be the cause of various diseases.

Previous research has shown that the composition of intestinal bacteria in patients with various diseases differs significantly from that of healthy individuals. However, the ideal composition of intestinal bacteria for healthy individuals remains unclear.

Faecalibacterium prausnitzii (hereafter sometimes referred to as F. prausnitzii), a type of intestinal bacteria, is found in large numbers in the intestines of healthy individuals, but it has been reported that its abundance is significantly reduced in the intestines of patients with various diseases. For this reason, F. prausnitzii has attracted attention as a possible biomarker for the intestinal bacteria of healthy humans.

In light of this background, F. prausnitzii is one of the intestinal bacteria that is currently attracting the most attention. For example, Non-Patent Document 1 reports that in many studies, the increase or decrease in F. prausnitzii is monitored by quantitative PCR or next-generation sequencer analysis.

On the other hand, it has been revealed that the species previously thought to be F. prausnitzii is actually composed of multiple diverse bacterial species (Non-Patent Document 2).

Mireia Lopes-Siles et al., Faecalibacterium prausnitzii: from microbiology to diagnostics and prognostics, The ISME Journal, 2017, 11, 841-852 Hiroki Tanno et al., 16S rRNA gene sequence diversity in Faecalibacterium prausnitzii-complex taxa has marked impacts on quantitative analysis, FEMS Microbiology Ecology, 2022, 98, 1-13

The method used in the study reported in Non-Patent Document 1 cannot accurately quantify F. prausnitzii and F. prausnitzii-related bacteria described in Non-Patent Document 2.

An object of one aspect of the present invention is to provide a method for quantifying F. prausnitzii and F. prausnitzii-related bacteria, a primer set, and a kit for quantifying F. prausnitzii and F. prausnitzii-related bacteria that can more accurately quantify F. prausnitzii and F. prausnitzii-related bacteria.

The present invention includes the following aspects.
[1] A method for quantifying F. prausnitzii and bacteria related to F. prausnitzii, comprising: carrying out a DNA amplification reaction using a sample containing nucleic acid as a template, and a primer set for amplifying at least a portion of a gene encoding an RNA polymerase of F. prausnitzii and bacteria related to F. prausnitzii, and quantifying F. prausnitzii and bacteria related to F. prausnitzii from the results of the DNA amplification reaction.
[2] The primer set includes an FG1F primer which is any one of a polynucleotide represented by SEQ ID NO: 1, a polynucleotide having the sequence of SEQ ID NO: 1 and 1 to 5 nucleotide bases bound to at least one of its 3' end and 5' end, and a polynucleotide having the sequence of SEQ ID NO: 1 lacking 1 to 5 nucleotide bases at least at either the 3' end or the 5'end; an FG1R primer which is any one of a polynucleotide represented by SEQ ID NO: 2, a polynucleotide having the sequence of SEQ ID NO: 2 and 1 to 5 nucleotide bases bound to at least one of its 3' end and 5' end, and a polynucleotide having the sequence of SEQ ID NO: 2 lacking 1 to 5 nucleotide bases at least at either the 3' end or the 5'end; a polynucleotide represented by SEQ ID NO: 3, a polynucleotide having the sequence of SEQ ID NO: 3 and 1 to 5 nucleotide bases bound to at least one of its 3' end and 5' end, and a polynucleotide having the sequence of SEQ ID NO: 2 lacking 1 to 5 nucleotide bases at least at either the 3' end or the 5'end; an FG2F primer which is any one of a polynucleotide having 1 to 5 nucleotide bases bound to at least one of the 3' and 5' ends, and a polynucleotide having 1 to 5 nucleotide bases deleted from at least one of the 3' and 5' ends of the sequence of SEQ ID NO:3; a polynucleotide represented by SEQ ID NO:4, a polynucleotide having 1 to 5 nucleotide bases bound to at least one of the 3' and 5' ends of the sequence of SEQ ID NO:4, and a FG2R primer which is any one of a polynucleotide having 1 to 5 nucleotide bases deleted from at least one of the 3' and 5' ends of the sequence of SEQ ID NO:4; a polynucleotide represented by SEQ ID NO:5, a polynucleotide having 1 to 5 nucleotide bases bound to at least one of the 3' and 5' ends of the sequence of SEQ ID NO:5; and an FG3F primer which is any one of the polynucleotides of SEQ ID NO:5 lacking 1 to 5 nucleotide bases at least at either the 3' end or the 5'end; a polynucleotide represented by SEQ ID NO:6, a polynucleotide having the sequence of SEQ ID NO:6 and 1 to 5 nucleotide bases bound to at least one of its 3' end or 5' end, and an FG3R primer which is any one of the polynucleotides of SEQ ID NO:6 lacking 1 to 5 nucleotide bases at either the 3' end or the 5' end, a polynucleotide represented by SEQ ID NO:7, a polynucleotide having the sequence of SEQ ID NO:7 and 1 to 5 nucleotide bases bound to at least one of its 3' end or 5' end, and a polynucleotide having the sequence of SEQ ID NO:7 lacking 1 to 5 nucleotide bases at either the 3' end or the 5' end a polynucleotide represented by SEQ ID NO: 8, a polynucleotide having the sequence of SEQ ID NO: 8 and 1 to 5 nucleotides bound to at least one of its 3' and 5' ends, and a polynucleotide having the sequence of SEQ ID NO: 8 deleted by 1 to 5 nucleotides at least one of its 3' and 5'ends; a polynucleotide represented by SEQ ID NO: 9, a polynucleotide having the sequence of SEQ ID NO: 9 and 1 to 5 nucleotides bound to at least one of its 3' and 5' ends, and a polynucleotide having the sequence of SEQ ID NO: 9 deleted by 1 to 5 nucleotides at least one of its 3' and 5'ends; a polynucleotide represented by SEQ ID NO:11, a polynucleotide represented by SEQ ID NO:11, a polynucleotide represented by SEQ ID NO:11, a polynucleotide represented by SEQ ID NO:11, a polynucleotide represented by SEQ ID NO:11, a polynucleotide represented by SEQ ID NO:11, a polynucleotide represented by SEQ ID NO:11, a polynucleotide represented by SEQ ID NO:11, a polynucleotide represented by SEQ ID NO:11, a polynucleotide represented by SEQ ID NO:11, a polynucleotide represented by SEQ ID NO:11, an FG6R primer which is any one of a polynucleotide having 5 nucleotide bases bound thereto, and a polynucleotide having 1 to 5 nucleotide bases deleted at least at one of the 3' and 5' ends of the sequence of SEQ ID NO: 12; a polynucleotide represented by SEQ ID NO: 13; a polynucleotide having 1 to 5 nucleotide bases bound to at least one of the 3' and 5' ends of the sequence of SEQ ID NO: 13; an FG7F primer which is any one of a polynucleotide having 1 to 5 nucleotide bases bound to at least one of the 3' and 5' ends of the sequence of SEQ ID NO: 13; a polynucleotide represented by SEQ ID NO: 14; a polynucleotide having 1 to 5 nucleotide bases bound to at least one of the 3' and 5' ends of the sequence of SEQ ID NO: 14; a polynucleotide having the sequence of SEQ ID NO:15 and 1 to 5 nucleotides bound to at least one of its 3' and 5' ends, and a polynucleotide having the sequence of SEQ ID NO:15 and 1 to 5 nucleotides bound to at least one of its 3' and 5' ends, respectively; an FG8F primer which is one of the polynucleotides having the sequence of SEQ ID NO:15 and 1 to 5 nucleotides deleted from at least one of its 3' and 5' ends, respectively; a polynucleotide having the sequence of SEQ ID NO:16 and 1 to 5 nucleotides bound to at least one of its 3' and 5' ends, respectively; and a polynucleotide having the sequence of SEQ ID NO:16 and 1 to 5 nucleotides deleted from at least one of its 3' and 5' ends, respectively. the FG8R primer being any one of the polynucleotides shown in SEQ ID NO:17, the polynucleotide having the sequence of SEQ ID NO:17 and 1 to 5 nucleotide bases bound to at least one of its 3' end and 5' end, and the polynucleotide having the sequence of SEQ ID NO:17 lacking 1 to 5 nucleotide bases at least at either the 3' end or the 5'end; and the FG9R primer being any one of the polynucleotide shown in SEQ ID NO:18, the polynucleotide having the sequence of SEQ ID NO:18 and 1 to 5 nucleotide bases bound to at least one of its 3' end and 5' end, and the polynucleotide having the sequence of SEQ ID NO:18 lacking 1 to 5 nucleotide bases at least at either the 3' end or the 5' end.
[3] The primer set includes an FG1F primer which is any one of a polynucleotide represented by SEQ ID NO: 1, a polynucleotide having the sequence of SEQ ID NO: 1 and 1 to 5 nucleotides bound to its 5' end, and a polynucleotide having the sequence of SEQ ID NO: 1 deleted from the 5' end of the sequence of SEQ ID NO: 1; an FG1R primer which is any one of a polynucleotide represented by SEQ ID NO: 2, a polynucleotide having the sequence of SEQ ID NO: 2 and 1 to 5 nucleotides bound to its 5' end, and a polynucleotide having the sequence of SEQ ID NO: 2 deleted from the 5' end of the sequence of SEQ ID NO: 2; a polynucleotide represented by SEQ ID NO: 3, a polynucleotide represented by SEQ ID NO:4, a polynucleotide represented by SEQ ID NO:4 and a polynucleotide having 1 to 5 nucleotide bases bound to its 5' end, and a polynucleotide having 1 to 5 nucleotide bases deleted from the 5' end of the sequence of SEQ ID NO:3; an FG2R primer which is any one of a polynucleotide represented by SEQ ID NO:4, a polynucleotide having 1 to 5 nucleotide bases bound to its 5' end, and a polynucleotide having 1 to 5 nucleotide bases deleted from the 5' end of the sequence of SEQ ID NO:4; a polynucleotide represented by SEQ ID NO:5, a polynucleotide having the sequence of SEQ ID NO:5 and a polynucleotide having 1 to 5 nucleotide bases bound to its 5' end, and a polynucleotide represented by SEQ ID NO:6, a polynucleotide having the sequence of SEQ ID NO:6 and 1 to 5 nucleotides bound to its 5' end, and a polynucleotide having the sequence of SEQ ID NO:6 and 1 to 5 nucleotides deleted from its 5'end; a FG3R primer, a polynucleotide represented by SEQ ID NO:7, a polynucleotide having the sequence of SEQ ID NO:7 and 1 to 5 nucleotides bound to its 5' end, and a polynucleotide having the sequence of SEQ ID NO:7 and 1 to 5 nucleotides deleted from its 5'end; an FG4F primer which is any one of the polynucleotides shown by SEQ ID NO:8, a polynucleotide having the sequence of SEQ ID NO:8 and 1 to 5 nucleotides bound to its 5' end, and a polynucleotide having the sequence of SEQ ID NO:8 deleted from 1 to 5 nucleotides at its 5'end; an FG5F primer which is any one of the polynucleotides shown by SEQ ID NO:9, a polynucleotide having the sequence of SEQ ID NO:9 and 1 to 5 nucleotides bound to its 5' end, and a polynucleotide having the sequence of SEQ ID NO:9 deleted from 1 to 5 nucleotides at its 5'end; a polynucleotide shown by SEQ ID NO:10 a polynucleotide represented by SEQ ID NO:11, a polynucleotide represented by SEQ ID NO:11, a polynucleotide represented by SEQ ID NO:11, a polynucleotide represented by SEQ ID NO:11, a polynucleotide represented by SEQ ID NO:11, a polynucleotide represented by SEQ ID NO:11, a polynucleotide represented by SEQ ID NO:11, a polynucleotide represented by SEQ ID NO:11, a polynucleotide represented by SEQ ID NO:11, a polynucleotide represented by SEQ ID NO:11, a polynucleotide represented by SEQ ID NO:11, a polynucleotide represented by SEQ ID NO:11, and a polynucleotide having 1 to 5 nucleotide bases deleted at the 5' end of the sequence of SEQ ID NO:12; an FG6R primer which is any one of a polynucleotide represented by SEQ ID NO:13, a polynucleotide having the sequence of SEQ ID NO:13 and 1 to 5 nucleotide bases bound to its 5' end, and a polynucleotide having 1 to 5 nucleotide bases deleted at the 5' end of the sequence of SEQ ID NO:13; an FG7F primer which is any one of a polynucleotide represented by SEQ ID NO:14, a polynucleotide having the sequence of SEQ ID NO:14 and 1 to 5 nucleotide bases bound to its 5' end, and a polynucleotide having 1 to 5 nucleotide bases deleted at the 5' end of the sequence of SEQ ID NO:14. an FG7R primer which is any one of the polynucleotides having 5 nucleotide bases deleted; a polynucleotide represented by SEQ ID NO:15, a polynucleotide having the sequence of SEQ ID NO:15 and 1 to 5 nucleotide bases bound to its 5' end, and a polynucleotide having the sequence of SEQ ID NO:15 and 1 to 5 nucleotide bases deleted at its 5'end; an FG8F primer which is any one of the polynucleotide represented by SEQ ID NO:16, a polynucleotide having the sequence of SEQ ID NO:16 and 1 to 5 nucleotide bases bound to its 5' end, and a polynucleotide having the sequence of SEQ ID NO:16 and 1 to 5 nucleotide bases deleted at its 5'end; The method for quantification according to [1] or [2], comprising an FG8R primer, an FG9F primer which is any one of a polynucleotide represented by SEQ ID NO:17, a polynucleotide having the sequence of SEQ ID NO:17 and 1 to 5 nucleotide bases bound to its 5' end, and a polynucleotide having the sequence of SEQ ID NO:17 lacking 1 to 5 nucleotide bases at the 5' end, and an FG9R primer which is any one of a polynucleotide represented by SEQ ID NO:18, a polynucleotide having the sequence of SEQ ID NO:18 and 1 to 5 nucleotide bases bound to its 5' end, and a polynucleotide having the sequence of SEQ ID NO:18 lacking 1 to 5 nucleotide bases at the 5' end.
[4] The primer set includes an FG1F primer which is a polynucleotide represented by SEQ ID NO: 1, an FG1R primer which is a polynucleotide represented by SEQ ID NO: 2, an FG2F primer which is a polynucleotide represented by SEQ ID NO: 3, an FG2R primer which is a polynucleotide represented by SEQ ID NO: 4, an FG3F primer which is a polynucleotide represented by SEQ ID NO: 5, an FG3R primer which is a polynucleotide represented by SEQ ID NO: 6, an FG4F primer which is a polynucleotide represented by SEQ ID NO: 7, an FG4R primer which is a polynucleotide represented by SEQ ID NO: 8, an FG5F primer which is a polynucleotide represented by SEQ ID NO: 9, and an FG6F primer which is a polynucleotide represented by SEQ ID NO: 10. The method for quantification according to any one of [1] to [3], comprising: an FG5R primer which is a polynucleotide represented by SEQ ID NO:11; an FG6F primer which is a polynucleotide represented by SEQ ID NO:12; an FG7F primer which is a polynucleotide represented by SEQ ID NO:13; an FG7R primer which is a polynucleotide represented by SEQ ID NO:14; an FG8F primer which is a polynucleotide represented by SEQ ID NO:15; an FG8R primer which is a polynucleotide represented by SEQ ID NO:16; an FG9F primer which is a polynucleotide represented by SEQ ID NO:17; and an FG9R primer which is a polynucleotide represented by SEQ ID NO:18.
[5] The sample includes a first to a ninth sample, and performing the DNA amplification reaction includes adding the FG1F primer and the FG1R primer to the first sample, adding the FG2F primer and the FG2R primer to the second sample, adding the FG3F primer and the FG3R primer to the third sample, adding the FG4F primer and the FG4R primer to the fourth sample, adding the FG5F primer and the FG5R primer to the fifth sample, adding the FG6F primer and the FG6R primer to the sixth sample, adding the FG7F primer and the FG7R primer to the seventh sample, adding the FG8F primer and the FG8R primer to the eighth sample, and adding the FG9F primer and the FG9R primer to the ninth sample. The quantitative method according to any one of [2] to [4].
[6] The method according to [5], wherein quantifying the F. prausnitzii and F. prausnitzii-related bacteria includes quantifying one of Faecalibacterium prausnitzii and F. prausnitzii-related bacteria in each of the first to ninth samples based on the results of the DNA amplification reaction in the first to ninth samples.
[7] The quantitative method according to any one of [1] to [6], further comprising extracting the nucleic acid from a specimen and preparing the sample containing the nucleic acid.
[8] The quantitative method according to [7], wherein the sample includes feces.
[9] A primer set for amplifying at least a part of a gene encoding an RNA polymerase of F. prausnitzii and bacteria related to F. prausnitzii, comprising an FG1F primer which is any one of a polynucleotide represented by SEQ ID NO: 1, a polynucleotide having the sequence of SEQ ID NO: 1 and 1 to 5 nucleotide bases bound to at least one of its 3' and 5' ends, and a polynucleotide having a deletion of at least 1 to 5 nucleotide bases from at least one of the 3' and 5' ends of the sequence of SEQ ID NO: 1; a polynucleotide represented by SEQ ID NO: 2, a polynucleotide having the sequence of SEQ ID NO: 2 and 1 to 5 nucleotide bases bound to at least one of its 3' and 5' ends, and a polynucleotide having a deletion of at least 1 to 5 nucleotide bases from at least one of the 3' and 5' ends of the sequence of SEQ ID NO: 2; an FG1R primer which is a polynucleotide represented by SEQ ID NO:3, a polynucleotide having the sequence of SEQ ID NO:3 and 1 to 5 nucleotide bases bound to at least one of its 3' end and 5' end, and a polynucleotide having the sequence of SEQ ID NO:3 lacking 1 to 5 nucleotide bases at least at either the 3' end or the 5'end; an FG2R primer which is a polynucleotide represented by SEQ ID NO:4, a polynucleotide having the sequence of SEQ ID NO:4 and 1 to 5 nucleotide bases bound to at least one of its 3' end and 5' end, and a polynucleotide having the sequence of SEQ ID NO:4 lacking 1 to 5 nucleotide bases at least at either the 3' end or the 5'end; a polynucleotide represented by SEQ ID NO:5, a polynucleotide having the sequence of SEQ ID NO:5 and 1 to 5 nucleotide bases bound to at least one of its 3' end and 5' end, and a polynucleotide having the sequence of SEQ ID NO:4 lacking 1 to 5 nucleotide bases at least at either the 3' end or the 5' end a polynucleotide having 1 to 5 nucleotide bases bound to at least one of the 3' and 5' ends of the sequence of SEQ ID NO:5; an FG3F primer which is any one of a polynucleotide represented by SEQ ID NO:6, a polynucleotide having 1 to 5 nucleotide bases bound to at least one of the 3' and 5' ends of the sequence of SEQ ID NO:6, and a polynucleotide having 1 to 5 nucleotide bases deleted from at least one of the 3' and 5' ends of the sequence of SEQ ID NO:6; an FG3R primer which is any one of a polynucleotide represented by SEQ ID NO:7, a polynucleotide having the sequence of SEQ ID NO:7 and 1 to 5 nucleotide bases bound to at least one of the 3' and 5' ends of the sequence of SEQ ID NO:7, and a polynucleotide having 1 to 5 nucleotide bases bound to at least one of the 3' and 5' ends of the sequence of SEQ ID NO:7. and a polynucleotide having 1 to 5 nucleotide bases deleted from at least one of the 5'termini; a polynucleotide represented by SEQ ID NO:8, a polynucleotide having the sequence of SEQ ID NO:8 and 1 to 5 nucleotide bases bound to at least one of its 3' termini and 5' termini, and a polynucleotide having 1 to 5 nucleotide bases deleted from at least one of the 3' termini and 5' termini of the sequence of SEQ ID NO:8; a polynucleotide represented by SEQ ID NO:9, a polynucleotide having the sequence of SEQ ID NO:9 and 1 to 5 nucleotide bases bound to at least one of its 3' termini and 5' termini, and a polynucleotide having 1 to 5 nucleotide bases deleted from at least one of the 3' termini and 5' termini of the sequence of SEQ ID NO:9. an FG5R primer which is any one of a polynucleotide represented by SEQ ID NO:10, a polynucleotide having the sequence of SEQ ID NO:10 and 1 to 5 nucleotides bound to at least one of its 3' and 5' ends, and a polynucleotide having 1 to 5 nucleotides deleted from at least one of the 3' and 5' ends of the sequence of SEQ ID NO:10; an FG6F primer which is any one of a polynucleotide represented by SEQ ID NO:11, a polynucleotide having the sequence of SEQ ID NO:11 and 1 to 5 nucleotides bound to at least one of its 3' and 5' ends, and a polynucleotide having 1 to 5 nucleotides deleted from at least one of the 3' and 5' ends of the sequence of SEQ ID NO:11; a polynucleotide represented by SEQ ID NO:12, a polynucleotide having the sequence of SEQ ID NO:12 and 1 to 5 nucleotides bound to at least one of its 3' and 5' ends, and a polynucleotide having 1 to 5 nucleotides deleted from at least one of the 3' and 5' ends of the sequence of SEQ ID NO:12; an FG6R primer which is any one of a polynucleotide having 1 to 5 nucleotide bases bound to at least one of its 3' and 5' ends, and a polynucleotide having 1 to 5 nucleotide bases deleted from at least one of the 3' and 5' ends of the sequence of SEQ ID NO: 12; a polynucleotide represented by SEQ ID NO: 13, a polynucleotide having 1 to 5 nucleotide bases bound to at least one of its 3' and 5' ends, and a FG7F primer which is any one of a polynucleotide having 1 to 5 nucleotide bases deleted from at least one of the 3' and 5' ends of the sequence of SEQ ID NO: 13; a polynucleotide represented by SEQ ID NO: 14, a polynucleotide having the sequence of SEQ ID NO: 14 and a polynucleotide having 1 to 5 nucleotide bases bound to at least one of its 3' and 5' ends, and and a polynucleotide having 1 to 5 nucleotide bases deleted from at least one of the 3' and 5' ends of the sequence of SEQ ID NO: 14; a polynucleotide represented by SEQ ID NO: 15, a polynucleotide having the sequence of SEQ ID NO: 15 and 1 to 5 nucleotide bases bound to at least one of its 3' and 5' ends, and a polynucleotide having 1 to 5 nucleotide bases deleted from at least one of the 3' and 5' ends of the sequence of SEQ ID NO: 15; a polynucleotide represented by SEQ ID NO: 16, a polynucleotide having the sequence of SEQ ID NO: 16 and 1 to 5 nucleotide bases bound to at least one of its 3' and 5' ends, and a polynucleotide having 1 to 5 nucleotide bases deleted from at least one of the 3' and 5' ends of the sequence of SEQ ID NO: 16. a polynucleotide having the sequence of SEQ ID NO:17 and 1 to 5 nucleotide bases bound to at least one of its 3' end and 5' end, and a polynucleotide having the sequence of SEQ ID NO:17 lacking 1 to 5 nucleotide bases at least at either the 3' end or the 5'end; and an FG9F primer which is a polynucleotide having the sequence of SEQ ID NO:17 and an FG9R primer which is a polynucleotide having the sequence of SEQ ID NO:18 and 1 to 5 nucleotide bases bound to at least one of its 3' end and 5' end, and a polynucleotide having the sequence of SEQ ID NO:18 lacking 1 to 5 nucleotide bases at least at either the 3' end or the 5' end.
[10] The primer set includes an FG1F primer which is any one of a polynucleotide represented by SEQ ID NO: 1, a polynucleotide having the sequence of SEQ ID NO: 1 and 1 to 5 nucleotides bound to its 5' end, and a polynucleotide having the sequence of SEQ ID NO: 1 deleted from the 5' end of the sequence of SEQ ID NO: 1; an FG1R primer which is any one of a polynucleotide represented by SEQ ID NO: 2, a polynucleotide having the sequence of SEQ ID NO: 2 and 1 to 5 nucleotides bound to its 5' end, and a polynucleotide having the sequence of SEQ ID NO: 2 deleted from the 5' end of the sequence of SEQ ID NO: 2; a polynucleotide represented by SEQ ID NO: 3, a polynucleotide represented by SEQ ID NO: an FG2F primer which is any one of a polynucleotide having the sequence of SEQ ID NO:3 and having 1 to 5 nucleotide bases bound to its 5' end, and a polynucleotide having 1 to 5 nucleotide bases deleted from the 5' end of the sequence of SEQ ID NO:3; an FG2R primer which is any one of a polynucleotide having the sequence of SEQ ID NO:4 and having 1 to 5 nucleotide bases bound to its 5' end, and a polynucleotide having 1 to 5 nucleotide bases deleted from the 5' end of the sequence of SEQ ID NO:4; a polynucleotide having the sequence of SEQ ID NO:5 and a polynucleotide having 1 to 5 nucleotide bases bound to its 5' end, and a polynucleotide having 1 to 5 nucleotide bases deleted at the 5' end of the sequence of SEQ ID NO:5; a polynucleotide represented by SEQ ID NO:6, a polynucleotide having the sequence of SEQ ID NO:6 and 1 to 5 nucleotide bases bound to its 5' end, and a polynucleotide having 1 to 5 nucleotide bases deleted at the 5' end of the sequence of SEQ ID NO:6; a polynucleotide represented by SEQ ID NO:7, a polynucleotide having the sequence of SEQ ID NO:7 and 1 to 5 nucleotide bases bound to its 5' end, and a polynucleotide having 1 to 5 nucleotide bases deleted at the 5' end of the sequence of SEQ ID NO:7. an FG4F primer which is any one of the polynucleotides shown by SEQ ID NO:8, a polynucleotide having the sequence of SEQ ID NO:8 and 1 to 5 nucleotides bound to its 5' end, and a polynucleotide having the sequence of SEQ ID NO:8 deleted from 1 to 5 nucleotides at its 5'end; an FG5F primer which is any one of the polynucleotides shown by SEQ ID NO:9, a polynucleotide having the sequence of SEQ ID NO:9 and 1 to 5 nucleotides bound to its 5' end, and a polynucleotide having the sequence of SEQ ID NO:9 deleted from 1 to 5 nucleotides at its 5'end; a polynucleotide shown by SEQ ID NO:10 a polynucleotide represented by SEQ ID NO:11, a polynucleotide represented by SEQ ID NO:11, a polynucleotide represented by SEQ ID NO:11, a polynucleotide represented by SEQ ID NO:11, a polynucleotide represented by SEQ ID NO:11, a polynucleotide represented by SEQ ID NO:11, a polynucleotide represented by SEQ ID NO:11, a polynucleotide represented by SEQ ID NO:11, a polynucleotide represented by SEQ ID NO:11, a polynucleotide represented by SEQ ID NO:11, a polynucleotide represented by SEQ ID NO:11, a polynucleotide represented by SEQ ID NO:13, a polynucleotide having the sequence of SEQ ID NO:13 and 1 to 5 nucleotides bound to its 5' end, and a polynucleotide having the sequence of SEQ ID NO:13 and 1 to 5 nucleotides missing from its 5'end; an FG7F primer, a polynucleotide represented by SEQ ID NO:14, a polynucleotide having the sequence of SEQ ID NO:14 and 1 to 5 nucleotides bound to its 5' end, and a polynucleotide having the sequence of SEQ ID NO:14 and 1 to 5 nucleotides missing from its 5'end; an FG7R primer which is any one of the polynucleotides having 1 to 5 nucleotide bases deleted from the 5'-terminus of the sequence of SEQ ID NO:15, a polynucleotide having the sequence of SEQ ID NO:15 and 1 to 5 nucleotide bases bound to its 5'-terminus, and an FG8F primer which is any one of the polynucleotides having the sequence of SEQ ID NO:15 and 1 to 5 nucleotide bases deleted from the 5'-terminus of the sequence of SEQ ID NO:15, a polynucleotide having the sequence of SEQ ID NO:16, a polynucleotide having the sequence of SEQ ID NO:16 and 1 to 5 nucleotide bases bound to its 5'-terminus, and a polynucleotide having the sequence of SEQ ID NO:16 and 1 to 5 nucleotide bases deleted from the 5'-terminus of the sequence of SEQ ID NO:16 the FG8R primer being any one of a polynucleotide represented by SEQ ID NO:17, a polynucleotide having the sequence of SEQ ID NO:17 and 1 to 5 nucleotide bases bound to its 5' end, and a polynucleotide having the sequence of SEQ ID NO:17 deleted from 1 to 5 nucleotide bases at the 5'end; and an FG9R primer being any one of a polynucleotide represented by SEQ ID NO:18, a polynucleotide having the sequence of SEQ ID NO:18 and 1 to 5 nucleotide bases bound to its 5' end, and a polynucleotide having the sequence of SEQ ID NO:18 deleted from 1 to 5 nucleotide bases at the 5' end.
[11] The primer set comprises an FG1F primer which is a polynucleotide represented by SEQ ID NO: 1, an FG1R primer which is a polynucleotide represented by SEQ ID NO: 2, an FG2F primer which is a polynucleotide represented by SEQ ID NO: 3, an FG2R primer which is a polynucleotide represented by SEQ ID NO: 4, an FG3F primer which is a polynucleotide represented by SEQ ID NO: 5, an FG3R primer which is a polynucleotide represented by SEQ ID NO: 6, an FG4F primer which is a polynucleotide represented by SEQ ID NO: 7, an FG4R primer which is a polynucleotide represented by SEQ ID NO: 8, an FG5F primer which is a polynucleotide represented by SEQ ID NO: 9, an FG6F primer which is a polynucleotide represented by SEQ ID NO: 10, and an FG7F primer which is a polynucleotide represented by SEQ ID NO: 11. the FG5R primer being a polynucleotide represented by SEQ ID NO:11, the FG6F primer being a polynucleotide represented by SEQ ID NO:12, the FG7F primer being a polynucleotide represented by SEQ ID NO:13, the FG7R primer being a polynucleotide represented by SEQ ID NO:14, the FG8F primer being a polynucleotide represented by SEQ ID NO:15, the FG8R primer being a polynucleotide represented by SEQ ID NO:16, the FG9F primer being a polynucleotide represented by SEQ ID NO:17, and the FG9R primer being a polynucleotide represented by SEQ ID NO:18.
[12] A quantitative determination kit for F. prausnitzii and bacteria related to F. prausnitzii, comprising the primer set according to any one of [9] to [11], deoxynucleotide triphosphates, DNA polymerase, and a pH buffer solution.

The above quantitative method, primer set, and quantitative kit allow for more accurate quantification of Faecalibacterium prausnitzii and bacteria related to Faecalibacterium prausnitzii.

1 is a dendrogram showing classification of F. prausnitzii and bacteria related to F. prausnitzii based on ANI values. 1 is a graph showing the results of quantitative PCR using specific primers for each bacterial group targeting the synthesized rpoA gene. 1 is a graph showing the results of quantitative PCR using specific primers for each bacterial group targeting the synthesized rpoA gene. 1 is a graph showing the results of quantitative PCR using specific primers for each bacterial group targeting the synthesized rpoA gene. 1 is a graph showing the results of quantitative PCR using specific primers for each bacterial group targeting the synthesized rpoA gene. 1 is a graph showing the results of quantitative PCR using specific primers for each bacterial group targeting the synthesized rpoA gene. 1 is a graph showing the results of quantitative PCR using synthetic primers targeting the 16S rRNA gene. 1 is a graph showing the results of quantitative PCR performed using specific primers for each bacterial group targeting the rpoA gene of the genome extracted from the sample. 1 is a graph showing the results of quantitative PCR performed using specific primers for each bacterial group targeting the rpoA gene of the genome extracted from the sample. 1 is a graph showing the results of quantitative PCR performed using specific primers for each bacterial group targeting the rpoA gene of the genome extracted from the sample.

Hereinafter, the embodiments will be described in detail with reference to the drawings.
In this specification, A, C, G and T in the base sequence of a nucleic acid respectively represent adenine base, cytosine base, guanine base and thymine base in deoxyribonucleotides.

<F. prausnitzii and related bacteria>
Figure 1 is a dendrogram in which F. prausnitzii and bacteria related to F. prausnitzii are classified based on the average nucleotide identity (ANI) value (cited with modification from Non-Patent Document 2). In Non-Patent Document 2, the inventors reclassified bacteria that had previously been classified as F. prausnitzii based on the homology of the entire genomic DNA as shown in the dendrogram in Figure 1. Details of the classification method are as described in "Acquisition of Genomic Data and Genomic Analysis" in Non-Patent Document 2. Group 1 to Group 8 in Figure 1 are classified into different phylogenetic groups based on the homology.

Group 1 in Figure 1 is F. prausnitzii. In this specification, bacteria classified into Groups 2 to 8 are defined as being included in bacteria related to F. prausnitzii. Bacteria classified into Groups 2 to 8 may be referred to as Groups 2 to 8 bacteria, respectively. Group 4 is classified as Faecalibacterium longum, Group 6 as Faecalibacterium duncaniae, and Group 7 as Faecalibacterium hattorii.

Furthermore, in this specification, Faecalibacterium butyicigenerans reported in Yuanqiang Zou et al., Scientific Reports, 2021,11, 11340 is also defined as being included in bacteria related to F. prausnitzii. In this specification, Faecalibacterium butyicigenerans may be referred to as Group 9 bacteria.

In other words, F. prausnitzii-related bacteria are bacteria that have previously been classified as F. prausnitzii or bacteria that are phylogenetically related to F. prausnitzii.

<Method for quantifying F. prausnitzii and bacteria related to F. prausnitzii>
In one embodiment of the present invention, a method for quantifying F. prausnitzii and bacteria related to F. prausnitzii includes carrying out a DNA amplification reaction using a sample containing nucleic acid and a primer set for amplifying at least a portion of a gene encoding an RNA polymerase of F. prausnitzii and bacteria related to F. prausnitzii as a template, and quantifying F. prausnitzii and bacteria related to F. prausnitzii from the results of the DNA amplification reaction.

In the present invention, the rpoA gene, which encodes an RNA polymerase, is used as a marker for quantifying F. prausnitzii and bacteria related to F. prausnitzii.

In general, the 16S rRNA gene is a gene that is highly conserved within a bacterial species. Therefore, it is a gene that is frequently used as a marker for the classification, identification, detection, and quantification of bacteria. However, in the Faecalibacterium genus, six copies of the 16S rRNA gene are conserved in the genome of the same strain. Furthermore, the homology of the 16S rRNA gene among the six copies is low, and some of the homology is so low that they can be considered to be different bacterial species. Therefore, if the 16S rRNA gene is used as a marker for quantifying bacteria of the Faecalibacterium genus, accurate quantification may not be possible.

The rpoA gene used in the present invention as a marker for quantifying F. prausnitzii and bacteria related to F. prausnitzii has a different sequence between species of F. prausnitzii and bacteria related to F. prausnitzii. SEQ ID NOs: 19 to 27 show the sequences of the rpoA1 gene to the rpoA9 gene of F. prausnitzii and bacteria of Groups 2 to 9, respectively. Since the rpoA gene sequence may partially differ between strains even within the same bacterial group, SEQ ID NOs: 19 to 27 show representative rpoA gene sequences for each species.

By using the rpoA gene as a marker, it is possible to individually quantify F. prausnitzii and eight species of F. prausnitzii-related bacteria contained in a sample. In addition, the total amount of F. prausnitzii and F. prausnitzii-related bacteria contained in a sample can be accurately quantified by summing the individual quantification values.

The primer set amplifies at least a portion of the rpoA gene of F. prausnitzii and bacteria related to F. prausnitzii.

The primer set consists of nine pairs of forward and reverse primers that amplify the rpoA gene of F. prausnitzii and eight species of F. prausnitzii-related bacteria.

The forward primer FG1F primer and reverse primer FG1R primer, which are composed of the polynucleotides shown in SEQ ID NO:1 and SEQ ID NO:2, respectively, are primers that amplify the rpoA1 gene, which is the rpoA gene of F. prausnitzii. The FG1F primer has a sequence complementary to the 367th to 387th base sequence of the rpoA1 gene, and the FG1R primer has a sequence complementary to the 498th to 518th base sequence of the rpoA1 gene.

The forward primer FG2F primer and reverse primer FG2R primer, which are composed of the polynucleotides shown in SEQ ID NO:3 and SEQ ID NO:4, respectively, are primers that amplify the rpoA2 gene, which is the rpoA gene of Group 2 bacteria. The FG2F primer has a sequence complementary to the 401-420th base sequence of the rpoA2 gene, and the FG2R primer has a sequence complementary to the 576-596th base sequence of the rpoA2 gene.

The forward primer FG3F primer and reverse primer FG3R primer, which are composed of the polynucleotides shown in SEQ ID NO:5 and SEQ ID NO:6, respectively, are primers that amplify the rpoA3 gene, which is the rpoA gene of Group 3 bacteria. The FG3F primer has a sequence complementary to the 467th to 487th base sequence of the rpoA3 gene, and the FG3R primer has a sequence complementary to the 598th to 618th base sequence of the rpoA3 gene.

The forward primer FG4F primer and reverse primer FG4R primer, which are composed of the polynucleotides shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively, are primers that amplify the rpoA4 gene, which is the rpoA gene of Group 4 bacteria. The FG4F primer has a sequence complementary to the 469th to 489th base sequence of the rpoA4 gene, and the FG4R primer has a sequence complementary to the 609th to 629th base sequence of the rpoA4 gene.

The forward primer FG5F primer and reverse primer FG5R primer, which are composed of the polynucleotides represented by SEQ ID NO:9 and SEQ ID NO:10, respectively, are primers that amplify the rpoA5 gene, which is the rpoA gene of Group 5 bacteria. The FG5F primer has a sequence complementary to the 229th to 249th base sequence of the rpoA5 gene, and the FG5R primer has a sequence complementary to the 450th to 469th base sequence of the rpoA5 gene.

The forward primer FG6F primer and reverse primer FG6R primer, which are composed of the polynucleotides represented by SEQ ID NO:11 and SEQ ID NO:12, respectively, are primers that amplify the rpoA6 gene, which is the rpoA gene of Group 6 bacteria. The FG6F primer has a sequence complementary to the 430-450th base sequence of the rpoA6 gene, and the FG6R primer has a sequence complementary to the 561-581st base sequence of the rpoA6 gene.

The forward primer FG7F primer and reverse primer FG7R primer, which are composed of the polynucleotides represented by SEQ ID NO: 13 and SEQ ID NO: 14, respectively, are primers that amplify the rpoA7 gene, which is the rpoA gene of Group 7 bacteria. The FG7F primer has a sequence complementary to the 367th to 388th base sequence of the rpoA7 gene, and the FG7R primer has a sequence complementary to the 495th to 515th base sequence of the rpoA7 gene.

The forward primer FG8F primer and reverse primer FG8R primer, which are composed of the polynucleotides represented by SEQ ID NO: 15 and SEQ ID NO: 16, respectively, are primers that amplify the rpoA8 gene, which is the rpoA gene of Group 8 bacteria. The FG8F primer has a sequence complementary to the 534th to 554th base sequence of the rpoA8 gene, and the FG8R primer has a sequence complementary to the 696th to 715th base sequence of the rpoA8 gene.

The forward primer FG9F primer and reverse primer FG9R primer, which are composed of the polynucleotides represented by SEQ ID NO:17 and SEQ ID NO:18, respectively, are primers that amplify the rpoA9 gene, which is the rpoA gene of Group 9 bacteria. The FG9F primer has a sequence complementary to the 544th to 564th base sequence of the rpoA9 gene, and the FG9R primer has a sequence complementary to the 696th to 717th base sequence of the rpoA9 gene.

The forward and reverse primers may each be a polynucleotide containing a sequence of SEQ ID NO: 1 to 18. The forward primer may be a polynucleotide having 1 to 5 bases, preferably 1 to 4 bases, more preferably 1 to 3 bases, complementary to each rpoA gene, bound to at least one of the 3' and 5' ends of the sequence of SEQ ID NO: 1 to 18. The forward primer may also be a polynucleotide having 1 to 5 bases, preferably 1 to 4 bases, more preferably 1 to 3 bases, deleted from at least one of the 3' and 5' ends of the sequence of SEQ ID NO: 1 to 18. The forward primer may be a polynucleotide having 1 to 5 bases, preferably 1 to 4 bases, more preferably 1 to 3 bases, complementary to each rpoA gene, bound to at least one of the 5' ends of the sequence of SEQ ID NO: 1 to 18. The forward primer may also be a polynucleotide having 1 to 5 bases, preferably 1 to 4 bases, more preferably 1 to 3 bases, deleted from at least one of the 5' ends of the sequence of SEQ ID NO: 1 to 18.

As mentioned above, the rpoA gene sequence may differ partially from strain to strain, even within the same bacterial group. Therefore, in the present invention, the base sequence data of a total of 200 or more strains of F. prausnitzii and bacteria related to F. prausnitzii are used, and within the same bacterial species (i.e., within any of F. prausnitzii and Group 2 to Group 9 bacteria species), even if the rpoA gene sequences are different, the parts that have perfect sequence homology and differ from the sequences of other bacterial species are determined as the primer sequences.

The forward and reverse primers may be labeled with a fluorescent substance, a binding affinity substance such as biotin or digoxin, an enzyme, a radioisotope, or a luminescent substance. Examples of fluorescent substances include SYBR green, fluorescamine, and fluorescein isothiocyanate. Examples of enzymes include peroxidase, alkaline phosphatase, malate dehydratase, α-glucosidase, and α-galactosidase. Examples of radioisotopes include ¹²⁵ I, ¹³¹ I, ³ H, and ¹⁴ C. Examples of luminescent substances include luciferin, lucigenin, luminol, and luminol derivatives.

The nine pairs of forward and reverse primers are added to different samples. That is, the nine pairs of forward and reverse primers are added to each of the first to ninth samples. Therefore, it is possible to quantify each of the F. prausnitzii and F. prausnitzii-related bacteria contained in the sample. Therefore, it is possible to know the quantitative balance of F. prausnitzii and F. prausnitzii-related bacteria in the intestines of the sample donor. As a result, it is possible to investigate the correlation between the respective proportions of F. prausnitzii and F. prausnitzii-related bacteria in the intestines and the health status and diseases of the sample donor. In addition, the results of this investigation can be used to develop foods and beverages that are more effective in improving health status and medicines that are more effective in treating illnesses.

The procedure for quantifying F. prausnitzii and bacteria related to F. prausnitzii will be described. First, nucleic acid is extracted from a sample. The sample may be feces of mammals, including humans. The sample may also be health foods, medicines, etc., containing F. prausnitzii and bacteria related to F. prausnitzii. From the viewpoint of being able to investigate the balance of intestinal bacteria, it is preferable that the sample is human feces.

The method for extracting nucleic acid from a specimen and preparing a sample is not particularly limited, and can be performed using a known method. Examples include the magnetic bead method, the phenol/chloroform method, the cetyltrimethylammonium bromide (CTAB) method, and combinations of these. A commercially available kit may be used to extract nucleic acid.

At least nine samples are prepared, and nine pairs of forward and reverse primers are used for each different sample. An amplification reaction is carried out using each forward and reverse primer with the nucleic acid contained in the sample as a template. The amplification reaction and quantification of each bacterium are carried out by quantitative PCR.

As described above, according to the method for quantifying F. prausnitzii and F. prausnitzii-related bacteria in one aspect of the present invention, it is possible to accurately quantify F. prausnitzii and F. prausnitzii-related bacteria. Furthermore, according to the method for quantifying F. prausnitzii and F. prausnitzii-related bacteria in one aspect of the present invention, it is possible to individually quantify F. prausnitzii and F. prausnitzii-related bacteria.

In the above embodiment, all nine pairs of primers are used, but the present invention is not limited to this. F. prausnitzii can be quantified using only one pair of primers, for example, FG1F primer and FG1R primer. In addition, at least two pairs of primers out of the nine pairs of primers described above can be used to quantify at least two types of bacteria among F. prausnitzii and bacteria related to F. prausnitzii. This type of quantification makes it possible to more easily quantify the target bacterial species.

<Primer set>
In one embodiment of the present invention, the primer set is a primer set for amplifying at least a part of a gene encoding an RNA polymerase of F. prausnitzii and bacteria related to F. prausnitzii, comprising a polynucleotide represented by SEQ ID NO:1, a polynucleotide having the sequence of SEQ ID NO:1 and 1 to 5 nucleotide bases bound to at least one of the 3' and 5' ends, and a polynucleotide having 1 to 5 nucleotide bases deleted from at least one of the 3' and 5' ends of the sequence of SEQ ID NO:1, a polynucleotide represented by SEQ ID NO:2, a polynucleotide having the sequence of SEQ ID NO:2 and 1 to 5 nucleotide bases bound to at least one of the 3' and 5' ends, and a polynucleotide having 1 to 5 nucleotide bases deleted from at least one of the 3' and 5' ends of the sequence of SEQ ID NO:2, an FG1R primer which is any one of a polynucleotide represented by SEQ ID NO:3, a polynucleotide having the sequence of SEQ ID NO:3 and 1 to 5 nucleotides bound to at least one of its 3' end and 5' end, and a polynucleotide having 1 to 5 nucleotides deleted from at least one of the 3' end and 5' end of the sequence of SEQ ID NO:3; an FG2R primer which is any one of a polynucleotide represented by SEQ ID NO:4, a polynucleotide having the sequence of SEQ ID NO:4 and 1 to 5 nucleotides bound to at least one of its 3' end and 5' end, and a polynucleotide having 1 to 5 nucleotides deleted from at least one of the 3' end and 5' end of the sequence of SEQ ID NO:4; a polynucleotide represented by SEQ ID NO:5, a polynucleotide having the sequence of SEQ ID NO:5 and 1 to 5 nucleotides bound to at least one of its 3' end and 5' end, and a polynucleotide having 1 to 5 nucleotides deleted from at least one of the 3' end and 5' end of the sequence of SEQ ID NO:5; a polynucleotide having 1 to 5 nucleotide bases bound to at least one of the 3' and 5' ends of the sequence of SEQ ID NO:5, and a polynucleotide having 1 to 5 nucleotide bases deleted from at least one of the 3' and 5' ends of the sequence of SEQ ID NO:5; an FG3R primer which is one of a polynucleotide represented by SEQ ID NO:6, a polynucleotide having 1 to 5 nucleotide bases bound to at least one of the 3' and 5' ends of the sequence of SEQ ID NO:6, and a polynucleotide having 1 to 5 nucleotide bases deleted from at least one of the 3' and 5' ends of the sequence of SEQ ID NO:6; a polynucleotide represented by SEQ ID NO:7, a polynucleotide having 1 to 5 nucleotide bases bound to at least one of the 3' and 5' ends of the sequence of SEQ ID NO:7, and a polynucleotide having 1 to 5 nucleotide bases bound to at least one of the 3' and 5' ends of the sequence of SEQ ID NO:7. an FG4F primer which is any one of a polynucleotide having 1 to 5 nucleotide bases deleted from at least one of the 3' and 5'ends; a polynucleotide represented by SEQ ID NO:8, a polynucleotide having the sequence of SEQ ID NO:8 and 1 to 5 nucleotide bases bound to at least one of its 3' and 5' ends, and a polynucleotide having the sequence of SEQ ID NO:8 and 1 to 5 nucleotide bases deleted from at least one of its 3' and 5'ends; an FG4R primer which is any one of a polynucleotide represented by SEQ ID NO:9, a polynucleotide having the sequence of SEQ ID NO:9 and 1 to 5 nucleotide bases bound to at least one of its 3' and 5' ends, and a polynucleotide having the sequence of SEQ ID NO:9 and 1 to 5 nucleotide bases deleted from at least one of its 3' and 5' ends an FG5R primer which is any one of a polynucleotide represented by SEQ ID NO:10, a polynucleotide having the sequence of SEQ ID NO:10 and 1 to 5 nucleotides bound to at least one of its 3' and 5' ends, and a polynucleotide having 1 to 5 nucleotides deleted from at least one of the 3' and 5' ends of the sequence of SEQ ID NO:10; an FG6F primer which is any one of a polynucleotide represented by SEQ ID NO:11, a polynucleotide having the sequence of SEQ ID NO:11 and 1 to 5 nucleotides bound to at least one of its 3' and 5' ends, and a polynucleotide having 1 to 5 nucleotides deleted from at least one of the 3' and 5' ends of the sequence of SEQ ID NO:11; a polynucleotide represented by SEQ ID NO:12, a polynucleotide having an FG6R primer which is any one of a polynucleotide having a sequence of SEQ ID NO:13, a polynucleotide having a sequence of SEQ ID NO:13 and a 1- to 5-base nucleotide bound to at least one of its 3' and 5' ends, and a polynucleotide having a deletion of at least 1- to 5-base nucleotides at the 3' and 5' ends of the sequence of SEQ ID NO:12; an FG7F primer which is any one of a polynucleotide having a sequence of SEQ ID NO:13 and a 1- to 5-base nucleotide bound to at least one of its 3' and 5' ends, and a polynucleotide having a deletion of at least 1- to 5-base nucleotides at the 3' and 5' ends of the sequence of SEQ ID NO:13; a polynucleotide having a sequence of SEQ ID NO:14, a polynucleotide having a sequence of SEQ ID NO:14 and a 1- to 5-base nucleotide bound to at least one of its 3' and 5' ends a polynucleotide represented by SEQ ID NO:15, a polynucleotide having the sequence of SEQ ID NO:15 and 1 to 5 nucleotides bound to at least one of its 3' and 5' ends, and a polynucleotide having the sequence of SEQ ID NO:15 and 1 to 5 nucleotides missing from at least one of its 3' and 5'ends; an FG8F primer, a polynucleotide represented by SEQ ID NO:16, a polynucleotide having the sequence of SEQ ID NO:16 and 1 to 5 nucleotides bound to at least one of its 3' and 5' ends, and a polynucleotide having the sequence of SEQ ID NO:16 and 1 to 5 nucleotides missing from at least one of its 3' and 5'ends; the FG8R primer being any one of a polynucleotide represented by SEQ ID NO:17, a polynucleotide having the sequence of SEQ ID NO:17 and 1 to 5 nucleotide bases bound to at least one of its 3' end and 5' end, and a polynucleotide having the sequence of SEQ ID NO:17 lacking 1 to 5 nucleotide bases at least at either the 3' end or the 5'end; and an FG9F primer being any one of a polynucleotide represented by SEQ ID NO:18, a polynucleotide having the sequence of SEQ ID NO:18 and 1 to 5 nucleotide bases bound to at least one of its 3' end and 5' end, and a polynucleotide having the sequence of SEQ ID NO:18 lacking 1 to 5 nucleotide bases at least at either the 3' end or the 5' end.

Each primer is the same as the primer described in <Quantitative method for F. prausnitzii and bacteria related to F. prausnitzii>, so explanation is omitted.

<Quantitative kit for F. prausnitzii and related bacteria>
In one embodiment of the present invention, the kit for quantifying F. prausnitzii and bacteria related to F. prausnitzii includes a primer set, deoxynucleotide triphosphates, a DNA polymerase, and a pH buffer solution. The primer set is the same as the primers described in the above-mentioned "Method for quantifying F. prausnitzii and bacteria related to F. prausnitzii," so the description of the primer set is omitted.

The quantification kit of this embodiment further includes deoxynucleotide triphosphates (dNTPs), a DNA polymerase, and a buffer solution. The quantification kit of this embodiment may further include at least one of distilled water, a salt, a protein, a surfactant, and the like.

The present invention will be described in detail below with reference to examples, but the present invention is not limited to the following description.

<Primer design>
Using the base sequence data of a total of 235 strains, primer sequences were designed that had complete sequence homology within the same species (i.e., within any of F. prausnitzii and Group 2 to Group 9 bacteria species) and were different from sequences in other species. The sequences of the FG1F to FG9F primers and the FG1R to FG9R primers are shown in Table 1.

<Evaluation of primers>
Example 1
Representative strains were selected from each of the bacterial groups F. prausnitzii and Group 2 to Group 9 bacteria. More specifically, three strains of F. prausnitzii (strain names APC918/95b, APC923/51-1, and ATCC27768), two strains of Group 6 (strain names A2165 and P9122), and one strain each of Groups 2 to 5 and 7 to 9 (strain names CNCM I 4541, CNCM I 4542, 942/30-2, APC942/8-14-2, APC922/41-1, MGYG-HGUT-T00195, and AF52-21) were selected, for a total of 12 strains. Based on the sequences of the selected strains, Eurofins Genomics was requested to synthesize rpoA gene DNA1 to 12. The rpoA1 gene DNA sequence of the ATCC27768 strain, which is F. prausnitzii, is as shown in SEQ ID NO: 19. The rpoA1 gene DNA sequences of APC918/95b and APC923/51-1 do not completely match SEQ ID NO: 19, but the sequence portions of the forward and reverse primers FG21F and FG1R are identical. The rpoA2 gene DNA sequence of the CNCM I 4541 strain, which is a Group 2 bacterium, is as shown in SEQ ID NO: 20. The rpoA3 gene DNA sequence of the CNCM I 4542 strain, which is a Group 3 bacterium, is as shown in SEQ ID NO: 21. The rpoA4 gene DNA sequence of the 942/30-2 strain, which is a Group 4 bacterium, is as shown in SEQ ID NO: 22. The rpoA5 gene DNA sequence of the APC942/8-14-2 strain, which is a Group 5 bacterium, is as shown in Sequence Listing 23. The rpoA6 gene DNA sequence of the A2165 strain, which is a Group 6 bacterium, is as shown in Sequence Listing 24. The rpoA6 gene DNA sequence of the P9122 strain does not completely match SEQ ID NO: 24, but the sequence portions of the forward and reverse primers FG6F and FG6R match. The rpoA7 gene DNA sequence of the APC922/41-1 strain, which is a Group 7 bacterium, is as shown in SEQ ID NO: 25. The rpoA8 gene DNA sequence of the MGYG-HGUT-T00195 strain, which is a Group 8 bacterium, is as shown in SEQ ID NO: 26. The rpoA9 gene DNA sequence of the AF52-21 strain, which is a Group 9 bacterium, is as shown in Sequence Listing 27.

The FG1F to FG9F primers and the FG1R to FG9R primers shown in Table 1 were created.

Samples containing 100 pg (=approximately ^107.5 copies) of each of the synthesized rpoA gene DNAs 1 to 12 were prepared. To each sample, one of the following primer combinations was added: FG1F and FG1R primer combination, FG2F and FG2R primer combination, FG3F and FG3R primer combination, FG4F and FG4R primer combination, FG5F and FG5R primer combination, FG6F and FG6R primer combination, FG7F and FG7R primer combination, FG8F and FG8R primer combination, or FG9F and FG9R primer combination, and quantitative PCR was performed under the following conditions.

(Experimental conditions)
Equipment used: Real-time PCR device (Roche, LightCycler 96 system)
Reagent: Real-time PCR reagent (Roche, FastStart Essential DNA Green Master Mix)
PCR conditions: 95°C for 10 minutes, followed by 45 cycles of [95°C for 10 seconds, 60°C for 10 seconds, 72°C for 15 seconds] Data analysis: Data analysis software (manufactured by Roche, software included with LightCycler 96 system)

Figures 2(a)-2(b) show the results of quantitative PCR using the combinations of FG1F and FG1R primers and FG2F and FG2R primers, respectively. Figures 3(a)-3(b) show the results of quantitative PCR using the combinations of FG3F and FG3R primers and FG4F and FG4R primers, respectively. Figures 4(a)-4(b) show the results of quantitative PCR using the combinations of FG5F and FG5R primers and FG6F and FG6R primers, respectively. Figures 5(a)-5(b) show the results of quantitative PCR using the combinations of FG7F and FG7R primers and FG8F and FG8R primers, respectively. Figure 6 shows the results of quantitative PCR using the combination of FG9F and FG9R primers. In Figures 2-6, F. prausnitzii is referred to as Group 1. Figures 2 to 6 show the rpoA gene copy numbers when using rpoA gene DNAs 1 to 12 as templates and each primer pair.

The quantitative PCR results shown in Figures 2 to 6 indicate that only the target bacterial species of each primer pair could be quantified as expected. On the other hand, the quantitative values of the non-target bacterial species were significantly lower than the target bacterial species. Even the highest quantitative value of the non-target bacterial species was only about 1% of the target bacterial species. These results demonstrate that the quantitative method and primer set of the present invention can be used to accurately quantify Faecalibacterium prausnitzii and Faecalibacterium prausnitzii-related bacteria.

(Comparative Example 1)
Quantitative PCR was carried out under the following conditions using known primers specific to the 16S rRNA gene sequence disclosed in Non-Patent Document 2 and samples containing rpoA gene DNAs 1 to 11 used in Example 1. The primer sequences are shown in Table 2.

(Experimental conditions)
Equipment used: Real-time PCR device (Roche, LightCycler 96 system)
Reagent: Real-time PCR reagent (Roche, FastStart Essential DNA Green Master Mix)
PCR conditions: 95°C for 10 minutes, followed by 45 cycles of [95°C for 10 seconds, 60°C for 10 seconds (58°C for 10 seconds when primer pair 2 was used), 72°C for 15 seconds]. Data analysis: Data analysis software (manufactured by Roche, software included with LightCycler 96 system).

Figure 7(a) shows the results of quantitative PCR using primer pair 1 in Table 2, and Figure 7(b) shows the results of quantitative PCR using primer pair 2. Figures 7(a) and 7(b) show the number of rpoA gene copies when primer pair 1 and primer pair 2 in Table 2 were used with rpoA gene DNAs 1 to 12 as templates. When primer pair 1 was used, only a portion of Faecalibacterium prausnitzii and Faecalibacterium prausnitzii-related bacteria were quantified. When primer pair 2 was used, almost the entire Faecalibacterium prausnitzii and Faecalibacterium prausnitzii-related bacteria could be quantified, but each species of Faecalibacterium prausnitzii and Faecalibacterium prausnitzii-related bacteria could not be quantified.

Quantitative analysis of Faecalibacterium prausnitzii and Faecalibacterium prausnitzii-related bacteria in samples
Example 2
Feces from seven subjects were used as specimens. DNA was extracted from 200 mg of the specimen using a kit (Qiagen, product number 51504), and 1 μl of the resulting DNA solution was used as a sample.

FG1F to FG9F primers and FG1R to FG9R primers were added to the prepared sample to form one set of forward and reverse primers, and quantitative PCR was performed under the same conditions as in Example 1.

Figures 8(a) to 8(c) show the results of quantitative PCR using combinations of FG1F and FG1R primers, FG2F and FG2R primers, and FG3F and FG3R primers, respectively. Figures 9(a) to 9(c) show the results of quantitative PCR using combinations of FG4F and FG4R primers, FG5F and FG5R primers, and FG6F and FG6R primers, respectively. Figures 10(a) to 10(c) show the results of quantitative PCR using combinations of FG7F and FG7R primers, FG8F and FG8R primers, and FG9F and FG9R primers, respectively. In Figures 8 to 10, F. Prausnitzii is referred to as Group 1. Figures 8 to 10 show the number of bacteria (each rpoA gene copy number) per gram of sample for each subject.

The quantitative PCR results shown in Figures 8 to 10 indicate that only the bacterial species targeted by each primer pair could be quantified as expected. These results demonstrate that the quantitative method and primer set of the present invention can be used to accurately quantify Faecalibacterium prausnitzii and Faecalibacterium prausnitzii-related bacteria.

Claims (12)

carrying out a DNA amplification reaction using a sample containing nucleic acid as a template, and a primer set for amplifying at least a part of a gene encoding an RNA polymerase of Faecalibacterium prausnitzii and a bacterium related to Faecalibacterium prausnitzii;
A method for quantifying Faecalibacterium prausnitzii and bacteria related to Faecalibacterium prausnitzii, comprising quantifying Faecalibacterium prausnitzii and bacteria related to Faecalibacterium prausnitzii from the results of the DNA amplification reaction. The primer set is
an FG1F primer which is any one of a polynucleotide represented by SEQ ID NO:1, a polynucleotide having the sequence of SEQ ID NO:1 and 1 to 5 nucleotides bound to at least one of the 3' and 5' termini, and a polynucleotide having the sequence of SEQ ID NO:1 lacking 1 to 5 nucleotides at at least one of the 3' and 5'termini;
an FG1R primer which is any one of a polynucleotide represented by SEQ ID NO: 2, a polynucleotide having the sequence of SEQ ID NO: 2 and 1 to 5 nucleotides bound to at least one of the 3' and 5' termini, and a polynucleotide having the sequence of SEQ ID NO: 2 lacking 1 to 5 nucleotides at least one of the 3' and 5'termini;
an FG2F primer which is any one of a polynucleotide represented by SEQ ID NO: 3, a polynucleotide having the sequence of SEQ ID NO: 3 and 1 to 5 nucleotides bound to at least one of the 3' and 5' termini, and a polynucleotide having the sequence of SEQ ID NO: 3 lacking 1 to 5 nucleotides at at least one of the 3' and 5'termini;
an FG2R primer which is any one of a polynucleotide represented by SEQ ID NO: 4, a polynucleotide having the sequence of SEQ ID NO: 4 and 1 to 5 nucleotides bound to at least one of the 3' and 5' termini, and a polynucleotide having 1 to 5 nucleotides deleted from at least one of the 3' and 5' termini of the sequence of SEQ ID NO: 4;
an FG3F primer which is any one of a polynucleotide represented by SEQ ID NO:5, a polynucleotide having the sequence of SEQ ID NO:5 and 1 to 5 nucleotides bound to at least one of the 3' and 5' termini, and a polynucleotide having 1 to 5 nucleotides deleted from at least one of the 3' and 5' termini of the sequence of SEQ ID NO:5;
an FG3R primer which is any one of a polynucleotide represented by SEQ ID NO:6, a polynucleotide having the sequence of SEQ ID NO:6 and 1 to 5 nucleotides bound to at least one of the 3' and 5' termini, and a polynucleotide having 1 to 5 nucleotides deleted from at least one of the 3' and 5' termini of the sequence of SEQ ID NO:6;
an FG4F primer which is any one of a polynucleotide represented by SEQ ID NO: 7, a polynucleotide having the sequence of SEQ ID NO: 7 and 1 to 5 nucleotides bound to at least one of the 3' and 5' termini, and a polynucleotide having 1 to 5 nucleotides deleted from at least one of the 3' and 5' termini of the sequence of SEQ ID NO: 7;
an FG4R primer which is any one of a polynucleotide represented by SEQ ID NO:8, a polynucleotide having the sequence of SEQ ID NO:8 and 1 to 5 nucleotides bound to at least one of the 3' and 5' termini, and a polynucleotide having 1 to 5 nucleotides deleted from at least one of the 3' and 5' termini of the sequence of SEQ ID NO:8;
an FG5F primer which is any one of a polynucleotide represented by SEQ ID NO:9, a polynucleotide having the sequence of SEQ ID NO:9 and 1 to 5 nucleotides bound to at least one of the 3' and 5' termini, and a polynucleotide having 1 to 5 nucleotides deleted from at least one of the 3' and 5' termini of the sequence of SEQ ID NO:9;
an FG5R primer which is any one of a polynucleotide represented by SEQ ID NO:10, a polynucleotide having the sequence of SEQ ID NO:10 and 1 to 5 nucleotides bound to at least one of the 3' and 5' termini, and a polynucleotide having 1 to 5 nucleotides deleted from at least one of the 3' and 5' termini of the sequence of SEQ ID NO:10;
an FG6F primer which is any one of a polynucleotide represented by SEQ ID NO:11, a polynucleotide having the sequence of SEQ ID NO:11 and 1 to 5 nucleotides bound to at least one of the 3' and 5' termini thereof, and a polynucleotide having 1 to 5 nucleotides deleted from at least one of the 3' and 5' termini of the sequence of SEQ ID NO:11;
an FG6R primer which is any one of a polynucleotide represented by SEQ ID NO:12, a polynucleotide having the sequence of SEQ ID NO:12 and 1 to 5 nucleotides bound to at least one of the 3' and 5' termini, and a polynucleotide having 1 to 5 nucleotides deleted from at least one of the 3' and 5' termini of the sequence of SEQ ID NO:12;
an FG7F primer which is any one of a polynucleotide represented by SEQ ID NO:13, a polynucleotide having the sequence of SEQ ID NO:13 and 1 to 5 nucleotides bound to at least one of the 3' and 5' termini thereof, and a polynucleotide having 1 to 5 nucleotides deleted from at least one of the 3' and 5' termini of the sequence of SEQ ID NO:13;
an FG7R primer which is any one of a polynucleotide represented by SEQ ID NO:14, a polynucleotide having the sequence of SEQ ID NO:14 and 1 to 5 nucleotides bound to at least one of the 3' and 5' termini, and a polynucleotide having 1 to 5 nucleotides deleted from at least one of the 3' and 5' termini of the sequence of SEQ ID NO:14;
an FG8F primer which is any one of a polynucleotide represented by SEQ ID NO:15, a polynucleotide having the sequence of SEQ ID NO:15 and 1 to 5 nucleotides bound to at least one of the 3' and 5' termini, and a polynucleotide having a deletion of 1 to 5 nucleotides from at least one of the 3' and 5' termini of the sequence of SEQ ID NO:15;
an FG8R primer which is any one of a polynucleotide represented by SEQ ID NO:16, a polynucleotide having the sequence of SEQ ID NO:16 and 1 to 5 nucleotides bound to at least one of the 3' and 5' termini, and a polynucleotide having 1 to 5 nucleotides deleted from at least one of the 3' and 5' termini of the sequence of SEQ ID NO:16;
2. The method for quantifying according to claim 1, comprising an FG9F primer which is any one of a polynucleotide represented by SEQ ID NO:17, a polynucleotide having the sequence of SEQ ID NO:17 and 1 to 5 nucleotide bases bound to at least one of its 3' end and 5' end, and a polynucleotide having 1 to 5 nucleotide bases deleted from at least one of the 3' end and 5' end of the sequence of SEQ ID NO:17; and an FG9R primer which is any one of a polynucleotide represented by SEQ ID NO:18, a polynucleotide having the sequence of SEQ ID NO:18 and 1 to 5 nucleotide bases bound to at least one of its 3' end and 5' end, and a polynucleotide having 1 to 5 nucleotide bases deleted from at least one of the 3' end and 5' end of the sequence of SEQ ID NO:18. The primer set is
an FG1F primer which is any one of a polynucleotide represented by SEQ ID NO:1, a polynucleotide having the sequence of SEQ ID NO:1 and 1 to 5 nucleotides linked to its 5'-terminus, and a polynucleotide having the sequence of SEQ ID NO:1 lacking 1 to 5 nucleotides at the 5'-terminus;
an FG1R primer which is any one of a polynucleotide represented by SEQ ID NO: 2, a polynucleotide having the sequence of SEQ ID NO: 2 and 1 to 5 nucleotides linked to its 5'-terminus, and a polynucleotide having the sequence of SEQ ID NO: 2 lacking 1 to 5 nucleotides at the 5'-terminus;
an FG2F primer which is any one of a polynucleotide represented by SEQ ID NO: 3, a polynucleotide having the sequence of SEQ ID NO: 3 and 1 to 5 nucleotides linked to its 5'-terminus, and a polynucleotide having the sequence of SEQ ID NO: 3 lacking 1 to 5 nucleotides at the 5'-terminus;
an FG2R primer which is any one of a polynucleotide represented by SEQ ID NO: 4, a polynucleotide having the sequence of SEQ ID NO: 4 and 1 to 5 nucleotides linked to its 5'-terminus, and a polynucleotide having the sequence of SEQ ID NO: 4 deleted from the 5'-terminus of 1 to 5 nucleotides;
an FG3F primer which is any one of a polynucleotide represented by SEQ ID NO:5, a polynucleotide having the sequence of SEQ ID NO:5 and 1 to 5 nucleotides linked to its 5'-terminus, and a polynucleotide having the sequence of SEQ ID NO:5 lacking 1 to 5 nucleotides at the 5'-terminus;
an FG3R primer which is any one of a polynucleotide represented by SEQ ID NO:6, a polynucleotide having the sequence of SEQ ID NO:6 and 1 to 5 nucleotides linked to its 5'-terminus, and a polynucleotide having the sequence of SEQ ID NO:6 lacking 1 to 5 nucleotides at the 5'-terminus;
an FG4F primer which is any one of a polynucleotide represented by SEQ ID NO: 7, a polynucleotide having the sequence of SEQ ID NO: 7 and 1 to 5 nucleotides bound to its 5'-terminus, and a polynucleotide having the sequence of SEQ ID NO: 7 deleted from the 5'-terminus of 1 to 5 nucleotides;
an FG4R primer which is any one of a polynucleotide represented by SEQ ID NO:8, a polynucleotide having the sequence of SEQ ID NO:8 and 1 to 5 nucleotides linked to its 5'-terminus, and a polynucleotide having the sequence of SEQ ID NO:8 lacking 1 to 5 nucleotides at the 5'-terminus;
an FG5F primer which is any one of a polynucleotide represented by SEQ ID NO:9, a polynucleotide having the sequence of SEQ ID NO:9 and 1 to 5 nucleotides bound to its 5'-terminus, and a polynucleotide having the sequence of SEQ ID NO:9 lacking 1 to 5 nucleotides at the 5'-terminus;
FG5R primer is any one of a polynucleotide represented by SEQ ID NO: 10, a polynucleotide having the sequence of SEQ ID NO: 10 and 1 to 5 nucleotides bound to the 5'-terminus thereof, and a polynucleotide having the sequence of SEQ ID NO: 10 lacking 1 to 5 nucleotides at the 5'-terminus thereof;
an FG6F primer which is any one of a polynucleotide represented by SEQ ID NO:11, a polynucleotide having the sequence of SEQ ID NO:11 and 1 to 5 nucleotides linked to its 5'-terminus, and a polynucleotide having the sequence of SEQ ID NO:11 deleted from the 5'-terminus of 1 to 5 nucleotides;
an FG6R primer which is any one of a polynucleotide represented by SEQ ID NO:12, a polynucleotide having the sequence of SEQ ID NO:12 and 1 to 5 nucleotides bound to its 5'-terminus, and a polynucleotide having the sequence of SEQ ID NO:12 lacking 1 to 5 nucleotides at the 5'-terminus;
an FG7F primer which is any one of a polynucleotide represented by SEQ ID NO:13, a polynucleotide having the sequence of SEQ ID NO:13 and 1 to 5 nucleotides bound to its 5'-terminus, and a polynucleotide having the sequence of SEQ ID NO:13 lacking 1 to 5 nucleotides at the 5'-terminus;
FG7R primer is any one of a polynucleotide represented by SEQ ID NO: 14, a polynucleotide having the sequence of SEQ ID NO: 14 and 1 to 5 nucleotides bound to its 5'-terminus, and a polynucleotide having the sequence of SEQ ID NO: 14 deleted from the 5'-terminus of 1 to 5 nucleotides;
an FG8F primer which is any one of a polynucleotide represented by SEQ ID NO:15, a polynucleotide having the sequence of SEQ ID NO:15 and 1 to 5 nucleotides bound to its 5'-terminus, and a polynucleotide having the sequence of SEQ ID NO:15 lacking 1 to 5 nucleotides at the 5'-terminus;
FG8R primer is any one of a polynucleotide represented by SEQ ID NO: 16, a polynucleotide having the sequence of SEQ ID NO: 16 and 1 to 5 nucleotides bound to its 5'-terminus, and a polynucleotide having the sequence of SEQ ID NO: 16 deleted from the 5'-terminus of 1 to 5 nucleotides;
2. The method for quantifying according to claim 1, comprising: an FG9F primer which is any one of a polynucleotide represented by SEQ ID NO:17, a polynucleotide having the sequence of SEQ ID NO:17 and 1 to 5 nucleotide bases bound to its 5' end, and a polynucleotide having 1 to 5 nucleotide bases deleted from the 5' end of the sequence of SEQ ID NO:17; and an FG9R primer which is any one of a polynucleotide represented by SEQ ID NO:18, a polynucleotide having the sequence of SEQ ID NO:18 and 1 to 5 nucleotide bases bound to its 5' end, and a polynucleotide having 1 to 5 nucleotide bases deleted from the 5' end of the sequence of SEQ ID NO:18. The primer set is
FG1F primer, which is a polynucleotide represented by SEQ ID NO:1;
FG1R primer, which is a polynucleotide represented by SEQ ID NO:2;
FG2F primer, which is a polynucleotide represented by SEQ ID NO:3;
FG2R primer, which is a polynucleotide represented by SEQ ID NO:4;
FG3F primer, which is a polynucleotide represented by SEQ ID NO:5;
FG3R primer, which is a polynucleotide represented by SEQ ID NO:6;
FG4F primer, which is a polynucleotide represented by SEQ ID NO:7;
FG4R primer, which is a polynucleotide represented by SEQ ID NO:8;
FG5F primer, which is a polynucleotide represented by SEQ ID NO:9;
FG5R primer, which is a polynucleotide represented by SEQ ID NO: 10;
FG6F primer, which is a polynucleotide represented by SEQ ID NO: 11;
FG6R primer, which is a polynucleotide represented by SEQ ID NO: 12;
FG7F primer, which is a polynucleotide represented by SEQ ID NO: 13;
FG7R primer, which is a polynucleotide represented by SEQ ID NO: 14;
FG8F primer, which is a polynucleotide represented by SEQ ID NO: 15;
FG8R primer, which is a polynucleotide represented by SEQ ID NO: 16;
The quantitative method according to claim 1, comprising: an FG9F primer which is a polynucleotide represented by SEQ ID NO:17; and an FG9R primer which is a polynucleotide represented by SEQ ID NO:18. The samples include samples 1 to 9,
The method for quantifying DNA according to any one of claims 2 to 4, wherein the DNA amplification reaction comprises adding the FG1F primer and the FG1R primer to the first sample, adding the FG2F primer and the FG2R primer to the second sample, adding the FG3F primer and the FG3R primer to the third sample, adding the FG4F primer and the FG4R primer to the fourth sample, adding the FG5F primer and the FG5R primer to the fifth sample, adding the FG6F primer and the FG6R primer to the sixth sample, adding the FG7F primer and the FG7R primer to the seventh sample, adding the FG8F primer and the FG8R primer to the eighth sample, and adding the FG9F primer and the FG9R primer to the ninth sample. The method according to claim 5, wherein quantifying the Faecalibacterium prausnitzii and Faecalibacterium prausnitzii-related bacteria includes quantifying one of Faecalibacterium prausnitzii and Faecalibacterium prausnitzii-related bacteria in each of the first to ninth samples based on the results of the DNA amplification reaction in the first to ninth samples. further comprising extracting the nucleic acid from the sample;
The method for quantifying a nucleic acid according to any one of claims 1 to 4, further comprising preparing the sample containing the nucleic acid. The quantitative method according to claim 7, wherein the sample includes feces. A primer set for amplifying at least a part of a gene encoding an RNA polymerase of Faecalibacterium prausnitzii and bacteria related to Faecalibacterium prausnitzii, comprising:
an FG1F primer which is any one of a polynucleotide represented by SEQ ID NO:1, a polynucleotide having the sequence of SEQ ID NO:1 and 1 to 5 nucleotide bases bound to at least one of the 3' and 5' termini, and a polynucleotide having the sequence of SEQ ID NO:1 lacking 1 to 5 nucleotide bases at at least one of the 3' and 5'termini;
an FG1R primer which is any one of a polynucleotide represented by SEQ ID NO: 2, a polynucleotide having the sequence of SEQ ID NO: 2 and 1 to 5 nucleotides bound to at least one of the 3' and 5' termini, and a polynucleotide having the sequence of SEQ ID NO: 2 lacking 1 to 5 nucleotides at least one of the 3' and 5'termini;
an FG2F primer which is any one of a polynucleotide represented by SEQ ID NO: 3, a polynucleotide having the sequence of SEQ ID NO: 3 and 1 to 5 nucleotides bound to at least one of the 3' and 5' ends thereof, and a polynucleotide having a deletion of 1 to 5 nucleotides from at least one of the 3' and 5' ends of the sequence of SEQ ID NO: 3;
an FG2R primer which is any one of a polynucleotide represented by SEQ ID NO: 4, a polynucleotide having the sequence of SEQ ID NO: 4 and 1 to 5 nucleotides bound to at least one of the 3' and 5' termini, and a polynucleotide having 1 to 5 nucleotides deleted from at least one of the 3' and 5' termini of the sequence of SEQ ID NO: 4;
an FG3F primer which is any one of a polynucleotide represented by SEQ ID NO:5, a polynucleotide having the sequence of SEQ ID NO:5 and 1 to 5 nucleotide bases bound to at least one of the 3' and 5' termini, and a polynucleotide having the sequence of SEQ ID NO:5 lacking 1 to 5 nucleotide bases at at least one of the 3' and 5'termini;
an FG3R primer which is any one of a polynucleotide represented by SEQ ID NO:6, a polynucleotide having the sequence of SEQ ID NO:6 and 1 to 5 nucleotides bound to at least one of the 3' and 5' termini, and a polynucleotide having 1 to 5 nucleotides deleted from at least one of the 3' and 5' termini of the sequence of SEQ ID NO:6;
an FG4F primer which is any one of a polynucleotide represented by SEQ ID NO: 7, a polynucleotide having the sequence of SEQ ID NO: 7 and 1 to 5 nucleotides bound to at least one of the 3' and 5' termini, and a polynucleotide having 1 to 5 nucleotides deleted from at least one of the 3' and 5' termini of the sequence of SEQ ID NO: 7;
an FG4R primer which is any one of a polynucleotide represented by SEQ ID NO:8, a polynucleotide having the sequence of SEQ ID NO:8 and 1 to 5 nucleotides bound to at least one of the 3' and 5' termini, and a polynucleotide having 1 to 5 nucleotides deleted from at least one of the 3' and 5' termini of the sequence of SEQ ID NO:8;
an FG5F primer which is any one of a polynucleotide represented by SEQ ID NO:9, a polynucleotide having the sequence of SEQ ID NO:9 and 1 to 5 nucleotides bound to at least one of the 3' and 5' termini, and a polynucleotide having 1 to 5 nucleotides deleted from at least one of the 3' and 5' termini of the sequence of SEQ ID NO:9;
an FG5R primer which is any one of a polynucleotide represented by SEQ ID NO:10, a polynucleotide having the sequence of SEQ ID NO:10 and 1 to 5 nucleotides bound to at least one of the 3' and 5' termini, and a polynucleotide having 1 to 5 nucleotides deleted from at least one of the 3' and 5' termini of the sequence of SEQ ID NO:10;
an FG6F primer which is any one of a polynucleotide represented by SEQ ID NO:11, a polynucleotide having the sequence of SEQ ID NO:11 and 1 to 5 nucleotides bound to at least one of the 3' and 5' termini thereof, and a polynucleotide having 1 to 5 nucleotides deleted from at least one of the 3' and 5' termini of the sequence of SEQ ID NO:11;
an FG6R primer which is any one of a polynucleotide represented by SEQ ID NO:12, a polynucleotide having the sequence of SEQ ID NO:12 and 1 to 5 nucleotides bound to at least one of the 3' and 5' termini, and a polynucleotide having 1 to 5 nucleotides deleted from at least one of the 3' and 5' termini of the sequence of SEQ ID NO:12;
an FG7F primer which is any one of a polynucleotide represented by SEQ ID NO:13, a polynucleotide having the sequence of SEQ ID NO:13 and 1 to 5 nucleotides bound to at least one of the 3' and 5' termini thereof, and a polynucleotide having 1 to 5 nucleotides deleted from at least one of the 3' and 5' termini of the sequence of SEQ ID NO:13;
an FG7R primer which is any one of a polynucleotide represented by SEQ ID NO:14, a polynucleotide having the sequence of SEQ ID NO:14 and 1 to 5 nucleotides bound to at least one of the 3' and 5' termini, and a polynucleotide having 1 to 5 nucleotides deleted from at least one of the 3' and 5' termini of the sequence of SEQ ID NO:14;
an FG8F primer which is any one of a polynucleotide represented by SEQ ID NO:15, a polynucleotide having the sequence of SEQ ID NO:15 and 1 to 5 nucleotides bound to at least one of the 3' and 5' termini, and a polynucleotide having a deletion of 1 to 5 nucleotides from at least one of the 3' and 5' termini of the sequence of SEQ ID NO:15;
an FG8R primer which is any one of a polynucleotide represented by SEQ ID NO:16, a polynucleotide having the sequence of SEQ ID NO:16 and 1 to 5 nucleotides bound to at least one of the 3' and 5' termini thereof, and a polynucleotide having 1 to 5 nucleotides deleted from at least one of the 3' and 5' termini of the sequence of SEQ ID NO:16;
A primer set consisting of an FG9F primer which is any one of a polynucleotide represented by SEQ ID NO:17, a polynucleotide having the sequence of SEQ ID NO:17 and 1 to 5 nucleotide bases bound to at least one of its 3' end and 5' end, and a polynucleotide having 1 to 5 nucleotide bases deleted from at least one of the 3' end and 5' end of the sequence of SEQ ID NO:17, and an FG9R primer which is any one of a polynucleotide represented by SEQ ID NO:18, a polynucleotide having the sequence of SEQ ID NO:18 and 1 to 5 nucleotide bases bound to at least one of its 3' end and 5' end, and a polynucleotide having 1 to 5 nucleotide bases deleted from at least one of the 3' end and 5' end of the sequence of SEQ ID NO:18. The primer set is
an FG1F primer which is any one of a polynucleotide represented by SEQ ID NO:1, a polynucleotide having the sequence of SEQ ID NO:1 and 1 to 5 nucleotides linked to its 5'-terminus, and a polynucleotide having the sequence of SEQ ID NO:1 lacking 1 to 5 nucleotides at the 5'-terminus;
an FG1R primer which is any one of a polynucleotide represented by SEQ ID NO: 2, a polynucleotide having the sequence of SEQ ID NO: 2 and 1 to 5 nucleotides linked to its 5'-terminus, and a polynucleotide having the sequence of SEQ ID NO: 2 lacking 1 to 5 nucleotides at the 5'-terminus;
an FG2F primer which is any one of a polynucleotide represented by SEQ ID NO: 3, a polynucleotide having the sequence of SEQ ID NO: 3 and 1 to 5 nucleotides linked to its 5'-terminus, and a polynucleotide having the sequence of SEQ ID NO: 3 lacking 1 to 5 nucleotides at the 5'-terminus;
an FG2R primer which is any one of a polynucleotide represented by SEQ ID NO: 4, a polynucleotide having the sequence of SEQ ID NO: 4 and 1 to 5 nucleotides linked to its 5'-terminus, and a polynucleotide having the sequence of SEQ ID NO: 4 deleted from the 5'-terminus of 1 to 5 nucleotides;
an FG3F primer which is any one of a polynucleotide represented by SEQ ID NO:5, a polynucleotide having the sequence of SEQ ID NO:5 and 1 to 5 nucleotides linked to its 5'-terminus, and a polynucleotide having the sequence of SEQ ID NO:5 lacking 1 to 5 nucleotides at the 5'-terminus;
an FG3R primer which is any one of a polynucleotide represented by SEQ ID NO:6, a polynucleotide having the sequence of SEQ ID NO:6 and 1 to 5 nucleotides linked to its 5'-terminus, and a polynucleotide having the sequence of SEQ ID NO:6 lacking 1 to 5 nucleotides at the 5'-terminus;
an FG4F primer which is any one of a polynucleotide represented by SEQ ID NO: 7, a polynucleotide having the sequence of SEQ ID NO: 7 and 1 to 5 nucleotides bound to its 5'-terminus, and a polynucleotide having the sequence of SEQ ID NO: 7 deleted from the 5'-terminus of 1 to 5 nucleotides;
an FG4R primer which is any one of a polynucleotide represented by SEQ ID NO:8, a polynucleotide having the sequence of SEQ ID NO:8 and 1 to 5 nucleotides linked to its 5'-terminus, and a polynucleotide having the sequence of SEQ ID NO:8 lacking 1 to 5 nucleotides at the 5'-terminus;
an FG5F primer which is any one of a polynucleotide represented by SEQ ID NO:9, a polynucleotide having the sequence of SEQ ID NO:9 and 1 to 5 nucleotides bound to its 5'-terminus, and a polynucleotide having the sequence of SEQ ID NO:9 lacking 1 to 5 nucleotides at the 5'-terminus;
FG5R primer is any one of a polynucleotide represented by SEQ ID NO: 10, a polynucleotide having the sequence of SEQ ID NO: 10 and 1 to 5 nucleotides bound to the 5'-terminus thereof, and a polynucleotide having the sequence of SEQ ID NO: 10 lacking 1 to 5 nucleotides at the 5'-terminus thereof;
an FG6F primer which is any one of a polynucleotide represented by SEQ ID NO:11, a polynucleotide having the sequence of SEQ ID NO:11 and 1 to 5 nucleotides linked to its 5'-terminus, and a polynucleotide having the sequence of SEQ ID NO:11 deleted from the 5'-terminus of 1 to 5 nucleotides;
an FG6R primer which is any one of a polynucleotide represented by SEQ ID NO:12, a polynucleotide having the sequence of SEQ ID NO:12 and 1 to 5 nucleotides bound to its 5'-terminus, and a polynucleotide having the sequence of SEQ ID NO:12 lacking 1 to 5 nucleotides at the 5'-terminus;
an FG7F primer which is any one of a polynucleotide represented by SEQ ID NO:13, a polynucleotide having the sequence of SEQ ID NO:13 and 1 to 5 nucleotides bound to its 5'-terminus, and a polynucleotide having the sequence of SEQ ID NO:13 lacking 1 to 5 nucleotides at the 5'-terminus;
FG7R primer is any one of a polynucleotide represented by SEQ ID NO: 14, a polynucleotide having the sequence of SEQ ID NO: 14 and 1 to 5 nucleotides bound to its 5'-terminus, and a polynucleotide having the sequence of SEQ ID NO: 14 deleted from the 5'-terminus of 1 to 5 nucleotides;
an FG8F primer which is any one of a polynucleotide represented by SEQ ID NO:15, a polynucleotide having the sequence of SEQ ID NO:15 and 1 to 5 nucleotides bound to its 5'-terminus, and a polynucleotide having the sequence of SEQ ID NO:15 lacking 1 to 5 nucleotides at the 5'-terminus;
FG8R primer is any one of a polynucleotide represented by SEQ ID NO: 16, a polynucleotide having the sequence of SEQ ID NO: 16 and 1 to 5 nucleotides bound to its 5'-terminus, and a polynucleotide having the sequence of SEQ ID NO: 16 deleted from the 5'-terminus of 1 to 5 nucleotides;
10. The primer set according to claim 9, comprising: an FG9F primer which is any one of a polynucleotide represented by SEQ ID NO:17, a polynucleotide having the sequence of SEQ ID NO:17 and 1 to 5 nucleotide bases bound to its 5' end, and a polynucleotide having 1 to 5 nucleotide bases deleted from the 5' end of the sequence of SEQ ID NO:17; and an FG9R primer which is any one of a polynucleotide represented by SEQ ID NO:18, a polynucleotide having the sequence of SEQ ID NO:18 and 1 to 5 nucleotide bases bound to its 5' end, and a polynucleotide having 1 to 5 nucleotide bases deleted from the 5' end of the sequence of SEQ ID NO:18. The primer set is
FG1F primer, which is a polynucleotide represented by SEQ ID NO:1;
FG1R primer, which is a polynucleotide represented by SEQ ID NO:2;
FG2F primer, which is a polynucleotide represented by SEQ ID NO:3;
FG2R primer, which is a polynucleotide represented by SEQ ID NO:4;
FG3F primer, which is a polynucleotide represented by SEQ ID NO:5;
FG3R primer, which is a polynucleotide represented by SEQ ID NO:6;
FG4F primer, which is a polynucleotide represented by SEQ ID NO:7;
FG4R primer, which is a polynucleotide represented by SEQ ID NO:8;
FG5F primer, which is a polynucleotide represented by SEQ ID NO:9;
FG5R primer, which is a polynucleotide represented by SEQ ID NO: 10;
FG6F primer, which is a polynucleotide represented by SEQ ID NO: 11;
FG6R primer, which is a polynucleotide represented by SEQ ID NO: 12;
FG7F primer, which is a polynucleotide represented by SEQ ID NO: 13;
FG7R primer, which is a polynucleotide represented by SEQ ID NO: 14;
FG8F primer, which is a polynucleotide represented by SEQ ID NO: 15;
FG8R primer, which is a polynucleotide represented by SEQ ID NO: 16;
The primer set according to claim 9, comprising an FG9F primer which is a polynucleotide represented by SEQ ID NO:17, and an FG9R primer which is a polynucleotide represented by SEQ ID NO:18. A quantitative determination kit for Faecalibacterium prausnitzii and bacteria related to Faecalibacterium prausnitzii, comprising the primer set according to any one of claims 9 to 11, deoxynucleotide triphosphates, DNA polymerase, and a pH buffer solution.

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