One or more primers or probes specific for Bacteroides fragilis

Abstract

To specifically detecting Bacteroides fragilis by avoiding non-specific acquisition of information from organisms other than Bacteroides fragilis (e.g., other closely related Bacteroidales bacterium, and host such as human).SOLUTION: Provided are one or more primers or probes specific for Bacteroides fragilis, which can detect one or more genes such as 3-oxo-5-α-steroid 4-dehydrogenase 1 (KO number: K12343); a method for detecting Bacteroides fragilis, which comprises detecting Bacteroides fragilis using the primer or probe; and a reagent for detecting Bacteroides fragilis, which comprises the primer or probe.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to one or more primers or probes specific to Bacteroides fragilis, etc.

Background Art

[0002] When comparing the composition and ratio of specific bacterial species, data from comprehensive analyses such as sequence analysis targeting 16S rRNA and shotgun metagenomic analysis are usually used. However, in addition to requiring enormous costs and complex information analysis processes, comprehensive analysis also has the problem of non-specifically obtaining information from organisms that are not the detection targets (e.g., related species, hosts).

[0003] In addition, since there are multiple copies of 16S rRNA in the bacterial genome, there is also a problem that the measured value based on the detection of 16S rRNA becomes a value obtained by multiplying the bacterial amount by the copy number.

[0004] In fact, it has been suggested that reported detection systems for the 16S rRNA gene non-specifically detect multiple bacteria, and it has been reported that the quantitative measurement value by quantitative PCR (qPCR) targeting the 16S rRNA gene is several times the actual number of bacteria (Patent Documents 1 and 2, and Non-Patent Document 1).

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Non-Patent Documents

[0006]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0007] Bacteroides fragilis is one of the major bacteria that make up the normal human gut microbiota. Depending on the health status, it can cause diseases in some cases, but it is also known to have a beneficial effect of regulating immunity and suppressing intestinal inflammation, and is regarded as an opportunistic bacterium. Therefore, the amount of Bacteroides fragilis present in the human intestine, etc. is considered useful as an indicator for evaluating the biological environment. The amount of Bacteroides fragilis can be indirectly determined by measuring the amount of Bacteroides fragilis present in samples such as human feces. Therefore, specific detection and quantification of Bacteroides fragilis present in human samples such as feces are desired.

[0008] An object of the present invention is to specifically detect Bacteroides fragilis by avoiding non - specific acquisition of information derived from organisms other than Bacteroides fragilis (e.g., other related Bacteroides species bacteria, hosts such as humans).

[0009] A further object of the present invention is to quantitatively detect Bacteroides fragilis so as to better reflect the actual number of bacteria in a quantitative method such as quantitative PCR (qPCR).

Means for Solving the Problems

[0010] As a result of intensive studies, the inventors conceived that the above problems can be solved by using a nucleic acid molecule that is specific to Bacteroides fragilis and can only be found in single copy as a detection target. Based on this concept, the inventors identified such a gene in Bacteroides fragilis, thereby providing a nucleic acid molecule that targets such a gene, and successfully detecting Bacteroides fragilis quantitatively using such a nucleic acid molecule, thus completing the present invention.

[0011] That is, the present invention is as follows. [1] One or more primers or probes specific to Bacteroides fragilis that can detect one or more genes selected from the group consisting of the following 1) to 49): 1) 3-oxo-5-α-steroid 4-dehydrogenase 1 (KO number: K12343); 2) CRP / FNR family transcriptional regulator, polysaccharide utilization system transcriptional regulator (KO number: K21556); 3) Chondroitin AC lyase (KO number: K19049); 4) Dipeptidyl-peptidase III (KO number: K01277); 5) α-N-acetylglucosaminidase (KO number: K01205); 6) (R)-citramalate synthase (KO number: K09011); 7) α-amylase (KO number: K07405); 8) 4-O-β-D-mannosyl-D-glucose phosphorylase (KO number: K16212); 9) N-acetylglucosamine 2-epimerase (KO number: K01787); 10) Glucan 1,4-α-glucosidase (KO number: K21574); 11) β-1,4-manno-oligosaccharide / β-1,4-mannosyl-N-acetylglucosamine phosphorylase (KO number: K18785); 12) Starch-binding outer membrane protein SusE / F (KO number: K21571); 13) Mannan endo-1,4-β-mannosidase (KO number: K19355); 14) 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (KO number: K15635); 15) N-succinyl-L-ornithine transcarbamylase (KO number: K13043); 16) TonB-dependent starch-binding outer membrane protein SusC (KO number: K21573); 17) UDP-3-O-[3-hydroxymyristoyl] N-acetylglucosamine deacetylase / 3-hydroxyacyl-[acyl-carrier-protein] dehydratase (KO number: K16363); 18) Cytochrome c nitrite reductase small subunit (KO number: K15876); 19) Histidine decarboxylase (KO number: K01590); 20) Uncharacterized protein (KO number: K07079); 21) Diphosphate-dependent phosphofructokinase (KO number: K00895); 22) Diaminopimelate dehydrogenase (KO number: K03340); 23) Uncharacterized protein (KO number: K01163); 24) Sialate O-acetyl esterase (KO number: K05970); 25) Aspartate 4-decarboxylase (KO number: K09758); 26) Hyaluronoglucosaminidase (KO number: K01197); 27) Trichorne protease (KO number: K08676); 28) Arabinan endo-1,5-α-L-arabinofuranosidase (KO number: K06113); 29) 2-oxoglutarate ferredoxin oxidoreductase subunit δ (KO number: K00176); 30) 2-Oxoglutarate ferredoxin oxidoreductase subunit γ (KO number: K00177); 31) Two-component system, NtrC family, response regulator (KO number: K02481); 32) α-L-Fucosidase 2 (KO number: K15923); 33) Mannan endo-1,4-β-mannosidase (KO number: K01218); 34) V / A-type H+ / Na+ transport ATPase subunit E (KO number: K02121); 35) Sialidase-1 (KO number: K01186); 36) V / A-type H+ / Na+ transport ATPase subunit D (KO number: K02120); 37) V / A-type H+ / Na+ transport ATPase subunit I (KO number: K02123); 38) V / A-type H+ / Na+ transport ATPase subunit A (KO number: K02117); 39) V / A-type H+ / Na+ transport ATPase subunit K (KO number: K02124); 40) V / A-type H+ / Na+ transport ATPase subunit B (KO number: K02118); 41) Tryptophan synthase β chain (KO number: K06001); 42) Pyruvate carboxylase subunit B (KO number: K01960); 43) Chorismate mutase (KO number: K04516); 44) Phosphate-selective porins OprO and OprP (KO number: K07221); 45) Phosphate butyryltransferase (KO number: K00634); 46) LL-Diaminopimelate aminotransferase (KO number: K10206); 47) 2-Oxoisovalerate dehydrogenase E1 component (KO number: K11381); 48) Dipeptidyl-peptidase 4 (KO number: K01278); and 49) α-L-fucosidase (KO number: K01206). 〔2〕The primer or probe according to 〔1〕, wherein the primer or probe contains a partial base sequence containing at least 15 consecutive nucleotide residues in one or more base sequences selected from the group consisting of the base sequences of SEQ ID NOs: 1 to 49, or a complementary base sequence thereof. 〔3〕The primer or probe according to 〔2〕, wherein the partial base sequence contains nucleotide residues in a partial region specific to one or more base sequences selected from the group consisting of the base sequences of SEQ ID NOs: 1 to 49 as at least 15 consecutive nucleotide residues. 〔4〕The primer or probe according to any one of 〔1〕 to 〔3〕, wherein the primer or probe is two or more primers. 〔5〕A method for detecting Bacteroides fragilis, comprising detecting Bacteroides fragilis in a sample containing Bacteroides fragilis obtained from a subject using the primer or probe according to any one of 〔1〕 to 〔4〕. 〔6〕The method according to 〔5〕, wherein the detection of Bacteroides fragilis is performed by a gene amplification method using two or more primers. 〔7〕The method according to 〔6〕, wherein the gene amplification method is PCR. 〔8〕The method according to any one of 〔5〕 to 〔7〕, wherein the detection is performed quantitatively. 〔9〕A detection reagent for Bacteroides fragilis, comprising the primer or probe according to any one of 〔1〕 to 〔4〕.

Advantages of the Invention

[0012] According to the present invention, since a gene specific to Bacteroides fragilis is the detection target, non-specific detection can be avoided. Further, since the present invention targets a single-copy gene, it is easy to quantitatively detect Bacteroides fragilis.

Brief Description of the Drawings

[0013]

Figure 1

Figure 2

Figure 3A

Figure 3B

Figure 4A

Figure 4B

Figure 5A

Figure 5B

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10A

Figure 10B

Figure 11

Figure 12

Figure 13

Figure 14

Figure 15

Figure 16A

Figure 16B

Figure 16C

Figure 17

Figure 18

Figure 19

Figure 20

Figure 21

Figure 22

Figure 23

Figure 24A

Figure 24B

Figure 25

Figure 26A

Figure 26B

Figure 27A

Figure 27B

Figure 27C

Figure 28

Figure 29

Figure 30

Figure 31

Figure 32A

Figure 32B

Figure 32C

Figure 33

Figure 34

Figure 35

Figure 36

Figure 37

Figure 38

Figure 39

Figure 40

Figure 41

Figure 42

Figure 43

Figure 44

Figure 45

Figure 46

Figure 47

Figure 48

Figure 49

Figure 50

Figure 51

Embodiments for Carrying Out the Invention

[0014] The present invention provides one or more primers or probes specific for Bacteroides fragilis. The primers or probes of the present invention are capable of detecting one or more genes selected from the group consisting of the above 1) to 49).

[0015] The primers or probes of the present invention may be single-stranded nucleic acids or double-stranded nucleic acids, with single-stranded nucleic acids being preferred. The nucleic acid molecule may also be DNA, RNA, or a modified nucleic acid, with DNA or RNA being preferred and DNA being more preferred.

[0016] In certain embodiments, the primer or probe may comprise a partial nucleotide sequence or its complementary nucleotide sequence that includes at least 15 consecutive nucleotide residues in one or more nucleotide sequences selected from the group consisting of the nucleotide sequences of SEQ ID NOs: 1 to 49.

[0017] In a preferred embodiment, the partial nucleotide sequence may include nucleotide residues in a partial region specific to one or more nucleotide sequences selected from the group consisting of the nucleotide sequences of SEQ ID NOs: 1 to 49 as at least 15 consecutive nucleotide residues.

[0018] As used herein, the expression "partial region specific to one or more base sequences selected from the group consisting of the base sequences of SEQ ID NOs: 1 to 49" refers to a region consisting of partial base sequences specific to Bacteroides fragilis, which are not found in genomic regions in microorganisms other than Bacteroides fragilis and are common among Bacteroides fragilis strains (see, for example, the strains listed as (1) to (7) in Example 1). Such partial regions correspond to the underlined regions in the base sequences of SEQ ID NOs: 1 to 49 as shown in FIGS. 1 to 49. Therefore, at least 15 consecutive nucleotide residues containing nucleotide residues in the partial region specific to one or more base sequences selected from the group consisting of the base sequences of SEQ ID NOs: 1 to 49 can be selected from the underlined regions consisting of at least 15 consecutive nucleotide residues among the underlined regions in the base sequences of SEQ ID NOs: 1 to 49 (see FIGS. 1 to 49).

[0019] More specifically, the underlined regions consisting of at least 15 consecutive nucleotide residues among the underlined regions in the base sequences of SEQ ID NOs: 1 to 49 are as follows.

[0020] 1) SEQ ID NO: 1 (see FIG. 1) Positions 14 to 32, 184 to 206, 317 to 335, 349 to 368, 512 to 527, 733 to 749

[0021] 2) SEQ ID NO: 2 (see FIG. 2) Positions 1 to 77, 82 to 104, 158 to 173, 199 to 230, 250 to 266, 274 to 296, 325 to 347, 607 to 626, 628 to 645, 647 to 662, 676 to 695

[0022] 3) SEQ ID NO: 3 (see FIG. 3) 84 - 98 bits, 160 - 218 bits, 259 - 290 bits, 301 - 330 bits, 411 - 425 bits, 456 - 482 bits, 484 - 506 bits, 514 - 528 bits, 565 - 581 bits, 586 - 605 bits, 607 - 641 bits, 691 - 712 bits, 724 - 752 bits, 765 - 785 bits, 802 - 829 bits, 895 - 911 bits, 970 - 986 bits, 1004 - 1025 bits, 1159 - 1173 bits, 1195 - 1217 bits, 1228 - 1250 bits, 1255 - 1283 bits, 1285 - 1301 bits, 1356 - 1373 bits, 1478 - 1499 bits, 1514 - 1532 bits, 1534 - 1572 bits, 1597 - 1642 bits, 1650 - 1676 bits, 1678 - 1700 bits, 1723 - 1748 bits, 1755 - 1772 bits, 1792 - 1819 bits, 1837 - 1873 bits, 1950 - 1981 bits

[0023] 4) SEQ ID NO: 4 (see Figure 4) 275 - 297 bits, 301 - 323 bits, 326 - 347 bits, 413 - 427 bits, 1573 - 1587 bits

[0024] 5) SEQ ID NO: 5 (see Figure 5) 1 - 29 bits, 91 - 125 bits, 166 - 182 bits, 218 - 239 bits, 259 - 275 bits, 376 - 393 bits, 395 - 410 bits, 484 - 509 bits, 544 - 570 bits, 637 - 653 bits, 700 - 725 bits, 857 - 878 bits, 943 - 962 bits, 1063 - 1085 bits, 1172 - 1187 bits, 1384 - 1415 bits, 1489 - 1545 bits, 1810 - 1826 bits, 1882 - 1898 bits, 1936 - 1958 bits, 2136 - 2150 bits

[0025] 6) SEQ ID NO: 6 (see Figure 6) 129 - 143 bits, 145 - 164 bits, 223 - 237 bits, 268 - 284 bits, 364 - 386 bits, 817 - 834 bits, 979 - 1002 bits, 1187 - 1217 bits, 1234 - 1274 bits, 1432 - 1451 bits

[0026] 7) SEQ ID NO: 7 (see Figure 7) 59 - 77 bits, 1197 - 1217 bits, 1252 - 1267 bits

[0027] 8) Sequence number 8 (see Figure 8) Positions 1 to 33, 38 to 54, 376 to 410, 524 to 539, 541 to 563, 592 to 617, 619 to 641, 646 to 662, 736 to 752, 754 to 770, 793 to 809, 857 to 872, 916 to 945, 1061 to 1076, 1130 to 1148

[0028] 9) Sequence number 9 (see Figure 9) Positions 1 to 18, 28 to 62, 64 to 101, 103 to 125, 154 to 218, 295 to 311, 328 to 350, 355 to 371, 437 to 452, 520 to 539, 631 to 651, 664 to 680, 700 to 716, 871 to 887, 907 to 929, 1012 to 1028, 1106 to 1127, 1129 to 1151, 1153 to 1169, 1171 to 1185

[0029] 10) Sequence number 10 (see Figure 10) Positions 49 to 78, 142 to 164, 170 to 197, 496 to 510, 1039 to 1061, 1200 to 1219, 1751 to 1766, 1834 to 1850, 1996 to 2012, 2142 to 2160

[0030] 11) Sequence number 11 (see Figure 11) Positions 11 to 38, 40 to 68, 70 to 95, 97 to 179, 184 to 203, 379 to 395, 481 to 506, 625 to 644, 646 to 662, 730 to 746, 787 to 806, 844 to 860, 874 to 899, 979 to 999

[0031] 12) Sequence number 12 (see Figure 12) 7 to 35 bits, 64 to 155 bits, 187 to 203 bits, 282 to 298 bits, 331 to 377 bits, 394 to 422 bits, 435 to 461 bits, 466 to 482 bits, 547 to 563 bits, 595 to 615 bits, 700 to 719 bits, 721 to 735 bits, 757 to 794 bits, 814 to 841 bits, 843 to 857 bits, 889 to 916 bits, 959 to 998 bits, 1045 to 1064 bits, 1099 to 1121 bits, 1135 to 1151 bits, 1258 to 1277 bits, 1279 to 1316 bits, 1318 to 1334 bits, 1385 to 1406 bits, 1408 to 1424 bits, 1471 to 1487 bits

[0032] 13) Sequence number 13 (see Figure 13) 88 to 102 bits, 109 to 131 bits, 151 to 167 bits, 385 to 404 bits, 424 to 449 bits, 454 to 470 bits, 494 to 512 bits, 519 to 533 bits, 664 to 683 bits, 868 to 902 bits, 966 to 981 bits, 1072 to 1086 bits, 1129 to 1151 bits, 1213 to 1241 bits, 1278 to 1308 bits

[0033] 14) Sequence number 14 (see Figure 14) 1 to 23 bits, 28 to 42 bits, 63 to 84 bits, 86 to 113 bits, 139 to 155 bits, 157 to 179 bits, 191 to 207 bits, 211 to 227 bits, 260 to 278 bits, 349 to 374 bits, 421 to 435 bits, 437 to 452 bits, 481 to 497 bits, 499 to 527 bits, 535 to 551 bits, 604 to 629 bits, 895 to 909 bits

[0034] 15) Sequence number 15 (see Figure 15) 53 to 72 bits, 178 to 200 bits, 595 to 611 bits

[0035] 16) Sequence number 16 (see Figure 16) 1 to 21 bits, 23 to 44 bits, 46 to 83 bits, 85 to 101 bits, 103 to 140 bits, 142 to 185 bits, 187 to 219 bits, 235 to 251 bits, 304 to 335 bits, 337 to 356 bits, 367 to 395 bits, 397 to 419 bits, 496 to 512 bits, 523 to 542 bits, 548 to 563 bits, 565 to 584 bits, 622 to 674 bits, 760 to 779 bits, 829 to 845 bits, 904 to 920 bits, 952 to 977 bits, 979 to 995 bits, 1021 to 1043 bits, 1066 to 1082 bits, 1111 to 1142 bits, 1144 to 1160 bits, 1162 to 1187 bits, 1198 to 1235 bits, 1242 to 1259 bits, 1273 to 1298 bits, 1321 to 1337 bits, 1339 to 1361 bits, 1442 to 1463 bits, 1474 to 1496 bits, 1541 to 1556 bits, 1573 to 1619 bits, 1678 to 1700 bits, 1702 to 1733 bits, 1735 to 1760 bits, 1852 to 1880 bits, 1906 to 1928 bits, 1972 to 1997 bits, 2017 to 2036 bits, 2038 to 2066 bits, 2068 to 2108 bits, 2110 to 2168 bits, 2209 to 2231 bits, 2242 to 2258 bits, 2320 to 2336 bits, 2353 to 2378 bits, 2446 to 2462 bits, 2470 to 2492 bits, 2578 to 2600 bits, 2611 to 2630 bits, 2638 to 2654 bits, 2689 to 2720 bits, 2725 to 2747 bits, 2752 to 2774 bits, 2776 to 2807 bits, 2809 to 2825 bits, 2860 to 2903 bits, 2917 to 2948 bits, 2953 to 2969 bits, 2971 to 3002 bits

[0036] 17) Sequence number 17 (see Figure 17) 68 to 89 bits, 98 to 117 bits, 174 to 188 bits, 221 to 237 bits, 413 to 428 bits, 460 to 474 bits, 622 to 639 bits, 752 to 767 bits, 1013 to 1029 bits, 1054 to 1068 bits, 1279 to 1298 bits

[0037] 18) Sequence number 18 (see Figure 18) 196 to 221 bits, 223 to 242 bits, 271 to 338 bits, 340 to 369 bits, 427 to 497 bits, 505 to 588 bits

[0038] 19) Sequence number 19 (see Figure 19) Positions 1 to 149, 158 to 197, 202 to 218, 223 to 242, 259 to 281, 298 to 326, 373 to 434, 436 to 452, 454 to 479, 481 to 497, 508 to 530, 532 to 578, 592 to 633, 673 to 740, 747 to 776, 782 to 845, 847 to 866

[0039] 20) SEQ ID NO: 20 (see Figure 20) Positions 19 to 39, 165 to 180, 412 to 428, 466 to 491, 511 to 545, 592 to 644, 729 to 747, 983 to 999, 1005 to 1026, 1148 to 1167, 1173 to 1188, 1194 to 1255, 1293 to 1367, 1369 to 1383

[0040] 21) SEQ ID NO: 21 (see Figure 21) Positions 1009 to 1028

[0041] 22) SEQ ID NO: 22 (see Figure 22) Positions 101 to 123, 154 to 168

[0042] 23) SEQ ID NO: 23 (see Figure 23) Positions 15 to 29, 34 to 50, 52 to 71, 109 to 140, 146 to 167, 343 to 365, 385 to 401, 403 to 419, 520 to 542, 598 to 617, 619 to 635, 661 to 689, 733 to 764, 766 to 795, 863 to 881, 895 to 918

[0043] 24) SEQ ID NO: 24 (see Figure 24) 1 to 38 bits, 52 to 66 bits, 139 to 161 bits, 166 to 200 bits, 208 to 230 bits, 244 to 266 bits, 273 to 291 bits, 294 to 341 bits, 343 to 365 bits, 367 to 381 bits, 390 to 434 bits, 436 to 518 bits, 523 to 557 bits, 559 to 602 bits, 604 to 656 bits, 712 to 737 bits, 739 to 775 bits, 850 to 864 bits, 866 to 881 bits, 883 to 899 bits, 901 to 929 bits, 931 to 945 bits, 949 to 1022 bits, 1056 to 1109 bits, 1165 to 1181 bits, 1183 to 1262 bits, 1273 to 1316 bits, 1333 to 1356 bits, 1359 to 1397 bits, 1399 to 1442 bits, 1447 to 1508 bits, 1510 to 1542 bits, 1552 to 1587 bits, 1591 to 1630 bits, 1642 to 1679 bits, 1681 to 1715 bits, 1717 to 1766 bits, 1780 to 1823 bits, 1825 to 1841 bits, 1900 to 1928 bits, 1930 to 1954 bits, 1969 to 2006 bits, 2011 to 2030 bits, 2044 to 2070 bits

[0044] 25) Sequence number 25 (see Figure 25) 4 to 59 bits, 64 to 78 bits, 90 to 107 bits, 133 to 158 bits, 193 to 216 bits, 283 to 305 bits, 370 to 392 bits, 481 to 500 bits, 526 to 542 bits, 562 to 617 bits, 619 to 650 bits, 667 to 713 bits, 734 to 761 bits, 793 to 875 bits, 922 to 944 bits, 988 to 1004 bits, 1030 to 1052 bits, 1057 to 1074 bits, 1099 to 1118 bits, 1144 to 1158 bits, 1348 to 1367 bits, 1457 to 1475 bits, 1477 to 1491 bits, 1513 to 1550 bits, 1555 to 1577 bits, 1588 to 1620 bits

[0045] 26) Sequence number 26 (see Figure 26) Positions 1 to 55, 69 to 92, 227 to 251, 448 to 470, 484 to 506, 535 to 554, 625 to 644, 655 to 677, 964 to 980, 1093 to 1110, 1144 to 1169, 1183 to 1202, 1210 to 1227, 1261 to 1280, 1351 to 1370, 1543 to 1565, 1660 to 1679, 1726 to 1757, 1861 to 1875, 1882 to 1904, 1924 to 1940, 1964 to 1985, 2062 to 2078, 2089 to 2108, 2171 to 2186, 2203 to 2219

[0046] 27) Sequence No. 27 (see Fig. 27) Positions 109 to 153, 175 to 245, 253 to 284, 316 to 332, 341 to 359, 460 to 478, 514 to 530, 532 to 554, 574 to 593, 601 to 618, 624 to 638, 643 to 707, 735 to 749, 775 to 794, 838 to 860, 874 to 891, 937 to 962, 985 to 1073, 1081 to 1172, 1186 to 1202, 1204 to 1364, 1366 to 1488, 1495 to 1616, 1618 to 1655, 1678 to 1727, 1729 to 1763, 1765 to 1802, 1804 to 1857, 1859 to 1946, 1948 to 2051, 2053 to 2122, 2124 to 2139, 2141 to 2189, 2191 to 2219, 2221 to 2252, 2276 to 2291, 2296 to 2336, 2338 to 2395, 2410 to 2435, 2437 to 2506, 2508 to 2555, 2557 to 2606, 2608 to 2624, 2626 to 2660, 2662 to 2804, 2806 to 2885, 2887 to 2954, 2956 to 2975, 3145 to 3180, 3200 to 3227

[0047] 28) Sequence No. 28 (see Fig. 28) Positions 11 to 30, 32 to 47, 49 to 74, 76 to 92, 94 to 125, 145 to 159, 163 to 215, 217 to 245, 247 to 269, 271 to 344, 383 to 398, 436 to 464, 493 to 512, 556 to 602, 655 to 693, 703 to 727, 729 to 749, 751 to 772, 805 to 834, 847 to 891, 893 to 911, 913 to 929, 940 to 956

[0048] 29) Sequence number 29 (see Fig. 28) Positions 1 to 35, 49 to 113, 115 to 179, 187 to 201

[0049] 30) Sequence number 30 (see Fig. 30) Positions 17 to 68, 88 to 125, 139 to 164, 211 to 233, 235 to 254, 340 to 356, 373 to 389, 436 to 450, 520 to 536

[0050] 31) Sequence number 31 (see Fig. 31) Positions 1 to 32, 34 to 84, 95 to 122, 124 to 176, 193 to 221, 223 to 242, 275 to 323, 385 to 402, 409 to 431, 433 to 479, 496 to 572, 574 to 596, 622 to 669, 671 to 731, 757 to 780, 782 to 821, 835 to 858, 860 to 905, 907 to 935, 937 to 965, 970 to 1001, 1039 to 1070, 1112 to 1141, 1183 to 1197, 1205 to 1265, 1321 to 1344

[0051] 32) Sequence number 32 (see Fig. 32) Positions 26 - 50, 85 - 110, 124 - 140, 208 - 224, 241 - 257, 262 - 281, 304 - 323, 349 - 371, 394 - 410, 418 - 452, 557 - 577, 616 - 644, 754 - 768, 791 - 830, 841 - 864, 904 - 920, 922 - 938, 973 - 1013, 1015 - 1035, 1038 - 1055, 1156 - 1175, 1242 - 1260, 1444 - 1458, 1693 - 1709, 1714 - 1739, 1846 - 1862, 1984 - 2009, 2011 - 2027, 2117 - 2132, 2308 - 2330, 2333 - 2351, 2353 - 2367

[0052] 33) Sequence number 33 (see Figure 33) Positions 1 - 68, 74 - 118, 121 - 206, 217 - 374, 376 - 518, 520 - 647, 652 - 722, 724 - 809, 811 - 879, 881 - 896, 898 - 993, 1003 - 1034, 1036 - 1064, 1096 - 1111

[0053] 34) Sequence number 34 (see Figure 34) Positions 70 - 84, 98 - 113, 244 - 260, 280 - 302, 377 - 392, 415 - 435

[0054] 35) Sequence number 35 (see Figure 35) Positions 1 - 61, 67 - 140, 142 - 167, 175 - 195, 197 - 248, 250 - 272, 274 - 308, 310 - 367, 369 - 399, 401 - 441, 443 - 476, 484 - 504, 506 - 533, 550 - 590, 592 - 644, 646 - 695, 697 - 880, 882 - 929, 940 - 977, 988 - 1088, 1090 - 1124, 1126 - 1226, 1228 - 1261, 1264 - 1358, 1362 - 1385, 1387 - 1436, 1438 - 1538, 1540 - 1592, 1594 - 1629

[0055] 36) SEQ ID NO: 36 (see FIG. 36) Positions 362 to 377, 379 to 393, 538 to 552

[0056] 37) SEQ ID NO: 37 (see FIG. 37) Positions 10 to 26, 28 to 55, 148 to 162, 292 to 316, 422 to 437, 439 to 458, 526 to 560, 610 to 638, 661 to 680, 844 to 860, 1099 to 1115, 1117 to 1148, 1185 to 1211, 1225 to 1241, 1309 to 1325, 1384 to 1415, 1448 to 1463, 1507 to 1529, 1619 to 1646, 1708 to 1733, 1750 to 1784, 1795 to 1817

[0057] 38) SEQ ID NO: 38 (see FIG. 38) None

[0058] 39) SEQ ID NO: 39 (see FIG. 39) None

[0059] 40) SEQ ID NO: 40 (see FIG. 40) None

[0060] 41) SEQ ID NO: 41 (see FIG. 41) Positions 1 to 26, 701 to 716

[0061] 42) SEQ ID NO: 42 (see FIG. 42) Positions 1 to 21, 48 to 75, 83 to 108, 161 to 240, 311 to 345, 353 to 486, 547 to 579, 587 to 610, 663 to 678, 684 to 750, 758 to 786, 850 to 911, 913 to 950, 952 to 995, 997 to 1058, 1060 to 1147, 1154 to 1260, 1266 to 1304, 1306 to 1430, 1441 to 1466, 1468 to 1553, 1567 to 1776

[0062] 43) SEQ ID NO: 43 (see FIG. 43) Positions 1 to 15, 34 to 53, 120 to 147, 169 to 185, 205 to 224, 340 to 359, 461 to 476, 614 to 634, 722 to 741, 862 to 896, 916 to 940, 961 to 987, 993 to 1042, 1048 to 1062

[0063] 44) Sequence number 44 (see Figure 44) Positions 1 to 53, 55 to 341, 343 to 473, 478 to 608, 613 to 647, 649 to 674, 676 to 743, 745 to 780, 782 to 824, 826 to 899, 925 to 980, 991 to 1013, 1015 to 1032, 1034 to 1079, 1081 to 1253, 1300 to 1320

[0064] 45) Sequence number 45 (see Figure 45) Positions 65 to 86, 88 to 105, 672 to 716, 724 to 740, 901 to 921, 923 to 942

[0065] 46) Sequence number 46 (see Figure 46) Positions 128 to 153, 236 to 269, 278 to 304, 443 to 459, 623 to 647, 662 to 682, 692 to 710, 914 to 936, 995 to 1011, 1061 to 1083

[0066] 47) Sequence number 47 (see Figure 47) Positions 13 to 38, 40 to 59, 127 to 146, 235 to 251, 316 to 371, 382 to 401, 478 to 500, 508 to 524, 589 to 605, 607 to 623, 661 to 680, 742 to 761, 1000 to 1019, 1414 to 1448, 1450 to 1481, 1495 to 1517, 1522 to 1541, 1576 to 1601, 1756 to 1770, 1812 to 1826, 1933 to 1965, 1984 to 2018

[0067] 48) Sequence number 48 (see Figure 48) 1 to 104 bits, 106 to 148 bits, 150 to 254 bits, 264 to 278 bits, 280 to 314 bits, 316 to 353 bits, 355 to 393 bits, 395 to 416 bits, 433 to 540 bits, 542 to 590 bits, 613 to 656 bits, 666 to 803 bits, 805 to 905 bits, 907 to 977 bits, 986 to 1010 bits, 1012 to 1064 bits, 1066 to 1104 bits, 1110 to 1145 bits, 1147 to 1298 bits, 1300 to 1341 bits, 1366 to 1440 bits, 1442 to 1464 bits, 1510 to 1524 bits, 1592 to 1611 bits, 1643 to 1661 bits, 1712 to 1743 bits, 1820 to 1836 bits, 1898 to 1917 bits, 2012 to 2028 bits, 2117 to 2145 bits

[0068] 49) SEQ ID NO: 49 (see FIG. 49) 59 to 109 bits, 115 to 173 bits, 216 to 236 bits, 242 to 363 bits, 370 to 398 bits, 400 to 448 bits, 455 to 500 bits, 509 to 529 bits, 551 to 616 bits, 624 to 638 bits, 640 to 668 bits, 685 to 705 bits, 712 to 727 bits, 729 to 764 bits, 775 to 839 bits, 845 to 968 bits, 973 to 992 bits, 1000 to 1033 bits, 1042 to 1119 bits, 1150 to 1176 bits, 1204 to 1220 bits, 1333 to 1350 bits

[0069] In one embodiment, the primer or probe of the present invention may be one or more primers. For example, such one or more primers of the present invention are excellent in specificity to Bacteroides fragilis and thus useful as sequencing primers.

[0070] When one or more primers of the present invention are sequencing primers, the sequencing primers may be composed of a partial base sequence containing at least 15 consecutive nucleotide residues in one or more base sequences selected from the group consisting of the base sequences of SEQ ID NOs: 1 to 49 or its complementary base sequence.

[0071] In another embodiment, one or more primers of the present invention may be a primer set containing two or more primers. For example, such a primer set is excellent in specificity for Bacteroides fragilis and is useful as a primer set for gene amplification.

[0072] When one or more primers of the present invention are a primer set for gene amplification, the primer set for gene amplification may include (a) a first primer containing a partial base sequence including at least 15 consecutive nucleotide residues in one or more base sequences selected from the group consisting of the base sequences of SEQ ID NOs: 1 to 49, and (b) a second primer containing a complementary base sequence of the partial base sequence including at least 15 consecutive nucleotide residues in one or more base sequences selected from the group consisting of the base sequences of SEQ ID NOs: 1 to 49 (wherein the SEQ ID NOs targeted in (a) and (b) are the same).

[0073] The number of primers required for gene amplification varies depending on the type of gene amplification method. For example, in PCR, the number of primers required for gene amplification is two. On the other hand, in LAMP (Loop-mediated isothermal amplification) (see, for example, International Publication No. 00 / 28082), the number of primers required for gene amplification is four or six. Therefore, when amplification by a gene amplification method requiring more than two primers is intended, the primer set may include additional primers.

[0074] The primer set including the above (a) and (b) is not particularly limited as long as the amplification product is set to a detectable size, but preferably, it can be set to generate an amplification product of any size that is easy to detect (for example, 150 bp or more, 200 bp or more, 250 bp or more, 300 bp or more, 350 bp or more, 400 bp or more, 450 bp or more, or 500 bp or more). Examples of gene amplification methods capable of generating the above amplification products include PCR, LAMP, TMA, ICAN, SDA, LCR, and NASBA.

[0075] In another embodiment, the primer or probe of the present invention may be one or more probes. For example, since one or more such probes of the present invention are excellent in specificity to Bacteroides fragilis, they are useful for detecting Bacteroides fragilis.

[0076] The probe of the present invention may be composed of a partial base sequence containing at least 15 consecutive nucleotide residues in one or more base sequences selected from the group consisting of the base sequences of SEQ ID NOs: 1 to 49 or its complementary base sequence.

[0077] The length of the primer or probe of the present invention is not particularly limited as long as one or more genes selected from the group consisting of the above 1) to 49) can be detected by one or more primers or probes. For example, it may contain at least 15 nucleotide residues. Preferably, the length of the nucleic acid molecule of the present invention may contain at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 nucleotide residues. The length of the primer or probe of the present invention may also contain 100 or less, 90 or less, 80 or less, 70 or less, 60 or less, 50 or less, 45 or less, or 40 or less nucleotide residues.

[0078] The present invention also provides a method for detecting Bacteroides fragilis. The method of the present invention includes detecting Bacteroides fragilis in a sample containing Bacteroides fragilis using the primer or probe of the present invention.

[0079] In one embodiment, the Bacteroides fragilis-containing sample may be a Bacteroides fragilis-containing sample obtained from a subject. The Bacteroides fragilis-containing sample obtained from a subject is not particularly limited as long as it contains Bacteroides fragilis cells, genomes, and / or transcripts of genes. Examples of the subject include animals such as mammals and birds, with mammals being preferred and humans being more preferred. Such a sample is preferably a minimally invasive sample. Examples of such samples include feces, rectal swabs, saliva, oral swabs, urine, blood, skin swabs, tears, nasal secretions, and nasal swabs.

[0080] In another embodiment, the Bacteroides fragilis-containing sample may be an environmental sample. Examples of the environmental sample include surfaces such as soil, sewage, rivers, seawater, walls, ceilings, floors, fixtures, furniture, fittings, machines, and the exterior and interior of vehicles that make up the built environment.

[0081] In the method of the present invention, genomic DNA or a transcript of a gene may be extracted from the sample. Such extraction can be performed by any method. The extracted genomic DNA or gene transcript can be subjected to an appropriate gene amplification method (e.g., PCR or LAMP) according to the size of the intended amplification product. Alternatively, the sample may be directly subjected to a gene amplification method (e.g., direct PCR).

[0082] Detection can be performed qualitatively or quantitatively. Preferably, detection can be performed quantitatively (e.g., quantitative PCR). Since one or more genes selected from the group consisting of the above 1) to 49), which are targeted in the present invention, can be found to have only a single copy in Bacteroides fragilis, the method of the present invention is excellent in the quantification of Bacteroides fragilis.

[0083] In certain embodiments, the detection may be performed in real time (e.g., real-time PCR). In this case, examples of methods that enable real-time detection include the intercalator method and the fluorescent substance-labeled probe method.

[0084] The present invention also provides a detection reagent for Bacteroides fragilis that contains the primer or probe of the present invention. According to the reagent of the present invention, the method of the present invention can be easily carried out.

[0085] The reagent of the present invention can contain one or more primers or probes of the present invention in the form of a powder (e.g., lyophilized) or a solution. The solution is preferably an aqueous solution. Examples of the aqueous solution include water (e.g., sterilized distilled water) and buffer solutions. Examples of the buffer solution include TE (Tris-EDTA) buffer solution, hydrochloric acid-potassium chloride buffer solution, glycine-hydrochloric acid buffer solution, citrate buffer solution, acetate buffer solution, citrate-phosphate buffer solution, phosphate buffer solution, Tris-hydrochloric acid buffer solution, glycine-sodium hydroxide buffer solution, carbonate-bicarbonate buffer solution, borate buffer solution, and tartrate buffer solution. When the reagent of the present invention is a solution containing one or more primers of the present invention, the concentration of the primer in the solution varies depending on factors such as the use of the primer and the dilution ratio during the use of the primer, but may be, for example, 0.1 to 100 mM, preferably 1 to 10 mM. The solution may contain other components such as a stabilizer.

[0086] The reagent of the present invention may be provided in the form of a kit. In such a case, the reagent of the present invention may contain (a) one or more primers or probes, and (b) a fluorescent substance or a fluorescent substance-labeled probe. Examples of the fluorescent substance include fluorescent substances used in the intercalator method (e.g., SYBR (registered trademark) Green I). Examples of the fluorescent substance-labeled probe include a probe in which a fluorescent substance is bound to one of the 5'-end or 3'-end and a quencher is bound to the other of the 5'-end or 3'-end (e.g., TaqMan (registered trademark) probe).

[0087] When the reagent of the present invention is a reagent for gene amplification, it may be provided in the form of a kit containing (a) one or more primers and (b) a polymerase. As the polymerase, an appropriate polymerase according to the type of gene amplification method can be used. For example, in the case of PCR, it is preferable to use a heat-resistant polymerase, and in the case of LAMP, it is preferable to use a strand-displacing polymerase. As the polymerase, a DNA polymerase is preferable.

[0088] In addition to (a) and (b), the kit of the present invention may further contain additional components. Such components include a deoxynucleoside triphosphate (dNTP) mixture, a reaction buffer, a molecular weight marker, a control (e.g., a standard of amplification products of various HLA alleles). When the kit of the present invention contains additional components, each component may be provided in a form isolated from each other, for example, stored in different containers (e.g., tubes), or may be provided in a pre-mixed form (e.g., PreMix) or the like.

Examples

[0089] The present invention will be described in detail by the following examples, but the present invention is not limited to the following examples.

[0090] Example 1: Extraction of a gene specific to Bacteroides fragilis A gene specific to B. fragilis was extracted according to the following procedures 1 to 4. [Procedure 1] First, a held gene data table was created according to the following procedures a to g.

[0091] [Procedure a] A total of 11,596 strains of microbial whole genome sequences were downloaded from the genome database publicly available from NCBI.

[0092] [Procedure b] Using the gene prediction software "Prokka", the gene region sequences on each of the downloaded whole genome sequences were predicted. As a result, a gene region was associated with each of the 11,596 whole genome sequences.

[0093] [Procedure c] Using the homology search software "DIAMOND", homology search of the sequences of each predicted gene region was performed against the gene sequence database "KO (KEGG Orthology) database" to annotate each gene region. As a result, a functional classification was associated with each of all the predicted gene regions.

[0094] [Procedure d] A gene ID was assigned to each functional classification of each gene region. As a result, a gene ID was associated with each of all the predicted gene regions.

[0095] [Procedure e] For each of the downloaded whole genome sequences, the number of copies of each of all the assigned gene IDs was tabulated.

[0096] [Procedure f] Referring to the Taxonomy database published by NCBI, taxonomic information (species name) was assigned to each of the downloaded whole genome sequences.

[0097] [Procedure g] A gene possession data table was created for each of the downloaded whole genome sequences, including the tabulated number of copies of each gene ID and the assigned taxonomic information.

[0098] Next, using a gene search device that stores the gene possession data table created in this way, 66 gene IDs were searched for that were commonly possessed by all strains of "B. fragilis" to be detected, and for which the possession rate of all microorganisms other than B. fragilis managed in the gene possession data table was less than 0.1 and each strain of B. fragilis had only 1 copy in the genome.

[0099] The "all strains of B. fragilis" to be detected were as follows. (1) Strain YCH46 (GenBank accession number: AP006841.1) (2) NCTC9343 strain (GenBank accession number: CR626927.1) (3) 638R strain (GenBank accession number: FQ312004.1) (4) BOB25 strain (GenBank accession number: CP011073.1) (5) BFBE1.1 strain (GenBank accession number: LN877293.1) (6) S14 strain (GenBank accession number: CP012706.1) (7) Q1F2 strain (GenBank accession number: CP018937.1)

[0100] [Procedure 2] The gene region sequences corresponding to the gene IDs searched in [Procedure 1] were extracted from the whole genome sequence of B. fragilis.

[0101] [Procedure 3] Using the multiple alignment analysis program "MAFFT" and the consensus sequence creation program "cons", a consensus sequence was created for the extracted gene region sequences corresponding to the gene IDs.

[0102] [Procedure 4] Using the primer design program "Primer3", a primer sequence set was created for the created consensus sequence. Forty-nine genes were extracted for which primers could be designed to bind to regions with low sequence diversity among B. fragilis strains and to obtain amplification products with a length of 150 to 200 base pairs. The results are shown in Table 1.

[0103]

Table 1-1

[0104]

Table 1-2

[0105] From the above, 49 genes specific to B. fragilis were extracted, which are highly conserved among B. fragilis strains and for which primer pairs capable of generating the desired gene amplification products can be created.

[0106] Example 2: Extraction of highly specific regions in genes specific to Bacteroides fragilis By performing the following procedures 5 to 7 after the above procedures 1 to 4, genes specific to B. fragilis were extracted.

[0107] [Procedure 5] Using the homology search program "Blast+", a homology search of the genes extracted in [Procedure 4] was performed using a database of a total of 11,596 whole genome sequences downloaded from the genome database publicly available from NCBI. Genomic regions of microorganisms other than B. fragilis having sequences partially identical to the extracted genes were extracted.

[0108] [Procedure 6] Using the multiple alignment tool "Clustal Omega", an alignment was performed between the B. fragilis-specific genes extracted in [Procedure 4] and the genomic regions of microorganisms other than B. fragilis having sequences partially identical to those extracted in [Procedure 5].

[0109] [Procedure 7] Regions in the sequences of the B. fragilis-specific genes that did not match the genomic regions of microorganisms other than B. fragilis were extracted as B. fragilis-specific gene sequences.

[0110] The results are shown in FIGS. 1 to 49. The left end of the figure indicates the 5'-end of the B. fragilis-specific gene, and the right side of the figure indicates the 3'-end of the B. fragilis-specific gene. In the upper part of the figure, black represents the region that matches the genomic region of microorganisms other than B. fragilis in the sequence of the B. fragilis-specific gene, and white represents the region that does not match the genomic region of microorganisms other than B. fragilis in the sequence of the B. fragilis-specific gene. In the lower part of the figure, black represents the region where the sequence is different from all the remaining B. fragilis genomes in at least one of the 38 B. fragilis genomes, and white represents the region where all 38 genomes have the same sequence. The regions that are represented by white in both the upper and lower parts, do not match the genomic regions of microorganisms other than B. fragilis, and have no inter-strain diversity are represented by the nucleotide sequences below the figure.

[0111] [Step 7] By using the highly specific region in the gene specific to Bacteroides fragilis extracted in [Step 7], it is possible to specifically and highly quantitatively identify and analyze B. fragilis by detecting B. fragilis-derived DNA with a nucleic acid probe.

[0112] Example 3: Verification of the specificity of primers designed based on the B. fragilis-specific gene sequence (in silico) Primers were designed for the B. fragilis-specific gene sequence, and the specificity of the primers was verified in silico. The verification was performed according to the following Steps 8 and 9.

[0113] [Step 8] Among the genes with the lowest retention rate in the non-detection target, for the gene ID "K21556", the primers for detecting the B. fragilis-specific gene sequence created in [Step 4] were used for verification. The forward primer sequence used was "5’-ATTGCAGAGATGTGGGCTCC-3’" (SEQ ID NO: 50), and the reverse primer sequence was "5’-CTTCTGCCTCCTACACCGTC-3’" (SEQ ID NO: 51). The nucleotide sequence of SEQ ID NO: 50 represents the nucleotide sequence of the forward primer for detecting the gene (gene ID "K21556") specific to B. fragilis. The nucleotide sequence of SEQ ID NO: 51 represents the nucleotide sequence of the reverse primer for detecting the gene (gene ID "K21556") specific to B. fragilis.

[0114] [Step 9] Using the homology search program "Blast+", a homology search of the designed primer sequences (SEQ ID NO: 50, SEQ ID NO: 51) and the primer sequences specific to B. fragilis for detecting existing 16S rRNA was performed using the 11,596 whole genome sequences downloaded in [Step 5] as a database. The forward primer sequence specific to B. fragilis for detecting existing 16S rRNA is "5’-ATAGCCTTTCGAAAGRAAGAT-3’" (SEQ ID NO: 52), and the reverse primer sequence is "5’-CCAGTATCAACTGCAATTTTA-3’" (SEQ ID NO: 53). As a result, it was confirmed that the designed primer sequences did not exist in the non-detection targets. On the other hand, it was confirmed that the primer sequences specific to B. fragilis for detecting 16S rRNA exist in Bacteroides ovatas, Bacteroides heparinolyticus, Bacteroides zoogleoformans, Bacteroides caccae, Bacteroides caecimuris, Bacteroides thetaiotamicron, Bacteroides cellulosilyticus, Bacteroides dorei, Bacteroides helcogenes. The results are shown in Fig. 50. In Fig. 50, the vertical axis represents the bacterial species in which the presence of the primer sequence was confirmed, and the horizontal axis represents the primer sequence. HU_F is the forward primer for detecting the gene specific to B. fragilis (gene ID "K21556"), HU_R is the reverse primer for detecting the gene specific to B. fragilis (gene ID "K21556"), g_Bfra_F3_F is the forward primer specific to B. fragilis for detecting existing 16S rRNA, and g_Bfra_F3_R is the reverse primer specific to B. fragilis for detecting existing 16S rRNA. In Fig. 50, black in the heat map indicates that the primer sequence and the bacterial genome are 100% identical or identical except for one base, and white indicates that there is no location where the primer sequence and the bacterial genome match.

[0115] From the above results of this example, it was confirmed that the primer sequences for detecting the B. fragilis - specific gene sequence do not exist outside of B. fragilis and non - specific detection is not expected, and that the existing 16S rRNA gene detection primer sequences exist in multiple Bacteroides species and non - specific detection of these bacterial species is expected.

[0116] Example 4: Verification of the specificity of primers designed based on the B. fragilis - specific gene sequence (in vitro) Using the primers for detecting the B. fragilis - specific gene sequence gene created in Example 3, it was verified in vitro whether the gene could actually be detected. The verification was performed according to the following procedures 10 to 12.

[0117] [Procedure 10] For the purpose of serving as a standard for absolute quantification in quantitative PCR, 10 μL of a PCR reaction solution was prepared using the genomic DNA of B. fragilis JCM 11019 strain. The components of the reaction solution are as follows. A total of 2 reaction solutions listed below were prepared.

[0118] <Components of the reaction solution> Template DNA: 1 μL 「NEBNext Q5 Hot Start HiFi 2X Master Mix」manufactured by New England Biolabs: 5 μL Forward primer prepared to 10 μM: 0.5 μL Reverse primer prepared to 10 μM: 0.5 μL Purified water: 3 μL

[0119] <Prepared reaction solution> (1) Reaction solution 1 using the genomic DNA of B. fragilis JCM 11019 strain and a primer set (SEQ ID NO: 50, SEQ ID NO: 51) for detecting a gene specific to B. fragilis (gene ID "K21556") (2) Reaction solution 2 using the genomic DNA of B. fragilis JCM 11019 strain and a primer set specific to B. fragilis for detecting 16S rRNA (SEQ ID NO: 52, SEQ ID NO: 53)

[0120] [Procedure 11] PCR was performed using the prepared PCR reaction solution. For PCR, a thermal cycler "Veriti" manufactured by Thermo Fisher Scientific was used. The PCR reaction conditions were as follows: after incubation at 98°C for 30 seconds, 35 cycles of thermal denaturation at 98°C for 10 seconds, annealing at 65°C for 15 seconds, and extension reaction at 72°C for 30 seconds were performed.

[0121] [Procedure 12] Using the genomic DNA of B. fragilis JCM 11019 strain, a 20 μL quantitative PCR reaction solution was prepared. Also, as a standard for absolute quantification, DNA solutions containing 10 3 , 10 4 , 10 5 , 10 6 , 10 7 copies of the PCR amplification product were used to prepare 20 μL quantitative PCR reaction solutions. The components of the reaction solution are as follows. A total of 4 reaction solutions listed below were prepared.

[0122] <Components of the reaction solution> Template DNA: 1 μL "2x KOD SYBR qPCR Mix" manufactured by Toyobo: 10 μL Forward primer prepared to 10 μM: 1 μL Reverse primer prepared to 10 μM: 1 μL ROX: 0.4 μL Purified water: 6.6 μL

[0123] <Prepared reaction solution> (1) Reaction solution 1 using the genomic DNA of B. fragilis JCM 11019 strain and a primer set for detecting a gene specific to B. fragilis (gene ID "K21556") (SEQ ID NO: 50, SEQ ID NO: 51) (2) Reaction solution 2 using the genomic DNA of B. fragilis JCM 11019 strain and a primer set specific to B. fragilis for detecting 16S rRNA (SEQ ID NO: 52, SEQ ID NO: 53) (3) As per [Procedures 10 - 11], a DNA solution containing the amplification product obtained by PCR amplification using the genomic DNA of B. fragilis JCM 11019 strain and a primer set for detecting a gene specific to B. fragilis (gene ID "K21556"), 10 3 or 10 4 or 10 5 or 10 6 or 10 7 copies of the PCR amplification product, and reaction solution 3 using a primer set for detecting a gene specific to B. fragilis (gene ID "K21556") (SEQ ID NO: 50, SEQ ID NO: 51) (4) As per [Procedures 10 - 11], a DNA solution containing the amplification product obtained by PCR amplification using the genomic DNA of B. fragilis JCM 11019 strain and a primer set specific to B. fragilis for detecting 16S rRNA, 10 3 or 10 4 or 10 5 or 10 6 or 10 7 copies of the PCR amplification product, and reaction solution 4 using a primer set for detecting a gene specific to B. fragilis (gene ID "K21556") (SEQ ID NO: 50, SEQ ID NO: 51)

[0124] [Procedure 13] Quantitative PCR was performed using the prepared quantitative PCR reaction solution. For quantitative PCR, "Mx3005P" manufactured by Agilent Technologies was used. The quantitative PCR reaction conditions were as follows: After incubation at 98°C for 2 minutes, 40 cycles of thermal denaturation at 98°C for 10 seconds, annealing at 60°C for 10 seconds, and extension reaction at 68°C for 30 seconds were carried out.

[0125] [Step 14] A calibration curve of gene copy number and Ct value was created from the obtained standard data, and the gene copy number in the template DNA was calculated from the obtained sample data. The calculation results were compared with the theoretical values calculated from the DNA concentration of the template DNA solution and the genome size of B. fragilis JCM 11019 strain. The comparison results are shown in Fig. 51. The experiment was carried out with 3 technical replicates within the experiment, and the average value was used for the analysis. In Fig. 51, the white graph shows the absolute quantification value of qPCR using a primer set (SEQ ID NO: 50, SEQ ID NO: 51) for detecting a gene specific to B. fragilis (gene ID "K21556"), the black graph shows the absolute quantification value of qPCR using a primer set specific to B. fragilis for detecting 16S rRNA (SEQ ID NO: 52, SEQ ID NO: 53), and the shaded graph shows the theoretical value calculated from the genome size of B. fragilis JCM 11019 strain. In Fig. 51, the apex of the bar graph indicates the average value of 3 technical replicates, and the error bar indicates the standard error of 3 technical replicates.

[0126] From the above results of this example, it was confirmed that B. fragilis can be actually detected with a primer for detecting a gene sequence specific to B. fragilis, and that the absolute quantification value is more accurate than the quantification by the existing 16S rRNA gene detection.

Claims

1. A primer pair comprising a first primer containing the nucleotide sequence of SEQ ID NO: 50 and a second primer containing the nucleotide sequence of SEQ ID NO: 51, which is specific for Bacteroides fragilis and can detect the gene of the CRP / FNR family transcriptional regulator and the polysaccharide utilization system transcriptional regulator (KO number: K21556).

2. A method for detecting Bacteroides fragilis, comprising detecting Bacteroides fragilis by a gene amplification method using the primer pair according to Claim 1 in a sample containing Bacteroides fragilis obtained from a subject.

3. The method according to Claim 2, wherein the gene amplification method is PCR.

4. The method according to Claim 2 or 3, wherein the detection is performed quantitatively.

5. A detection reagent for Bacteroides fragilis, comprising the primer pair according to Claim 1.

Images