Patent

1. A primer set for detection of Listeria monocytogenes , comprising any of the following primer sets for amplification of a partial region of lmo0084 gene or lmo2736 gene of Listeria monocytogenes:

(A-1) a set of a forward primer containing in its 3′-side the base sequence of SEQ ID NO:26 or a sequence which is the same as said base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in said base sequence, and a reverse primer containing in its 3′-side the base sequence of SEQ ID NO:30 or a sequence which is the same as said base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in said base sequence;

(A-2) a set of a forward primer containing in its 3′-side the base sequence of SEQ ID NO:26 or a sequence which is the same as said base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in said base sequence, and a reverse primer containing in its 3′-side the base sequence of SEQ ID NO:31 or a sequence which is the same as said base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in said base sequence;

(A-3) a set of a forward primer containing in its 3′-side the base sequence of SEQ ID NO:27 or a sequence which is the same as said base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in said base sequence, and a reverse primer containing in its 3′-side the base sequence of SEQ ID NO:30 or a sequence which is the same as said base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in said base sequence;

(A-4) a set of a forward primer containing in its 3′-side the base sequence of SEQ ID NO:27 or a sequence which is the same as said base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in said base sequence, and a reverse primer containing in its 3′-side the base sequence of SEQ ID NO:31 or a sequence which is the same as said base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in said base sequence;

(A-5) a set of a forward primer containing in its 3′-side the base sequence of SEQ ID NO:28 or a sequence which is the same as said base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in the base sequence, and a reverse primer containing in its 3′-side the base sequence of SEQ ID NO:30 or a sequence which is the same as said base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in said base sequence;

(A-6) a set of a forward primer containing in its 3′-side the base sequence of SEQ ID NO:28 or a sequence which is the same as said base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in said base sequence, and a reverse primer containing in its 3′-side the base sequence of SEQ ID NO:31 or a sequence which is the same as said base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in said base sequence;

(A-7) a set of a forward primer containing in its 3′-side the base sequence of SEQ ID NO:29 or a sequence which is the same as said base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in said base sequence, and a reverse primer containing in its 3′-side the base sequence of SEQ ID NO:30 or a sequence which is the same as said base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in said base sequence;

(A-8) a set of a forward primer containing in its 3′-side the base sequence of SEQ ID NO:29 or a sequence which is the same as said base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in said base sequence, and a reverse primer containing in its 3′-side the base sequence of SEQ ID NO:31 or a sequence which is the same as said base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in said base sequence;

(B-1) a set of a forward primer containing in its 3′-side the base sequence of SEQ ID NO:58 or a sequence which is the same as said base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in said base sequence, and a reverse primer containing in its 3′-side the base sequence of SEQ ID NO:59 or a sequence which is the same as said base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in said base sequence;

(C-1) a set of a forward primer containing in its 3′-side the base sequence of SEQ ID NO:32 or a sequence which is the same as said base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in said base sequence, and a reverse primer containing in its 3′-side the base sequence of SEQ ID NO:37 or a sequence which is the same as said base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in said base sequence;

(D-1) a set of a forward primer containing in its 3′-side the base sequence of SEQ ID NO:33 or a sequence which is the same as said base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in said base sequence, and a reverse primer containing in its 3′-side the base sequence of SEQ ID NO:38 or a sequence which is the same as said base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in said base sequence;

(E-1) a set of a forward primer containing in its 3′-side the base sequence of SEQ ID NO:34 or a sequence which is the same as said base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in the base sequence, and a reverse primer containing in its 3′-side the base sequence of SEQ ID NO:38 or a sequence which is the same as said base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in said base sequence;

(F-1) a set of a forward primer containing in its 3′-side the base sequence of SEQ ID NO:34 or a sequence which is the same as said base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in said base sequence, and a reverse primer containing in its 3′-side the base sequence of SEQ ID NO:39 or a sequence which is the same as said base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in said base sequence;

(G-1) a set of a forward primer containing in its 3′-side the base sequence of SEQ ID NO:34 or a sequence which is the same as said base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in said base sequence, and a reverse primer containing in its 3′-side the base sequence of SEQ ID NO:40 or a sequence which is the same as said base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in said base sequence;

(H-1) a set of a forward primer containing in its 3′-side the base sequence of SEQ ID NO:35 or a sequence which is the same as said base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in the base sequence, and a reverse primer containing in its 3′-side the base sequence of SEQ ID NO:39 or a sequence which is the same as said base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in said base sequence;

(I-1) a set of a forward primer containing in its 3′-side the base sequence of SEQ ID NO:35 or a sequence which is the same as said base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in said base sequence, and a reverse primer containing in its 3′-side the base sequence of SEQ ID NO:40 or a sequence which is the same as said base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in said base sequence;

(I-2) a set of a forward primer containing in its 3′-side the base sequence of SEQ ID NO:60 or a sequence which is the same as said base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in said base sequence, and a reverse primer containing in its 3′-side the base sequence of SEQ ID NO:61 or a sequence which is the same as said base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in said base sequence;

(I-3) a set of a forward primer containing in its 3′-side the base sequence of SEQ ID NO:62 or a sequence which is the same as said base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in said base sequence, and a reverse primer containing in its 3′-side the base sequence of SEQ ID NO:61 or a sequence which is the same as said base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in said base sequence;

(J-1) a set of a forward primer containing in its 3′-side the base sequence of SEQ ID NO:35 or a sequence which is the same as said base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in said base sequence, and a reverse primer containing in its 3′-side the base sequence of SEQ ID NO:41 or a sequence which is the same as said base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in said base sequence;

(K-1) a set of a forward primer containing in its 3′-side the base sequence of SEQ ID NO:36 or a sequence which is the same as said base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in said base sequence, and a reverse primer containing in its 3′-side the base sequence of SEQ ID NO:39 or a sequence which is the same as said base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in the base sequence;

(L-1) a set of a forward primer containing in its 3′-side the base sequence of SEQ ID NO:36 or a sequence which is the same as said base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in said base sequence, and a reverse primer containing in its 3′-side the base sequence of SEQ ID NO:40 or a sequence which is the same as said base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in said base sequence; and

(M-1) a set of a forward primer containing in its 3′-side the base sequence of SEQ ID NO:63 or a sequence which is the same as said base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in said base sequence, and a reverse primer containing in its 3′-side the base sequence of SEQ ID NO:64 or a sequence which is the same as said base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in said base sequence wherein the primer set further comprises a probe containing an oligonucleotide portion, wherein the ends of the oligonucleotide portion are modified with a fluorescent substance and a quencher substance, respectively.

2. A method of detecting Listeria monocytogenes , comprising a step of amplifying a partial region of lmo0084 gene or lmo2736 gene by a nucleic acid amplification method using the primer set according to claim 1 .

3. The method according to claim 2 , wherein the amplification of the partial region is carried out by a PCR method or an isothermal amplification method.

4. The method according to claim 3 , wherein the isothermal amplification method is a loop-mediated isothermal amplification method.

5. The method according to claim 3 , wherein the PCR method is a real-time PCR method.

6. A primer set for detection of Listeria monocytogenes , comprising any of the following sets:

(A-9) a set of a mixed forward primer containing in its 3′-side the base sequence of SEQ ID NO:67, and a mixed reverse primer containing in its 3′-side the base sequence of SEQ ID NO:69;

(A-10) a set of a mixed forward primer containing in its 3′-side the base sequence of SEQ ID NO:68, and a mixed reverse primer containing in its 3′-side the base sequence of SEQ ID NO:69;

(D-2) a set of a mixed forward primer containing in its 3′-side the base sequence of SEQ ID NO:70, and a mixed reverse primer containing in its 3′-side the base sequence of SEQ ID NO:74;

(E-2) a set of a mixed forward primer containing in its 3′-side the base sequence of SEQ ID NO:71, and a mixed reverse primer containing in its 3′-side the base sequence of SEQ ID NO:74;

(F-2) a set of a mixed forward primer containing in its 3′-side the base sequence of SEQ ID NO:71, and a mixed reverse primer containing in its 3′-side the base sequence of SEQ ID NO:75;

(G-2) a set of a mixed forward primer containing in its 3′-side the base sequence of SEQ ID NO:71, and a mixed reverse primer containing in its 3′-side the base sequence of SEQ ID NO:76;

(H-2) a set of a mixed forward primer containing in its 3′-side the base sequence of SEQ ID NO:72, and a mixed reverse primer containing in its 3′-side the base sequence of SEQ ID NO:75;

(I-4) a set of a mixed forward primer containing in its 3′-side the base sequence of SEQ ID NO:72, and a mixed reverse primer containing in its 3′-side the base sequence of SEQ ID NO:76;

(J-2) a set of a mixed forward primer containing in its 3′-side the base sequence of SEQ ID NO:72, and a reverse primer containing in its 3′-side the base sequence of SEQ ID NO:41 or a sequence which is the same as said base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in said base sequence;

(K-3) a set of a mixed forward primer containing in its 3′-side the base sequence of SEQ ID NO:73, and a mixed reverse primer containing in its 3′-side the base sequence of SEQ ID NO:75;

(L-2) a set of a mixed forward primer containing in its 3′-side the base sequence of SEQ ID NO:73, and a mixed reverse primer containing in its 3′-side the base sequence of SEQ ID NO:76;

(N-1) a set of a mixed forward primer containing in its 3′-side the base sequence of SEQ ID NO:71, and a reverse primer containing in its 3′-side the base sequence of SEQ ID NO:41 or a sequence which is the same as said base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in said base sequence; and

(O-1) a set of a mixed forward primer containing in its 3′-side the base sequence of SEQ ID NO:73, and a reverse primer containing in its 3′-side the base sequence of SEQ ID NO:41 or a sequence which is the same as said base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in said base sequence wherein the primer set further comprises a probe containing an oligonucleotide portion, wherein the ends of the oligonucleotide portion are modified with a fluorescent substance and a quencher substance, respectively.

7. A method of detecting Listeria monocytogenes , comprising a step of amplifying a partial region of lmo0084 gene or lmo2736 gene by a nucleic acid amplification method using the primer set according to claim 6 .

8. A probe for detection of Listeria monocytogenes by real-time PCR, comprising an oligonucleotide portion having the base sequence of SEQ ID NO:77, SEQ ID NOs:80 to 82, SEQ ID NO:85, wherein in SEQ ID NO:85 ngaan is tgaaa or cgaac; (or SEQ ID NO:86, wherein in SEQ ID NO:86, ngcaan is ggcaag or cgcaac; and wherein the oligonucleotide portion has ends modified with a fluorescent substance and a quencher substance, respectively.

9. A primer-probe set for real-time PCR for detection of Listeria monocytogenes , comprising any of the following sets of primers and a probe:

[1] a set of a forward primer containing in its 3′-side the base sequence of SEQ ID NO:67, a reverse primer containing in its 3′-side the base sequence of SEQ ID NO:69, and a probe containing an oligonucleotide portion having the base sequence of SEQ ID NO:77 or 80;

[2] a set of a forward primer containing in its 3′-side the base sequence of SEQ ID NO:67, a reverse primer containing in its 3′-side the base sequence of SEQ ID NO:69, and a mixed probe containing an oligonucleotide portion having the base sequence of SEQ ID NO:85, wherein in SEQ ID NO:85 ngaan is tgaaa or cgaac;

[3] a set of a forward primer containing in its 3′-side the base sequence of SEQ ID NO:68, a reverse primer containing in its 3′-side the base sequence of SEQ ID NO:69, and a probe containing an oligonucleotide portion having the base sequence of SEQ ID NO:77 or 80;

[4] a set of a forward primer containing in its 3′-side the base sequence of SEQ ID NO:68, a reverse primer containing in its 3′-side the base sequence of SEQ ID NO:69, and a mixed probe containing an oligonucleotide portion having the base sequence of SEQ ID NO:85, wherein in SEQ ID NO:85 ngaan is tgaaa or cgaac;

[5] a set of a forward primer containing in its 3′-side the base sequence of SEQ ID NO:32, a reverse primer containing in its 3′-side the base sequence of SEQ ID NO:37, and a probe containing an oligonucleotide portion having the base sequence of SEQ ID NO:81;

[6] a set of a forward primer containing in its 3′-side the base sequence of SEQ ID NO:70, a reverse primer containing in its 3′-side the base sequence of SEQ ID NO:74, and a probe containing an oligonucleotide portion having the base sequence of SEQ ID NO:82; and

[7] a set of a forward primer containing in its 3′-side the base sequence of SEQ ID NO:72, a reverse primer containing in its 3′-side the base sequence of SEQ ID NO:76, and a mixed probe containing an oligonucleotide portion having the base sequence of SEQ ID NO:86, wherein in SEQ ID NO:86, ngcaan is ggcaag or cgcaac; and

wherein each of the oligonucleotide portions of the probes has ends modified with a fluorescent substance and a quencher substance, respectively.