Rapid detection of the “high-virulent” ST-17 clone of group B
View Patent ↗The present invention relates to polynucleotides enabling the rapid, simple and specific detection of Group B Streptococcus highly-virulent ST-17 clones. The present invention also relates to the polypeptides encoded by the polynucleotides, as well as to antibodies directed or raised against the polypeptides. The present invention also relates to kits and methods for the specific detection of Group B Streptococcus highly-virulent ST-17 clones, using the polynucleotides, the polypeptides or the antibodies according to the invention.
1. A primer set, suitable for use in a process for the detection of DNA of a GBS strain of the ST-17 clone or for the detection of products derived from such DNA, wherein said primer set comprises at least one of the following primer pairs:
a) ST-17S having the sequence of SEQ ID NO: 33 and ST-17AS having sequence of SEQ ID NO: 34;
b) ST-17S having the sequence of SEQ ID NO: 33 and O12 having sequence of SEQ ID NO: 28;
c) O11 having the sequence of SEQ ID NO: 27 and O12 having sequence of SEQ ID NO: 28;
d) O12 having the sequence of SEQ ID NO: 28 and O13 having sequence of SEQ ID NO: 29;
e) O11 having the sequence of SEQ ID NO: 27 and ST-17AS having sequence of SEQ ID NO: 34;
f) O13 having the sequence of SEQ ID NO: 29 and ST-17AS having sequence of SEQ ID NO: 34;
g) a primer pair which is fully complementary to a primer pair in one of a) to f); and
h) a primer pair which hybridized to a primer pair in a) to f) under stringent conditions wherein said stringent conditions comprise hybridization at a temperature from about 35 to 65° C. in a solution comprising SSC 6×, SDS 0.5%, Denhardt's solution 5× and 100 μg of non-specific DNA, or a solution of equivalent ionic strength, and then at least one wash step conducted at about 65° C. in a solution comprising at most 0.2×SSC and at most 0.1% SDS, or a solution of equivalent ionic strength,
wherein the polynucleotides making up primer pairs a) to h) are labeled to enable detection.
2. The primer set of claim 1 which consists of ST-17S having the sequence of SEQ ID NO: 33 and ST-17AS having sequence of SEQ ID NO: 34.
3. An amplimer consisting of a product of the amplification of a DNA of a GBS strain of the ST-17 clone with the primer set of claim 1 .
4. The amplimer according to claim 3 , wherein said amplification product is selected from the group consisting of:
the polynucleotide of sequence ST-17S/ST-17AS of SEQ ID NO: 35, obtained with the primer set consisting of ST-17S of SEQ ID NO: 33 and ST-17AS of SEQ ID NO: 34;
the polynucleotide of sequence O13/ST-17AS of SEQ ID NO: 36 obtained with the primer set consisting of O13 of SEQ ID NO: 29 and ST17-AS of SEQ ID NO: 34;
the polynucleotide of sequence O13/O12 of SEQ ID NO: 37 obtained with the primer set consisting of O13 of SEQ ID NO: 29 and O12 of SEQ ID NO: 28;
the polynucleotide of sequence ST-17S/O12 of SEQ ID NO: 38, obtained with the primer set consisting of ST-17S of SEQ ID NO: 33 and O12 of SEQ ID NO: 28;
the polynucleotide of sequence O11/ST-17AS of SEQ ID NO: 39, obtained with the primer set consisting of O11 of SEQ ID NO: 27 and ST17-AS of SEQ ID NO: 34;
the polynucleotide of sequence O11/O12 of SEQ ID NO: 40, obtained with the primer set consisting of O11 of SEQ ID NO: 27 and O12 of SEQ ID NO: 28.
5. A kit for the in vitro detection of an infection by a GBS strain in a biological sample, which comprises a primer set and means for the detection of an amplification product obtained with said primer set, wherein said primer set comprises at least one of the following primer pairs:
a) ST-17S having the sequence of SEQ ID NO: 33 and ST-17AS having sequence of SEQ ID NO: 34;
b) ST-17S having the sequence of SEQ ID NO: 33 and O12 having sequence of SEQ ID NO: 28;
c) O11 having the sequence of SEQ ID NO: 27 and O12 having sequence of SEQ ID NO: 28;
d) O12 having the sequence of SEQ ID NO: 28 and O13 having sequence of SEQ ID NO: 29;
e) O11 having the sequence of SEQ ID NO: 27 and ST-17AS having sequence of SEQ ID NO: 34;
f) O13 having the sequence of SEQ ID NO: 29 and ST-17AS having sequence of SEQ ID NO: 34;
g) a primer pair which is fully complementary to a primer pair in one of a) to f); and
h) a primer pair which hybridized to a primer pair in a) to f) under stringent conditions wherein said stringent conditions comprise hybridization at a temperature from about 35 to 65° C. in a solution comprising SSC 6×, SDS 0.5%, Denhardt's solution 5× and 100 μg of non-specific DNA, or a solution of equivalent ionic strength, and then at least one wash step conducted at about 65° C. in a solution comprising at most 0.2×SSC and at most 0.1% SDS, or a solution of equivalent ionic strength.
6. The kit according to claim 5 which further comprises a primer set suitable for the amplification of the DNA of GBS strains, said primer set comprising at least two oligonucleotides, wherein at least one oligonucleotide is a sense primer and at least one oligonucleotide is an anti-sense primer, said oligonucleotides being selected from the group consisting of:
dltRS having sequence of SEQ ID NO: 31;
dltRAS having sequence of SEQ ID NO: 32;
O1 having sequence of SEQ ID NO: 17;
O2 having sequence of SEQ ID NO: 18;
O3 having sequence of SEQ ID NO: 19;
O4 having sequence of SEQ ID NO: 20;
O5 having sequence of SEQ ID NO: 21;
O6 having sequence of SEQ ID NO: 22;
O7 having sequence of SEQ ID NO: 23;
O8 having sequence of SEQ ID NO: 24;
O9 having sequence of SEQ ID NO: 25;
O10 having sequence of SEQ ID NO: 26; and
O14 having sequence of SEQ ID NO: 30.
7. The kit according to claim 6 , wherein the oligonucleotides are labeled to enable detection.