Methods for detection and identification of methicillin-resistant
View Patent ↗The present invention describes novel SCCmec right extremity junction sequences for the detection of methicillin-resistant Staphyloccocus aureus (MRSA). It relates to the use of these DNA sequences for diagnostic purposes.
1. A method to detect the presence of a methicillin-resistant Staphylococcus aureus (MRSA) strain in a specimen, comprising:
obtaining a sample from said specimen to be analyzed for the presence of a MRSA strain that includes an SCCmec insert containing a mecA gene, said SCCmec being inserted into chromosomal DNA, thereby generating a polymorphic right extremity junction (MREJ) region sequence that includes sequence from both the SCCmec insert right extremity and chromosomal DNA adjoining that right extremity;
contacting the sample with at least two primers, wherein the at least two primers amplify the MREJ region sequence of at least one of SEQ ID NOs: 42, 43, 44, 45, 46, 51, 47, 48, 49, 50,171,165,166,167 and/or 168 under conditions of 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 2.5 mM MgCl 2 at 55° C., and wherein said contacting takes place under annealing conditions wherein an amplicon is produced if any of said SEQ ID NOs are present in said sample, thereby generating an amplicon if a MRSA strain of at least one of MREJ types iv-ix is present in the sample; and
detecting said amplicon as indicative of the presence of a MRSA strain in said specimen.
2. The method of claim 1 , wherein said at least two primers further include at least two primers that amplify the MREJ region sequence of any one of SEQ ID NOs: 1, 20, 21, 22, 23, 24, 25, 41, 199, 2, 17, 18, 19, 26, 40, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 185, 186, 197, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 104, 184, and 198 under conditions of 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 2.5 mM MgCl 2, at 55° C.
3. The method of claim 1 , wherein said at least two primers include SEQ ID NO: 64 and at least one of the following primers: SEQ ID NO: 79 for the detection of MREJ type iv; SEQ ID NO: 80 for the detection of MREJ type v; SEQ ID NO: 204 for the detection of MREJ type vi; SEQ ID NO: 112 for the detection of MREJ type vii; SEQ ID NO: 113 for the detection of MREJ type vii; SEQ ID NO: 115 for the detection of MREJ type viii; SEQ ID NO: 116 for the detection of MREJ type viii; and SEQ ID NO: 109 for the detection of MREJ type ix.
4. The method of claim 1 , further comprising detecting the presence of SEQ ID NO: 172 as indicative of the presence of MREJ type x.
5. A method for classifying a MRSA strain comprising performing the method of claim 1 and determining which of SEQ ID NOs: 42, 43, 44, 45, 46, 51, 47, 48, 49, 50, 171, 165, 166, 167 and/or 168 is present in said sample, wherein the presence of any of SEQ ID NOs: 42-46 and 51 is indicative of MREJ type iv, the presence of any of SEQ ID NOs: 47-50 is indicative of MREJ type v, the presence of SEQ ID NO: 171 is indicative of the presence of MREJ type vi, the presence of either of SEQ ID NOs: 165 and 166 is indicative of the presence of MREJ type vii, the presence of SEQ ID NO: 167 is indicative of the presence of MREJ type viii, and the presence of SEQ ID NO: 168 is indicative of the presence of MREJ type ix.