Detection of methicillin-resistant
The present methods pertain to amplifying and/or detecting Staphylococcus aureus (“SA”) and methicillin-resistant Staphylococcus aureus (“MRSA”) nucleic acids based on a combined detection of ldh1 as a SA marker and mecA as a MRSA marker. In certain embodiments the methods also pertain to amplifying and/or detecting one or more SCCmec integration sites or bridge regions. Primers and probes are suitable to be used in the present methods to detect SA and MRSA simultaneously in a single reaction or in separate reactions. The amplified nucleic acid can be detected by a variety of state of the art methods, including fluorescence resonance energy transfer (“FRET”), radiolabels, enzyme labels, and the like.
1. A method for detecting a methicillin-resistant Staphylococcus aureus (“MRSA”) in a sample containing nucleic acids, comprising:
contacting the nucleic acids in the sample with one or more primers, wherein at least one of the primers is selected from the group consisting of:
a primer comprising the nucleic acid sequence of SEQ ID NO:20,
a primer comprising the nucleic acid sequence of SEQ ID NO:21,
a primer comprising the nucleic acid sequence of SEQ ID NO:23,
a primer comprising the nucleic acid sequence of SEQ ID NO: 24,
a primer comprising the nucleic acid sequence of SEQ ID NO:25, and
a primer comprising the nucleic acid sequence of SEQ ID NO:26; and
detecting nucleic acids in the sample comprising amplified mecA, ldh1 and mecA LGA251 genes wherein the presence of amplified mecA and ldh1 genes in approximately 1:1 ratio or amplified mecA LGA251 and ldh1 genes in approximately 1:1 ratio indicates the presence of methicillin-resistant Staphylococcus aureus , wherein the nucleic acids in the sample comprising amplified mecA, ldh1 and mecA LGA251 genes are detected in a single reaction by one or more fluorescence-generating probes, wherein at least one of the fluorescence-generating probes is selected from the group consisting of:
a fluorescence-generating probe comprising the nucleic acid sequence of SEQ ID NO:27, Ra-G*TAAAAGGTGTA*CTGTTGC-Rb,
a fluorescence-generating probe comprising the nucleic acid sequence of SEQ ID NO:28, Ra-G*ATAAAAT*T*T*GTA*TA*GG-Rb, and
a fluorescence-generating probe comprising the nucleic acid sequence of SEQ ID NO:29, Ra-FAM-AAAT*T*T*CAAATCACTAC-Rb,
wherein Ra is independently selected from (M) a -Fl and (M) a -Q, Rb is independently selected from (M) a -Fl and (M) a -Q, G* is 6-amino-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one, A* is 4-(4,6-Diamino-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-but-3-yn-1-ol, T* is 5-(4-hydroxy-but-1-ynyl)-1H-pyrimidine-2,4-dione, M is a minor groove binder, a is 0 or 1, Fl is a fluorophore with emission wavelength between about 400 and 900 nm, and Q is a non-fluorescent quencher, wherein one of Ra and Rb comprises a fluorophore and the other comprises a quencher, and
wherein the quencher allows quenching of fluorescence when the probes are unhybridized.
2. The method of claim 1 , wherein the primers are fluorescence-generating primers.
3. The method of claim 1 , further comprising the step of detecting nucleic acids in the sample comprising at least one amplified SCCmec integration site or bridge region.
4. A method for detecting Staphylococcus aureus (“SA”), methicillin-resistant Staphylococcus aureus LGA251 (“LGA251”) and methicillin-resistant Staphylococcus aureus (“MRSA”) nucleic acids in a sample using combined detection of ldh1, mecA LGA251 and mecA genes, comprising:
(a) contacting the sample with a first primer and a second primer having the formula:
5′-(X) n Y-3′ (I),
wherein X represents a 5′ portion of the primers that is non-complementary to the ldh1 gene, n is 0 or 1, Y represents a 3′ portion of the primers that is complementary to the ldh1 gene, and X is about 3-30 nucleotides in length;
(b) contacting the sample after step (a) with a third primer and a fourth primer having the formula:
5′-(X) n Y′-3′ (I),
wherein X represents a 5′ portion of the primers that is non-complementary to the mecA gene, n is 0 or 1, Y′ represents a 3′ portion of the primers that is complementary to the mecA gene, and X is about 3-30 nucleotides in length;
(c) contacting the sample after step (b) with a fifth primer and a sixth primer having the formula:
5′-(X) n Y′-3′ (I),
wherein X represents a 5′ portion of the primers that is non-complementary to the mecA LGA251 gene, n is 0 or 1, Y′ represents a 3′ portion of the primers that is complementary to the mecA LGA251 gene, and X is about 3-30 nucleotides in length;
(d) incubating the sample following steps (a), (b) and (c) under conditions sufficient to amplify the ldh1, mecA and mecA LGA251 genes; and
(e) detecting amplified ldh1, mecA and mecA LGA251 genes in the sample, wherein the amplified mecA LGA251 genes in the sample are detected in a single reaction by one or more fluorescence generating probes, wherein at least one of the fluorescence-generating probes is selected from the group consisting of:
a fluorescence-generating probe comprising the nucleic acid sequence of SEQ ID NO:27, Ra-G*TAAAAGGTGTA*CTGTTGC-Rb,
a fluorescence-generating probe comprising the nucleic acid sequence of SEQ ID NO:28, Ra-G*ATAAAAT*T*T*GTA*TA*GG-Rb, and
a fluorescence-generating probe comprising the nucleic acid sequence of SEQ ID NO:29, Ra-FAM-AAAT*T*T*CAAATCACTAC-Rb,
wherein Ra is independently selected from (M) a -Fl and (M) a -Q, Rb is independently selected from (M) a -Fl and (M) a -Q, G* is 6-amino-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one, A* is 4-(4,6-Diamino-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-but-3-yn-1-ol, T* is 5-(4-hydroxy-but-1-ynyl)-1H-pyrimidine-2,4-dione, M is a minor groove binder, a is 0 or 1, Fl is a fluorophore with emission wavelength between about 400 and 900 nm, and Q is a non-fluorescent quencher, wherein one of Ra and Rb comprises a fluorophore and the other comprises a quencher, and
wherein the quencher allows quenching of fluorescence when the probes are unhybridized.
5. The method of claim 4 , wherein the fifth primer and the sixth primer are selected from the group consisting of:
a fifth primer comprising the nucleic acid sequence of SEQ ID NO:20 and a sixth primer comprising the nucleic acid sequence of SEQ ID NO:21,
a fifth primer comprising the nucleic acid sequence of SEQ ID NO:23 and a sixth primer comprising the nucleic acid sequence of SEQ ID NO: 24, and
a fifth primer comprising the nucleic acid sequence of SEQ ID NO:25 and a sixth primer comprising the nucleic acid sequence of SEQ ID NO:26.