Detection of methicillin-resistant
Disclosed are methods of identifying a methicillin-resistant Staphylococcus aureus (MRSA) in a sample wherein the methods involve detecting a S. aureus -specific nucleic acid sequence, mecA and mecC, in the sample. Kits for determining the presence of MRSA in a sample are also provided.
1 . A method for determining the presence or absence of methicillin-resistant Staphylococcus aureus (MRSA) in a biological sample comprising:
(a) contacting the biological sample with:
(i) a first primer pair that specifically hybridizes under stringent conditions to a segment of a target nucleic acid specific for Staphylococcus aureus , if present, wherein at least one primer of the first primer pair is associated with a fluorophore label,
(ii) a second primer pair that specifically hybridizes under stringent conditions to a segment of a target mecC nucleic acid, if present, wherein one primer of the second primer pair is a primer-probe that (i) is associated with a fluorophore label and (ii) comprises a probe sequence element of SEQ ID NO: 5 at the 5′ end of the primer-probe and SEQ ID NO: 6 at the 3′ end of the primer-probe, wherein the probe sequence element further comprises a quencher, and
(iii) a third primer pair that specifically hybridizes under stringent conditions to a segment of a target mecA nucleic acid, if present, wherein at least one primer of the third primer pair is associated with a fluorophore label, to produce a reaction-sample mixture,
(b) subjecting the reaction-sample mixture to real-time polymerase chain reaction (PCR) conditions under which each of the target nucleic acids present in the biological sample is amplified to produce a fluorescent signal,
(c) measuring the amount of fluorescent signal produced by each fluorophore label, and
(d) determining the presence or absence of MRSA by comparing the amount of fluorescence from the S. aureus -specific target nucleic acid and the mecA and mecC target nucleic acids.
2 . The method of claim 1 , wherein the first primer pair consists of two primers and one primer of the first primer pair comprises SEQ ID NO: 1 and one primer of the first primer pair comprises an oligonucleotide comprising SEQ ID NO: 3 or a primer-probe that (i) is associated with a fluorophore label and (ii) comprises a probe sequence element of SEQ ID NO: 2 at the 5′ end of the primer-probe and a primer sequence element comprising SEQ ID NO: 3 at the 3′ end of the primer-probe, wherein the probe sequence element further comprises a quencher and is covalently linked to the primer sequence element via a polymerase-blocking group.
3 . The method of claim 1 , wherein the second primer pair consists of a first primer comprising SEQ ID NO: 4 and the primer-probe.
4 . The method of claim 1 , wherein the third primer pair consists of two primers and one primer of the third primer pair comprises SEQ ID NO: 7 and one primer of the third primer pair comprises either an oligonucleotide comprising SEQ ID NO: 9 or a primer-probe that (i) is associated with a fluorophore label and (ii) comprises a probe sequence element of SEQ ID NO: 8 at the 5′ end of the primer-probe and a primer sequence element comprising SEQ ID NO: 9 at the 3′ end of the primer-probe, wherein the probe sequence element further comprises a quencher and is covalently linked to the primer sequence element via a polymerase-blocking group.
5 . The method of claim 1 , wherein one primer of the first primer pair and one primer of the third primer pair is a primer-probe that (i) is associated with the fluorophore label and (ii) comprises a probe sequence element at the 5′ end of the primer-probe, wherein the probe sequence element further comprises a quencher, and wherein the fluorophore label of the mecA primer and the fluorophore label of the mecC primer are identical.
6 . The method of claim 5 , wherein the primer-probe of the first primer pair comprises a fluorescein amidite fluorophore and the primer-probes of the second and third primer pairs comprise a xanthene dye that fluoresces in the red region of the visible spectrum.
7 . The method of claim 1 , wherein all primer pairs are contained together in an amplification master mix further comprising DNA polymerase, dNTPs and PCR buffer prior to contacting with the biological sample.
8 . The method of claim 1 , wherein step (a) further comprises contacting the biological sample with a fourth primer pair that specifically hybridizes under stringent conditions to a segment of a control target nucleic acid.
9 . A detection method comprising:
(a) contacting a biological sample with:
(i) a first primer pair that specifically hybridizes under stringent conditions to a segment of a target nucleic acid specific for Staphylococcus aureus , if present, wherein at least one primer of the first primer pair is associated with a fluorophore label,
(ii) a second primer pair that specifically hybridizes under stringent conditions to a segment of a target mecC nucleic acid, if present, wherein one primer of the second primer pair is a primer-probe that (i) is associated with a fluorophore label and (ii) comprises a probe sequence element of SEQ ID NO: 5 at the 5′ end of the primer-probe and SEQ ID NO: 6 at the 3′ end of the primer-probe, wherein the probe sequence element further comprises a quencher, and
(iii) a third primer pair that specifically hybridizes under stringent conditions to a segment of a target mecA nucleic acid, if present, wherein at least one primer of the third primer pair is associated with a fluorophore label, to produce a reaction-sample mixture,
(b) subjecting the reaction-sample mixture to real-time polymerase chain reaction (PCR) conditions under which each of the target nucleic acids present in the biological sample is amplified to produce a fluorescent signal, and
(c) measuring the amount of fluorescent signal produced by each fluorophore label.
10 . The method of claim 9 , wherein the first primer pair consists of two primers and one primer of the first primer pair comprises SEQ ID NO: 1 and one primer of the first primer pair comprises an oligonucleotide comprising SEQ ID NO: 3 or a primer-probe that (i) is associated with a fluorophore label and (ii) comprises a probe sequence element of SEQ ID NO: 2 at the 5′ end of the primer-probe and a primer sequence element comprising SEQ ID NO: 3 at the 3′ end of the primer-probe, wherein the probe sequence element further comprises a quencher and is covalently linked to the primer sequence element via a polymerase-blocking group.
11 . The method of claim 9 , wherein the second primer pair consists of a first primer comprising SEQ ID NO: 4 and the primer-probe.
12 . The method of claim 9 , wherein the third primer pair consists of two primers and one primer of the third primer pair comprises SEQ ID NO: 7 and one primer of the third primer pair comprises either an oligonucleotide comprising SEQ ID NO: 9 or a primer-probe that (i) is associated with a fluorophore label and (ii) comprises a probe sequence element of SEQ ID NO: 8 at the 5′ end of the primer-probe and a primer sequence element comprising SEQ ID NO: 9 at the 3′ end of the primer-probe, wherein the probe sequence element further comprises a quencher and is covalently linked to the primer sequence element via a polymerase-blocking group.
13 . The method of claim 9 , wherein one primer of the first primer pair and one primer of the third primer pair is a primer-probe that (i) is associated with the fluorophore label and (ii) comprises a probe sequence element at the 5′ end of the primer-probe, wherein the probe sequence element further comprises a quencher, and wherein the fluorophore label of the mecA primer and the fluorophore label of the mecC primer are identical.
14 . The method of claim 13 , wherein the primer-probe of the first primer pair comprises a fluorescein amidite fluorophore and the primer-probes of the second and third primer pairs comprise a xanthene dye that fluoresces in the red region of the visible spectrum.
15 . The method of claim 9 , wherein:
MRSA is determined to be present in the biological sample when a fluorescent signal is detected for both the S. aureus -specific target nucleic acid and the mecA and/or mecC nucleic acids and (1) the cycle threshold (Ct) from the S. aureus -specific target nucleic acid minus the Ct from the mecA and/or mecC nucleic acids ≤1.9 and ≥−1.9, or (2) the Ct from the S. aureus -specific target nucleic acid minus the Ct from the mecA and/or mecC nucleic acids >1.9;
S. aureus and a methicillin-resistance gene are determined to be present in the biological sample when a Ct is detected for both the S. aureus -specific target nucleic acid and the mecA and/or mecC nucleic acids, and the Ct from the S. aureus -specific target nucleic acid plus 1.9<the Ct from the mecA and/or mecC nucleic acids; or
S. aureus is determined to be present and MRSA is determined to be absent in the biological sample if a fluorescent signal is detected for the S. aureus -specific target nucleic acid sequence but no fluorescent signal is detected for mecA and mecC.