Detection of methicillin-resistant and methicillin-sensitive Staphylococcus aureus in biological samples
AssigneeQuest Diagnostics Investments Incorporated
InventorsLarry McCarthy, Lilly Kong, Michelle Tabb, Ming-Chou Lee
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Abstract
Disclosed are methods of identifying a methicillin-resistant Staphylococcus aureus (MRSA) or methicillin-sensitive Staphylococcus aureus (MSSA) in a sample. The present invention provides a diagnostic method comprising modification of sequences of S. aureus by converting non-methylated cytosine residues ultimately into thymidine residues in the target nucleic acid. The invention further provides for the detection of modified sequences derived from the spa gene, the mecA gene, and the integrated SCCmec cassette of S. aureus.
Claims29
A method for detecting Staphylococcus aureus in a biological sample comprising:
- (a) converting the non-methylated cytosines present in the nucleic acids contained in a biological sample, to uracils to produce sequence-modified nucleic acids,
- (b) bringing the biological sample containing the sequence modified nucleic acids in contact with:
- (i) a first primer pair which is complementary to a segment of a marker gene specific for Staphylococcus aureus of the sequence modified nucleic acids;
- (ii) a second primer pair which is complementary to a segment of the mecA gene of the sequence modified nucleic acids;
- and
- (iii) a third primer pair, one primer of which is complementary to a segment of SCCmec of the sequence modified nucleic acids and the other primer of which is complementary to a segment of the orfx gene of the sequence modified nucleic acids;
- under conditions wherein the primers specifically hybridize and amplification products of the sequence-modified nucleic acids are produced;
- and
- (c) identifying the modified nucleic acids from Staphylococcus aureus by detecting the amplification product produced by one or more of the primer pairs.
A method for determining if a biological sample from an individual contains methicillin resistant Staphylococcus aureus (MRSA) or methicillin sensitive Staphylococcus aureus (MSSA), comprising:
- (a) converting the non-methylated cytosines present in the nucleic acids contained in the biological sample, to uracils to produce sequence-modified nucleic acids,
- (b) bringing the biological sample containing the sequence modified nucleic acids in contact with:
- (i) a first primer pair which is complementary to a segment of a marker gene specific for Staphylococcus aureus of the sequence modified nucleic acids;
- (ii) a second primer pair which is complementary to a segment of the mecA gene of the sequence modified nucleic acids;
- and
- (iii) a third primer pair, one primer of which is complementary to a segment of SCCmec of the sequence modified nucleic acids and the other primer of which is complementary to a segment of the orfx gene of the sequence modified nucleic acids;
- under conditions wherein the primers specifically hybridize and amplification products of the sequence-modified nucleic acids are produced;
- and
- (c) identifying the modified nucleic acids from Staphylococcus aureus by detecting the amplification product produced by one or more of the primer pairs, wherein
- (i) amplification of all three sequence-modified nucleic acids indicates MRSA in the sample;
- and
- (ii) amplification of the S. aureus specific marker gene alone, or a segment of SCCmec and the S. aureus specific marker gene, but not mecA, indicates MSSA in the sample.
A method of identifying a methicillin-resistant Staphylococcus aureus (MRSA) or methicillin sensitive Staphylococcus aureus (MSSA), if present, in a biological sample, comprising:
- (a) converting the non-methylated cytosines present in the nucleic acids contained in a biological sample, to uracils to produce sequence-modified nucleic acids,
- (b) bringing the biological sample in contact with:
- (i) a first primer pair which is complementary to a marker gene specific for Staphylococcus aureus;
- (ii) a second primer pair which is complementary to the mecA gene;
- and
- (iii) a third primer pair, one primer of which is complementary to SCCmec and the other primer of which is complementary to the orfx gene;
- under conditions wherein the primers specifically hybridize and amplify the marker gene, mecA gene, SCCmec and orfx gene, and
- (c) identifying the MSSA and/or MRSA by detecting an amplification product produced by all of the three primer pairs, wherein
- (i) amplification of all three sequence-modified nucleic acids indicates MRSA in the sample;
- and
- (ii) amplification of the S. aureus specific marker gene alone, or a segment of SCCmec and the S. aureus specific marker gene, but not mecA, indicates MSSA in the sample.
Dependencies are inferred from the claim text.