Methods, kits, and compositions for detection of MRSA
The present invention provides multiplex assays, methods and kits that may be used to detect and confirm the presence of MRSA in a sample. The methods include real-time PCR assays, and the kits and compositions include oligonucleotides used as primers and probes. The present invention further comprises assays useful to identify and differentiate MRSA, MSSA, MRSE, MSSE, MRCNS and MSCNS in a sample.
1. A multiplex assay for identifying and differentiating combinations of methicillin-resistant Staphylococcus aureus (MRSA), methicillin-sensitive Staphylococcus aureus (MS SA), methicillin-resistant Staphylococcus epidermidis (MRSE), methicillin-sensitive Staphylococcus epidermidis (MSSE), methicillin-resistant coagulase-negative staphylococci (MRCNS), and/or methicillin-sensitive coagulase-negative staphylococci (MSCNS) in a sample, comprising:
performing a PCR-based mecA assay, a PCR-based tuf-Sa assay, and at least one PCR-based assay selected from a femA Se assay and a tuf-CNS assay;
wherein said PCR-based assays comprise at least one target gene and at least one reference gene; the primers for the mecA assay comprise SEQ ID NO: 16 and 17 and the probe for the mecA assay comprises SEQ ID NO: 18; the primers for the tuf-Sa assay comprise SEQ ID NO: 10 and 11 and the probe for the tuf-Sa assay comprises SEQ ID NO: 12; the primers for the femA-Se assay comprise SEQ ID NO: 7 and 8 and the probe for the femA-Se assay comprises SEQ ID NO: 9; and the primers for the tuf-CNS assay comprise SEQ ID NO: 13 and 14 and the probe for the tuf-CNS assay comprises SEQ ID NO: 15; and
wherein the PCR-based assays are performed with quantitative PCR, quantitative real time PCR, quantitative reverse transcription PCR, or quantitative real time reverse transcription PCR.
2. The multiplex assay of claim 1 , further comprising at least one PCR-based assay to detect the presence or absence of a nuc-Sa gene or a femA-Sa gene.
3. The multiplex assay of claim 2 , wherein the primer set associated with the PCR-based nuc-Sa assay comprises SEQ ID NO: 1 and 2.
4. The multiplex assay of claim 3 , further comprising a probe associated with the PCR-based nuc-Sa assay comprising SEQ ID NO: 3.
5. The multiplex assay of claim 2 , wherein the primer set associated with the PCR-based femA-Sa assay comprises SEQ ID NO: 4 and 5.
6. The multiplex assay of claim 5 , further comprising a probe associated with the PCR-based femA-Sa assay comprising SEQ ID NO: 6.
7. The multiplex assay of claim 1 , wherein the PCR-based assays are configured to analyze expression of the target gene in relation to the reference gene according to relative quantification of crossing points values or cycle threshold values using additive effects of relative cycle threshold values contributed from different strains within the sample.
8. The multiplex assay of claim 1 , wherein the PCR-based mecA assay, the PCR-based tuf-Sa assay, and the PCR-basedfemA-Se assay are performed.
9. The multiplex assay of claim 1 , wherein the PCR-based mecA assay, the PCR-based tuf-Sa assay, and the PCR-based tuf-CNS assay are performed.
10. The multiplex assay of claim 1 , wherein the PCR-based assays are performed with quantitative real time PCR.
11. The multiplex assay of claim 1 , wherein the step of performing the PCR-based assays further comprises:
a. determining the presence of a mecA gene within the sample to detect the presence of at least one of MRSA, MRCNS and MRSE;
b. determining the presence of a tuf- S. aureus gene to detect the presence of at least one of MRSA and MSSA;
c. determining the presence of a tuf-CNS assay gene to detect the presence of at least one of MRCNS and MSCNS; and/or
d. determining the presence of a femA- S. epidermis gene to detect the presence of at least one of MRSE and MS SE.
12. A method for identifying combinations of MRSA, MSSA, MRSE, MSSE, MRCNS, and/or MSCNS in a sample, comprising
a. obtaining a sample;
b. screening the sample by conducting a PCR-based mecA assay, a PCR-based tuf-Sa assay, and at least one PCR-based assay selected from a femA-Se assay and a tuf-CNS assay,
wherein the PCR-based assays comprise at least one target gene and at least one reference gene; the primers for the mecA assay comprise SEQ ID NO: 16 and 17 and the probe for the mecA assay comprises SEQ ID NO: 18; the primers for the tuf-Sa assay comprise SEQ ID NO: 10 and 11 and the probe for the tuf-Sa assay comprises SEQ ID NO: 12; the primers for the femA-Se assay comprise SEQ ID NO: 7 and 8 and the probe for the femA-Se assay comprises SEQ ID NO: 9; and the primers for the tuf-CNS assay comprise SEQ ID NO: 13 and 14 and the probe for the tuf-CNS assay comprises SEQ ID NO: 15; and
wherein the PCR-based assays are performed with quantitative PCR, quantitative real time PCR, quantitative reverse transcription PCR, or quantitative real time reverse transcription PCR;
c. analyzing results obtained from the PCR-based assays to determine the presence or absence of a mecA gene, a tuf- S. aureus gene, and at least one gene selected from a femA- S. epidermis gene and a tuf-CNS gene;
d. identifying combinations of MRSA, MSSA, MRSE, MSSE, MRCNS and/or MSCNS based on the results of the PCR-based assays.
13. The method of claim 12 , wherein the step of analyzing results obtained from the PCR-based assays further comprises:
a. determining the presence of said mecA gene within the sample to detect the presence of at least one of MRSA, MRCNS and MRSE;
b. determining the presence of said tuf- S. aureus gene to detect the presence of at least one of MRSA and MSSA;
c. determining the presence of said tuf-CNS assay gene to detect the presence of at least one of MRCNS and MSCNS; and/or
d. determining the presence of said femA- S. epidermis gene to detect the presence of at least one of MRSE and MSSE.
14. The method of claim 12 , wherein the PCR-based assays are configured to analyze expression of the target gene in relation to the reference gene according to relative quantification of crossing points values or cycle threshold values using additive effects of relative cycle threshold values contributed from different strains within the sample.
15. The method of claim 12 , wherein screening the sample comprises conducting the PCR-based mecA assay, the PCR-based tuf-Sa assay, and the PCR-basedfemA-Se assay.
16. The method of claim 12 , wherein screening the sample comprises conducting the PCR-based mecA assay, the PCR-based tuf-Sa assay, and the PCR-based tuf-CNS assay.
17. The method of claim 12 , wherein the PCR-based assays are performed with quantitative real time PCR.
18. The method of claim 12 , wherein the sample is a fluid sample comprising peripheral blood, lymph fluid, ascites, serous fluid, pleural effusion, sputum, bronchial wash, bronchioalveolar lavage fluid (BALF), cerebrospinal fluid, semen, amniotic fluid, lacrimal fluid, stool, or urine.
19. The method of claim 12 , wherein the sample is derived from an agricultural or environmental source.