Detection of antibiotic-resistant microorganisms
View Patent ↗Method of detecting methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) in a nucleic acid coamplification assay. The invention advantageously reduces the incidence of false-positive MRSA determinations in real-time assays by requiring satisfaction of a threshold criterion that excludes certain co-infections from the MRSA determination. The invention further provides for determination of MSSA, even when the MSSA is present in combination with methicillin-resistant coagulase-negative (MR-CoNS) bacteria at high or low levels.
1. A method of establishing whether a nasal swab sample contains methicillin-sensitive S. aureus bacteria or methicillin-resistant S. aureus bacteria, said method comprising the steps of:
(a) obtaining nucleic acids from the nasal swab sample;
(b) coamplifying a S. aureus -specific target sequence and a target sequence specific for methicillin resistance in an in vitro nucleic acid amplification reaction performed using nucleic acids obtained in step (a) as templates,
wherein each of said target sequences is included among nucleic acids obtained in step (a), and
wherein amplification products for both of said target sequences are produced and detected in the in vitro nucleic acid amplification reaction;
(c) determining time-dependent indicia of amplification for the S. aureus -specific target sequence and for the target sequence specific for methicillin resistance that coamplified in step (b);
(d) calculating a numerical value that is a function of both of said time-dependent indicia of amplification determined in step (c); and
(e) establishing that
(1) said biological sample contains methicillin-sensitive S. aureus bacteria if the numerical value calculated in step (d) is less than a threshold criterion value that distinguishes the coamplification kinetics of nucleic acids obtained from
(i) methicillin-resistant S. aureus bacteria, and
(ii) a mixture of methicillin-sensitive S. aureus bacteria and methicillin-resistant coagulase-negative bacteria, and
(2) said biological sample contains methicillin-resistant S. aureus bacteria for all values of the numerical value calculated in step (d) that are greater than said threshold criterion value.
2. The method of claim 1 , wherein the threshold criterion value of step (e) is an empirically determined threshold cut-off value.
3. The method of claim 1 , wherein the numerical value calculated in step (d) is a numerical ΔCt value calculated as a difference between the time-dependent indicia of amplification determined in step (c).
4. The method of claim 3 ,
wherein the numerical ΔCt value of step (d) is calculated by subtracting the time-dependent indicia of amplification determined for the mecA target sequence from the time-dependent indicia of amplification determined the S. aureus -specific target sequence.
5. The method of claim 1 , wherein the obtaining step comprises obtaining genomic DNA.
6. The method of claim 1 , wherein the in vitro nucleic acid amplification reaction in step (b) comprises a reverse transcriptase.
7. The method of claim 1 , wherein the S. aureus -specific target sequence is a S. aureus ribosomal nucleic acid sequence.
8. The method of claim 1 , wherein the determining step comprises determining the time at which a predetermined level of a detectable signal indicative of amplicon production is achieved.
9. The method of claim 1 , wherein the target sequence specific for methicillin resistance comprises a mecA target sequence.
10. The method of claim 9 , wherein the numerical value calculated in step (d) is a numerical ΔCt value calculated as a difference between the time-dependent indicia of amplification determined in step (c).
11. The method of claim 10 , wherein the threshold criterion value of step (e) is an empirically determined upper threshold cut-off value.
12. The method of claim 10 ,
wherein the numerical ΔCt value of step (d) is calculated by subtracting the time-dependent indicia of amplification determined for the mecA target sequence from the time-dependent indicia of amplification determined the S. aureus -specific target sequence.