We first established a qPCR assay for S. aureus based on the single copy loci lysS and proS. qPCR of both target genes yielded amplimers of the expected molecular weight on gel electrophoresis (data not shown) and showed a direct correlation between critical cycle threshold and substrate concentration. Appropriate sigmoidal curves for amplimer accumulation were observed in all instances.
We next determined what degree of amplification might be afforded by MDA on the naked DNA template used to validate our S. aureus-specific assays. Pre-incubation with the GenomiPhi enzyme resulted in a vast increase in the amount of target S. aureus DNA present, reducing the threshold critical cycle from ~ 31.7 to ~16.9. This represents a theoretical amplification (assuming a two-fold exponential increase with each cycle reduced, and allowing for the 20-fold dilution introduced for experimental purposes) of > 214 × 20, or over 300 thousand-fold (Table ).
Quantitation of S. aureus genomic DNA (gDNA) by real time PCR
We next examined what effect MDA pre-treatment would have on clinical samples of joint aspirates recovered from a well-characterized infected patient after total elbow arthroplasty; both samples stored in RNAlater and also snap-frozen directly were tested. In order to make available DNA for amplification without need for extensive specimen handling, a simple protocol for alkali/heat lysis was introduced. Results are shown in Table .
qPCR assay of aspirated synovial fluid, +/- lysis and MDA
When aspirate samples were unlysed there was no signal accumulation above the detection threshold, consistent with the presence of inhibitors since these same materials have yielded PCR amplimers successfully after purification of nucleic acids within. Aspirate stored in RNA later as well as stored directly, when lysed, did give a positive signal on direct qPCR assay but only when 0.1 μl of material was used, not when greater quantities of starting material were present, again consistent with the presence of inhibitors. In contrast, all lysed aspirates subjected to MDA gave convincingly positive qPCR signals for both proS and lysS, demonstrating that pre-amplification with MDA expands the window of detectability in these joint aspirate samples by at least 100-fold (from 0.1 μl to 10 μl inclusive). Use of RNAlater as a storage medium had no effect on the initial detection or ultimate amplification yield.
We next used real-time PCR to quantify the amount of S. aureus and human genomic DNA present in synovial fluid samples. Assaying two independently obtained samples from the same patient, we found that S. aureus DNA was present at 0.014 μg/ml in one sample, and at 0.020 μg/ml in the other. Human genomic DNA was present in over a thousand-fold excess by comparison, measured at 33.6 μg/ml and 79.4 μg/ml respectively.
In order to verify that MDA could function to amplify minor quantities of bacterial DNA in the presence of large (but physiologically relevant) quantities of human DNA, we next mixed small quantities of purified S. aureus DNA (in roughly the quantity we would expect to find in a small volume of synovial fluid) with increasing amounts of purified human genomic DNA. Samples were either treated +/- MDA; the resulting mixes were diluted and then assayed by quantitative PCR. Results are shown in Table .
qPCR assay of purified S. aureus and human DNA (hDNA) mix, with and without MDA pre-treatment
Using 0.01 ng of S. aureus DNA as a starting template, we were unable to detect any accumulation signal in our real-time assay under any condition when pre-treatment with MDA had not been applied. In contrast, when pre-treatment with MDA was effected, all samples gave convincing positive results, despite the additional dilution. Results were uniformly positive when up to 10 ng of human DNA were in the initial mix, representing a thousand-fold excess. Results were still mostly positive, in 10 out of 12 trials, when 100 ng of human DNA were in the initial template mix, raising the possibility that a > 104-fold excess of human DNA may itself begin to exert some inhibitory effect on the ability of MDA to amplify minor bacterial components in the mix. However, in general these results support our direct evidence from the clinical samples that MDA has the potential to be a useful adjunct to PCR examination of synovial aspirates with its ability to amplify small quantities of bacterial DNA in complex mixes.