Table 1.
List of synthetic template, primer and probe sequences used in this study.
Fig 1.
A schematic diagram demonstrating the principle of suppressor eliminating false positive results.
A. In the case there is only contaminant in the reaction (no suppressor and no true target), if the contaminating Staphylococcus species level is higher than LOD, it will be amplified and detected on the array as a false positive (red spot). B. If the suppressor is also present in the reaction (but no true target), it will be amplified and may optionally be detected by a suppressor-specific probe on the array (blue spot). At the same time, the amplification of the contaminant is suppressed and will not be amplified to a detectable level. Therefore, the false positive is eliminated and the result will be negative (as there is no true target). C. In the case wherein there is also a target truly present in the reaction in addition to the contaminant and suppressor, if the level of the true target input is higher than LOD, it will be amplified and detected on the array as a true positive (green spot) whereas the amplification of the contaminant will be suppressed due to competition and will not be amplified to a detectable level.
Fig 2.
A mathematic model of the effect of amplification rate on suppressor input requirements.
An equation is used to determine how many copies of suppressor input would be required for a specified threshold amount (Ta) with various suppressor amplification doubling rates at a fixed amplification rate for the target organism (k1).
Fig 3.
Effect of suppressor input on the limit of detection for MSSA.
Staphylococcus muscae cells are used to suppress the low copy amplification of the tuf gene using primer pair tuf430L/tuf527v1. The probe map is shown in the right panel. Fid = Fiducial control; HC = Hybridization control; DC = Detection control, Gen = Staphylococcus genus probe, Sau = Staphylococcus aureus specific probe; Smu = Staphylococcus muscae. Others are blank or irrelevant species probes. The red rectangles indicate the Staphylococcus aureus tuf specific probe reacted, generating detectable signal. The blue ovals show the detectable signal with the suppressor (Staphylococcus muscae) specific probe.
Fig 4.
tuf gene amplicon region sequence alignment.
The tuf gene amplicon sequence was aligned with seven clinically relevant Staphylococcus species commonly detected in human bloodstream infection and two species used as suppressors in this study. The sequences aligned from top to bottom are Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus warneri, Staphylococcus hominis, Staphylococcus haemolyticus, Staphylococcus capitis, Staphylococcus lugdunensis; Staphylococcus muscae and Staphylococcus succinus. The bpHDA amplification primers are shown on the top of the alignment (red arrows), which are perfect match with the corresponding regions of Staphylococcus aureus tuf gene amplicon (the top sequence in the alignment). The two mismatch bases at the 3’ end flap of the forward primer tuf430L with Staphylococcus succinus amplicon are indicated within the red rectangle (the bottom sequence in the alignment), which dramatically slows down the amplification rate of Staphylococcus succinus. The dots in the alignment indicate identical nucleotides with the top sequence of Staphylococcus aureus tuf gene amplicon in the alignment.
Table 2.
MSSA limit of detection with different primer pairs and different suppressor cell inputs.
Table 3.
Effect of multiple synthetic templates as suppressors on assay limit of detection.
Table 4.
Impact of competitive organism cells on the limit of detection for different Staphylococcus species.
Table 5.
Impact of synthetic template suppressor on assay contamination rate.