To test the specificity of our molecular probes, we reacted the probe set with different “dropout” pools of genomic DNAs (see Materials and Methods in the supplemental material). Each of the 36 pools was lacking one of the 36 target DNAs recognized by our probe set. The positive control contained all DNAs. For example, the data for the Bifidobacterium longum DNA dropout pool is shown in Fig. . The only negative probes were the five probes directed against B. longum DNA. These molecular probes could not hybridize to the absent B. longum DNA and did not hybridize to any of the other 35 prokaryotic genomic DNAs in the dropout pool. Included in these tests for specificity were molecular probes for different species of the same genus. As one example, Neisseria gonorrhoeae probes did not hybridize to Neisseria meningitidis DNA and, conversely, N. meningitidis probes did not hybridize to N. gonorrhoeae DNA (data not shown). As a second example, there were three species of the Pseudomonas genus in the probe mix and the dropout pools: P. aeruginosa, P. fluorescens, and P. putida. The molecular probes for each species hybridized only to the species for which they were designed and not to the DNA of the other two species in the genus (data not shown).
FIG. 3. Results of a representative dropout experiment demonstrating the specificity of the B. longum molecular probes. One-hundred-twenty-two molecular probes were reacted with a pool of DNA representing 31 microbial targets and an identical pool that lacked (more ...)
Because of the importance of Lactobacillus bacteria in the adult vagina, additional experiments were performed to test the specificity of the Lactobacillus species probes. As an example, a mixture of six L. gasseri molecular probes was hybridized separately to each of seven Lactobacillus DNAs: L. acidophilus, L. brevis, L. crispatus, L. delbrueckii, L. gasseri, L. jensenii, and L. plantarum. The results are shown in Fig. . The only strong fluorescence resulted from each molecular probe reacted with L. gasseri genomic DNA.
FIG. 4. Histogram of the amount of fluorescence produced by each of six L. gasseri probes against seven Lactobacillus DNAs. A mixture of the six L. gasseri molecular probes was separately hybridized to 10 ng of each of seven genomic Lactobacillus DNAs, L. acidophilus (more ...) The possible complication of the presence of human DNA in the target.
When collecting vaginal swabs, human cells are sometimes inadvertently also collected, leading to the presence of contaminating human DNA. During our annealing reaction, we did not want any of our molecular probes to hybridize to human DNA, since such hybridization would compromise the interpretation of the data. Therefore, three tests were undertaken. (i) All potential 40-base homers were BLASTed against the human DNA sequence in GenBank. Any potential homer with more than negligible homology was discarded (Materials and Methods). (ii) The sequences of the two universal amplification primers were BLASTed against the human DNA sequence in GenBank. “No significant similarity” was found. (iii) An experiment was undertaken to determine if human DNA would act as a target for any of the molecular probes (see Materials and Methods in the supplemental material). The resulting fluorescence signals are depicted in Fig. S1 in the supplemental material. No probe produced significant signal consistently with all three amounts of human DNA. Only one probe was found to react significantly with the highest concentration of human DNA. However, even this probe yielded a very low signal. Therefore, we concluded that the reactivity of our molecular probes with the assayed amounts of human DNA was negligible.
Minimum detection limit.
Fig. S2 in the supplemental material presents a histogram of the minimum detection limit for the molecular probes (see Materials and Methods in the supplemental material). Notably, molecular probes targeting the same whole-genome amplified (WGA) microbial DNA did not necessarily have the same minimum detection limit. Among the molecular probes with a minimum detection limit of, at most, 0.5 pg were probes for Burkholderia mallei, Corynebacterium glutamicum, L. brevis, L. gasseri, Stenotrophomonas maltophilia, and Treponema pallidum.
ATCC genomic Escherichia coli DNA was employed to measure the minimum detection limit without the complication of WGA. A mixture of the three E. coli molecular probes (ED35, ED36, and ED39) was hybridized to decreasing concentrations of E. coli DNA and taken through our procedure. The Tag4 array results are shown in Fig. . ED35 had a minimum detection limit of 1 pg (326 yoctomol; 2,000 molecules). ED36 and ED39 had a minimum detection limit of 10 pg (3.26 zeptomol; 20,000 molecules). For the same amount of E. coli genomic DNA, the three molecular probes produced a different amount of fluorescence. For example, at apparent saturation (100 ng), ED35 produced 3,984 fluorescence units (FU), ED36 produced 5,513 FU, and ED39 produced 2,914 FU (Fig. ).
FIG. 5. Minimum detection limit for three molecular probes for E. coli DNA. A mixture of the three E. coli molecular probes was annealed to decreasing amounts of ATCC E. coli genomic DNA, including a negative control containing no DNA. In the annealing reaction, (more ...) Use of molecular probes with simulated clinical samples.
To simulate the total DNA derived from vaginal swabs, we constructed five mixtures of DNAs wherein each mixture had very different mass amounts of 3 to 5 WGA genomic DNAs relevant to the human vagina, called simulated clinical samples A through E (see Table S3 in the supplemental material). As an example, the data for simulated clinical sample E is shown in Fig. . The data for the other four simulated clinical samples are shown in Fig. S3 to S6 in the supplemental material. In all cases, the molecular probes detected only the DNAs known to be in the simulated clinical sample. There were no false positives, and there were no false negatives.
FIG. 6. Characterization of simulated clinical sample E. The background-subtracted fluorescence is on the left ordinate and shown in blue. The abscissa lists the target 31 microbes in alphabetical order. Following the microbe names, the numbers in parentheses (more ...)
While there was excellent qualitative agreement, quantitative agreement was elusive. For example, in simulated sample E (Fig. ), there were 0.45 ng (0.15 attomol) of WGA E. coli DNA and 10 ng (13 attomol) of WGA T. pallidum DNA. The range of fluorescence achieved for the five T. pallidum probes extends both higher and lower than the range of fluorescence of the three E. coli probes (Fig. ), despite the ~100-fold difference in the molar amount of target DNA. The explanation probably lies in the well-known facts that standard PCR is not quantitative and fluorescence intensity is not a linear function of mass.