We have introduced an assay that uses MultiCode-RTx genotyping technology for the simultaneous analysis of mixed targets that differ by a single nucleotide. The approach combines a type of competitive allele-specific PCR and an additional base pair. The additional base pair allows for site-specific quencher incorporation opposite a variety of fluorophores in order to simultaneously observe fluorescent change in multiple channels over the time course of the assay. The number of PCR cycles required to observe signal change below a threshold is proportional to target concentration. Thus, PCR cycle threshold data can be used to develop quantitation curves and determine the levels of targets in unknown samples.
Using this method, we demonstrated the ability to quantitate mixed populations of nucleic acid targets specific to HIV-1 and drug-resistant variants of HIV-1 that differ by a single nucleotide change, even when the minority species is present at 1 part in 10,000. The method was tested on two of the most important drug selected mutations, M184V and K65R. The ability to quantitate the K65R mutation even when it is a minor species is of particular importance since most of the nucleoside and nucleotide inhibitors select for this mutation. The method was also shown to be extremely robust. Data using four different cycling parameters for both assays demonstrated high levels of specificity. In addition when the total concentration of targets fluctuated from 103 to 107, the difference in Ct values for any given mixed population stayed constant. Combined, the results indicate that the method may have uses in a wide variety of applications.
These preliminary results led us to test the RTx method on a set of 13 samples from HIV-1-infected individuals. The results were evaluated by comparison to those previously determined using LiPA. Ten of the RTx results showed 100% concordance with LiPA, while 3 showed some minor inconsistencies in population percentages. Reasons for these population differences are unclear, since the level of mixtures should be within the range of both methodologies. Further studies are under way to perform single-genome sequencing on the samples.
Other methods for biallelic polymorphism testing from pooled samples exist. The first was discussed by Kwok et al. (13
). Since then, a large number of other techniques have been proposed to further simplify mixed-population studies—some more complicated then others (1
). But easy-to-use and ultraspecific methodologies do not seem to be available for mixed-population quantitative testing. One method called “needle-in-a-haystack” exceeds the specificity described here with sensitivities down to 1 in 106
yet requires complicated multiple steps (27
). Other real-time PCR methods that are easy to use such as SYBR green have been used for mixed-population analysis (4
). However, SYBR green methodology does not allow for the simultaneous real-time detection of multiple targets which limits the specificity. For example, SNP allele frequency was measured be dividing the pools between two PCR each containing a primer pair specific to one or the other variant. Mismatch amplification under these conditions is typically delayed by 10 cycles. Mixtures of 19:1 could be identified using the multiwell approach. This level in mixture detection range may be due to well-to-well variation, lack of competition between primers for the target, or reaction conditions. There are other techniques that enable differential fluorescent labeling of the primers (15
). These should also allow for mixed-population genotyping, yet to our knowledge there are no published reports that demonstrate specificity near what is described for the RTx method. Other techniques employing amplicon-specific hybridization probes are well entrenched within the science community (10
). Ironically this is due to the perception that probes are needed to gain specificity. Yet no literature reports showing single-base specificity in mixed populations below the standard 50:50 SNP studies could be found. This is not to say that these probe-based systems could not be used in such studies, but the limits placed on hybridization methods may make this difficult (12
). This being said, an approach which combines allele-specific PCR and the TaqMan real-time probe hybridization system was able to detect a single base change in a mutant DNA target in a sample with 1,000-fold-greater wild-type targets. Yet both targets (wild type and mutant) could not be detected simultaneously since the hybridization probe was not discriminatory and ratios of the two species were not determined (6
). Previously, our group has used a method called LiPA to analyze mixed populations of HIV-1 (9
). LiPA, which requires post-PCR handling, has the benefit of analyzing multiple targets simultaneously. Yet we found that LiPA was unable to detect the K65R target and could not reproducibly detect subpopulations below 4%. The MultiCode-RTx system is a faster method and allows for a higher level of analytical specificity. In addition, we have shown that the RTx system can quantitate both wild-type and mutant populations. The LiPA system is a solid-phase post-PCR methodology and therefore is semiquantitative.
The MultiCode-RTx method presented is not a substitute for sequencing of course. Since only one site can be analyzed per assay, the RTx method is not appropriate for scanning entire genomes and the mutation tested must be previously known. The method also cannot determine the linkage of mutations present at low frequency on the same genome as heteroduplex tracking assays can. Yet RTx should be useful in a wide variety of other applications, such as early detection of emerging drug-resistant strains, determining allele frequency of biallelic polymorphisms in pooled samples, and early detection of cancer in patients. Our results show that RTx may have utility in drug development or clinical testing when it relates to HIV resistant strain emergence. It should be noted, however, that the primers and targets used to construct the results did not take into account the diversity of all the HIV targets that could be found in a clinical setting. Efforts are under way to address the diversity issue using two approaches. The first employs primers that bind noncontiguous regions of the HIV genome. This approach allows highly polymorphic regions to be bypassed. At lower temperatures where hybridization of noncontiguous regions to the target occurs, primer extension can take place. After duplex formation, priming sites that are identical to the primer are formed. In subsequent rounds of PCR, the annealing temperature is increased to the Tm of the primers and exponential amplification begins. The second and more standard approach is introduction of degenerate bases; particularly at the third position of the amino acid codon. The specificity of either approach still needs to be determined using larger sample numbers, but the data so far show promise for RTx HIV-1 mixed-population analysis.