In this study, we quantitatively evaluated the impacts of mismatches between the primers and probes and their binding sites on the detection of HPV16 DNA integration by real-time PCR. Because the numbers of E2 and E7 gene copies within the plasmid construct are known to be equal, comparisons of ratios of E2 to E7 gene copies estimated by the specific versus the degenerate sets of primers and probes reflect to what extent the physical status of the virus would be misinterpreted simply because of base mismatches.
We have assumed that the estimates derived by using primers and probes that were fully complementary to the targets were the actual copy numbers. As expected, the specific sets of primers and probes, which had no mismatches with the E variant construct, gave a ratio of E2 to E7 gene copies of close to 1 for this construct. The ratio of 1 for the E variant construct, derived by using the degenerate sets of primers and probes, suggested that the incorporation of degenerate bases, relative to those fully complementary, did not appreciably affect the determination of copy number. Impressively, results for the three non-E variant constructs differed substantially between tests that used the specific versus the degenerate sets of primers and probes. While the ratio estimates remained close to 1 for all except the Af2 variant construct with the degenerate sets of primers and probes, the estimates were significantly different from 1 with the specific sets of primers and probes. These differences cannot be explained by variations in specimen loading or any other assay procedures, because the aliquots for E2 and E7 that were tested on the same plate were from the same premix and the results from five independent runs were comparable.
The departure from the ratio of 1 for the Af1 and AA variant constructs with the specific sets of primers and probes is likely to be a result of the mismatch-related reduction of amplification efficiency (32
). For example, the ratio of <1 for the Af1 plasmid construct could be explained by the presence of mismatches in the E2 forward primer and probe binding sites but not in the E7 primer and probe binding sites. The AA variant had a G-to-A change in the E2 probe target and a T-to-C change in the E7 probe target. Both mismatches reduced the amplification efficiency, but the impact of the E7 mismatch was more substantial than that of the E2 mismatch, thereby leading to the reduced copy numbers of both E2 and E7 genes and the ratio of >1. The ratio of <1 for the Af2 variant construct that was derived by using the degenerate sets of primers and probes was unexpected because this variant had only a single mismatch in the E2 probe binding site, the one that was also present in the Af1 and AA variant constructs. The ratio of close to 1 obtained by testing with the modified degenerate sets of primers and probes suggested that the underestimation of E2 gene copies by the degenerate set may be attributed to a nucleotide alteration at the position immediately following the 3′ end of the E2 forward primer: from guanine at position 3431 in the prototype that was used to generate the standard curve to adenine in the Af2 variant construct. Knowing about this pattern, although the underlying mechanism is unclear, is useful for a better design of primers and probes for real-time PCR.
The present study further showed evidence of the variant-associated misinterpretation of HPV16 integration by testing a set of cervical swab samples. Because proportions of HPV16 non-E variants are related to geography and vary from one population to another, the findings of this study may in part explain the wide range of integration rates reported so far. Given that HPV16 variants differ biologically and etiologically (31
), the variant-related base mismatches may somewhat bias estimates of the integration-related risk of cervical neoplasia. We noticed a ratio of <1 estimated by the modified degenerate sets of primers and probes for a clinical sample that was positive for the HPV16 Af2 variant. One interpretation for this result is that the variant might have existed in both episomal and integrated forms. Alternatively, this variant may carry additional nucleotide alterations that were not covered by our modified degenerate sets of primers and probes.
To our knowledge, this is one of the first studies, if not the first, to quantitatively address the impact of HPV16 variants on the detection of viral integration. Although the influence of HPV16 E2 polymorphisms on the detection of HPV16 integration was reported previously (4
), the findings of the study were limited by the fact that the evaluation was based only on clinical samples in which a true integration status was unknown. The use of plasmid constructs for illustration avoided any integration-related misinterpretation that might arise with the use of clinical samples. We are, however, aware that our degenerate sets of primers and probes, although better than the specific sets, were designed based on limited sequence information. In natural infections, some HPV16 variants may carry more nucleotide alterations than those currently identified. Because of the intratypic diversity of many HPV types, the careful design of primers and probes is a prerequisite for a valid assay to test the integration of the HPV genome. One way to achieve this design is to choose a conserved region as the target (4
). However, it is sometimes difficult to find such a region, particularly when one needs to take into account the appropriate amplicon size (usually less than 100 bp for real-time PCR) and comparable efficiencies of amplification. Alternatively, as shown in the present study, we may incorporate degenerate bases into the primers and probes at sites where nucleotides differ among the variants.
The implications of the results of this study go beyond studying viral DNA integration. Currently, quantitative analysis of the HPV DNA load by real-time PCR has been widely used in many clinical and epidemiological investigations. The primers and probes for the assay are usually designed based on the prototype sequence. It is likely that errors in viral load estimation may arise if the sample is positive for nonprototype HPV variants.
In summary, our data indicate that mismatches between the primers and probe and their targets can introduce significant errors into the determination of copy number. Incorporating degenerate bases into the primers and probe can sufficiently compensate for the mismatch-reduced efficiency of amplification.