Sequence Diversity in the Glycoprotein B Gene Complicates Real-Time PCR Assays for Detection and Quantification of Cytomegalovirus

Abstract

Real-time quantitative PCR systems (Q-PCRIn an effort to evaluate the impact of CMV sequence variants in our patient population by use of a similar Q-PCR assay, we surveyed 54 isolates of CMV, each from a different patient. We detected evidence for the C630T variant in 4 of 54 (7.4%) patients. Furthermore, isolates from two additional patients were completely negative in the test. Sequencing of these false-negative isolates revealed multiple mutations within the probe hybridization sites. A Q-PCR that targeted the CMV polymerase gene instead of gB detected all 54 isolates. We suggest that Q-PCR assays for viral load be rigorously tested on large panels of viral isolates to assess the impact of sequence diversity on detection as well as quantification.Real-time quantitative PCR (Q-PCR) is currently the method of choice for the rapid detection and quantification of cytomegalovirus (CMV) in clinical specimens (10, 12). The glycoprotein B gene (gB, UL55) is, overall, one of the less variable regions of the CMV genome (11, 17), except for two variable subregions (4, 16), and has been frequently used as a target for Q-PCR assays (5,6,(8)(9)(10)19). Schaade et al. (14) described a Q-PCR assay using fluorescence energy transfer technology with two fluorophore-labeled hybridization probes within the gB gene and concluded that their assay was superior in terms of sensitivity, specificity, linear range, and efficiency to the commercially available COBAS Amplicor CMV Monitor assay (Roche), which uses the CMV DNA polymerase gene (pol) as a target. Other groups, using modifications of the assay described by Schaade et al. (14), have reported similar experiences (8, 12).However, CMV variants that could not be quantified using this Q-PCR have been reported (15). In the initial report (15), 912 clinical specimens were tested, of which 197 were positive for CMV DNA, but for 12 specimens (6.1% of the positive specimens) a viral load could not be determined. The specimens that were detected but not quantified did not produce a crossing point or any evidence of amplification during the PCR. However, in the melting curve analysis following the PCR, a signal was detected with a melting temperature of 53.1°C, which is below the 59.2°C melting temperature found for wild-type CMV. Sequence analysis revealed a single base pair mutation, C630T, in the target sequence of the downstream probe (15). This sequence variant prevented the appearance of a fluorescent signal under the cycling conditions of the assay, which included an annealing temperature of 58°C (15). The melting curve employed a lower annealing temperature (45°C), which allowed hybridization of the probe with the C630T variant (15). Thus, for the C630T gB variant, the assay developed by Schaade et al. (14) was only sufficient to provide qualitative results.To evaluate the impact of sequence variants in our patient population using a similar Q-PCR assay, we studied 54 CMV isolates, each from a different patient. We found two isolates that were not detectable. Sequence analysis o...

Marked Variability of BK Virus Load Measurement Using Quantitative Real-Time PCR among Commonly Used Assays

“…Only in the last few years have a substantial number of full-genome BKV DNA sequences been published (3,10,17,27,29); thus, the extent of BKV genetic diversity is not necessarily reflected in the design of commonly used PCR reagents. The importance of locating real-time PCR primers and probes within well-conserved genomic regions is well known (15,19). The experiments in this study illustrate some potential manifestations of primer/probe mismatches.…”

Impact of Genomic Sequence Variability on Quantitative PCR Assays for Diagnosis of Polyomavirus BK Infection

“…Prototypes 2 thru 14 correspond to a broad spectrum of sequences that includes genotypes II, III, and IV at coverage frequencies that are summarized in Table 2. It is apparent that prototypes 1,2,6,7,8,9,10,12,13, and 14 cover primarily genotype I strains. Genotype II is represented by prototypes 5 and 11, genotype III by prototype 5, and genotype IV by prototypes 3 and 4.…”

“…Analogous findings have been reported in the literature. Thus, single-nucleotide polymorphisms have been reported to not always be detrimental to assay performance (5), while 2 mismatches at the 3Ј end of a PCR primer can result in up to 2-log-unit differences in the calculated cytomegalovirus load (9). Large numbers of mismatches result in complete lack of amplification of the viral target sequence (a false-negative result) (15).…”

Active CMV disease does not always correlate with viral load detection

“…Large multicentre comparisons of PBSCT and BMT have shown no significant evidence of increase in CMV infection following PBSCT, but they have not specifically looked at CMV as a single entity. [24][25][26] In more than 500 patients undergoing SCT at a single centre, the introduction of CMV PCR monitoring and preemptive therapy has been associated with a low incidence of CMV end-organ disease and related mortality, with the incidence of CMV pneumonia reduced particularly. Notably, we observed negative CMV PCR in association with active CMV organ disease in half of the eight affected patients, although reactivation had been observed previously in these patients.…”