Effect of Sequence Polymorphisms on Performance of Two Real-Time PCR Assays for Detection of Herpes Simplex Virus

Abstract

Herpes simplex virus (HSV) is the most common cause of acquired, sporadic encephalitis in the UnitedStates. PCR identification of HSV in spinal fluid has become the diagnostic gold standard due to its sensitivity and potential for speed, replacing other methods such as culture. We developed a real-time PCR assay to detect HSV, using a new type of hybridization probe, the Eclipse probe. In this study, we ran 323 samples (171 positives and 152 negatives) with the Eclipse real-time PCR assay and compared these results with another PCR assay using gel detection. The real-time assay agreed with our reference method for 319 out of the 323 samples tested (99%). Using two different real-time PCR platforms, we discovered that SNPs within the amplicon's probe binding region that are used to distinguish HSV-1 from HSV-2 can decrease assay sensitivity. This problem is potentially a general one for assays using fluorescent probes to detect target amplification in a real-time format. While real-time PCR can be a powerful tool in the field of infectious disease, careful sequence evaluation and clinical validation are essential in creating an effective assay.PCR is now accepted as the gold standard for diagnosis of herpes simplex virus (HSV) infections in the central nervous system (CNS), exhibiting a high degree of specificity and a sensitivity superior to that of culture or DFA (3,7,10,11,17,18). While viral culture for HSV encephalitis is still routinely employed in the clinical lab, it is only capable of detecting virus in less than 5% of patients with CNS infections (10, 14). Detection of low viral titers often found in spinal fluid requires the high level of sensitivity that is provided by amplificationbased assays (13).In recent years, real-time PCR has made testing more convenient. Real-time PCR is more amenable to automation, allowing higher throughputs and decreased turnaround times. In addition, the implementation of a one-tube closed reaction greatly reduces the possibility for contamination. Besides the potential for sensitivity roughly equivalent to gel detection, the use of target-specific probes in real-time PCR offers an additional level of specificity. These probes also allow the detection of single nucleotide polymorphisms (SNPs) using melt curve analysis, permitting typing of different viral species.While real-time PCR has been widely adopted for molecular infectious disease testing, real-time technology has often been compared to traditional methods such as culture, with a predictable enhancement in sensitivity (4,6,7,12). The current literature is more limited on the comparative performance of real-time PCR with established PCR formats. There is little definitive published evidence that real-time detection using fluorescent probes is more sensitive than end detection by gel, raising the question of whether real-time PCR is necessarily more sensitive than conventional PCR with gel detection. Therefore, it is essential during the validation of any real-time assay to compare its performance to something in ad...

Unlabeled Probes for the Detection and Typing of Herpes Simplex Virus

“…Aberrant melting curves were observed approximately 5% of the time, most often in positive samples. In comparison, the reference assay, which initially processes samples as single reactions, would have a repeat rate of 74% based on the number of positive clinical samples processed, since all positive samples with a C T Ͼ30 cycles are reanalyzed to confirm an HSV-positive sample (17 ).…”

“…Samples were extracted using a 96-well DNA extraction set (Qiagen) following the manufacturer's protocol. The internal control (IC), a Caenorhabditis elegans::green fluorescent protein transcriptional fusion plasmid (17 ), was added to AVL lysis buffer (Qiagen) to obtain an expected concentration of 1 ϫ 10 2 copies/L in eluted nucleic acid.…”

“…For comparison, we used an in-house reference assay, an Epoch minor groove binding probe-based assay (Nanogen) (17 ). The minor groove binding probe was 100% complementary to a region of the glycoprotein D gene in HSV-1 and -2.…”

“…Herpes simplex virus (HSV) was chosen to develop an unlabeled probe assay based on the availability of quantified DNA targets for HSV-1 and HSV-2, characterized clinical samples, the necessity of low-level detection (13)(14)(15)(16), and an established in-house reference assay (17 ).…”

Description and Validation of a Novel Real-Time RT-PCR Enterovirus Assay

“…The RT-PCR primer and probe target sequences within this region, although not perfectly homologous, are to a great extent conserved among the human enteroviruses (11 ). With real-time RT-PCR testing, however, false negative results can occur when single nucleotide polymorphisms (SNPs) located beneath the probe reduce or prevent probe hybridization and fluorescence during detection (12)(13)(14)(15).…”