Real-Time PCR for detection of herpes simplex virus without nucleic acid extraction

Abstract

Background: The speed and sensitivity of real-time polymerase chain reaction (PCR) have made it a popular method for the detection of microbiological agents in both research and clinical specimens. For the detection and genotyping of herpes simplex virus (HSV) in clinical specimens, real-time PCR has proven to be faster, more sensitive and safer than earlier methods which included isolation of the virus in cell culture followed by immunofluorescence microscopy. While PCR-based assays for HSV detection posses clear advantages over these earlier techniques, certain aspects of the PCR method remain onerous. The process of extraction and purification of nucleic acid from clinical specimens prior to PCR is particularly cumbersome. Nucleic acid extraction is expensive, time-consuming and provides a step whereby specimens can become contaminated prior to their analysis. Herein, we investigate the necessity of nucleic acid extraction from swab-based clinical specimens for HSV detection by real-time PCR. We find that nucleic acid extraction is unnecessary for specific and sensitive detection of HSV in clinical specimens using real-time PCR.

Methods: Prospective (n = 36) and retrospective (n = 21) clinical specimens from various anatomical sites were analyzed for the presence of herpes simplex virus 1 or 2 by real-time PCR using the RealArt HSV 1/2 LC PCR Kit. Specimens were analyzed by PCR both before and following automated nucleic acid extraction. PCR using extracted and unextracted specimens was also compared to cell culture as a means of detecting HSV.

Results: Detection of HSV 1/2 DNA in clinical specimens by real-time PCR did not require that the specimen be subjected to nucleic acid extraction/purification prior to analysis. Each specimen that was detectable by real-time PCR when analyzed in the extracted form was also detectable when analyzed in the unextracted form using the methods herein. The limit of detection of HSV-1 and HSV-2 particles when analyzed in the unextracted form was found to be approximately 17 and 32 virus particles respectively, compared to a sensitivity of 10 copies, for analysis of purified DNA. Omission of the nucleic acid extraction step shortened both the assay time and cost.

Conclusion: Omission of the nucleic acid extraction step prior to real-time PCR for detection of herpes simplex virus resulted in a more rapid and cost-effective assay, with little impact upon the sensitivity of detection.

Figure 1

HSV Clinical Specimens, Analyzed by Real-Time PCR Using Extracted and Unextracted Specimens. Clinical specimens found to be positive for HSV-1 (A), HSV-2 (B), or found to be negative for HSV (C) when analyzed by cell culture and immunofluorescence microscopy were analyzed by real-time PCR using nucleic acid-extracted and unextracted samples of each specimen. The amplification curves for the internal controls of the reactions shown in A, B and C are shown in D, E and F respectively. For extracted samples, 200 μl of a clinical specimen (in viral transport buffer) was subjected to automated total nucleic acid extraction with an elution volume of 50 μl; 5 μl of the eluted sample was analyzed. For unextracted samples, 5 μl, 2.5 μl and 1 μl of straight clinical specimen were analyzed. The specimens were taken by swab of genital lesion of a male (A, D) or female (B, C, E, F). The swabs were placed in viral transport buffer and stored frozen (-35°C) until analysis. Probes hybridizing to HSV DNA were detected in the 640 nm channel of a Light Cycler 2.0. Probes specific for internal control DNA were detected in the 705 nm channel. The positive real-time PCR control in Figure 1C was 10,000 copies of a purified plasmid containing HSV-2 DNA target fragment (provided by the real-time PCR kit).