Development of a novel internal control for a real-time PCR for HSV DNA types 1 and 2
Introduction
Improved sensitivity and specificity for detecting herpes simplex virus (HSV) DNA in genital samples by polymerase chain reaction (PCR) compared with culture and antigen detection has been demonstrated in a number of reports (Burrows et al., 2002, Stránská et al., 2004, Mengelle et al., 2004). There are also reports describing the use of internal controls to monitor the effect of inhibitors in samples, which by suppressing amplification, might result in a false negative result (Burggraf and Olgemöller, 2004, Hoorfar et al., 2004, Stöcher et al., 2004). PCR reactions are susceptible to inhibition by a variety of substances in specimens collected from patients including haemoglobin, urea, bile salts, and heparin (Wilde et al., 1990, Gerritsen et al., 1991, Akane et al., 1994, Satsangi et al., 1994). Reagents such as ethanol and SDS, used in nucleic acid extraction procedures, may also be inhibitory. Inhibition can be detected by setting up parallel reactions containing the test samples spiked with a positive control (Fout et al., 2003). However, this is laborious and increases the potential for contamination by making the test procedure more involved. An alternative method is to add an internal PCR inhibition control (IC) of non-related DNA with the test sample into the same reaction tube, this also ensures that reaction conditions for both the IC and test sample are identical (Cone et al., 1992).
One approach is to produce an IC with identical primer binding sites to the diagnostic target DNA but with internal sequences derived from plasmid DNA (Sachadyn and Kur, 1998). Using a relatively straightforward two-step PCR technique we have produced such an IC suitable for use in a routine diagnostic LightCycler real-time PCR assay to detect herpes virus types 1 and 2 DNA.
Section snippets
Construction and production of the PCR inhibition control
The 190 bp PCR IC consisted of a sequence of DNA derived from the pGEM-T Vector plasmid (Promega, Southampton, UK) flanked by the HSV forward and reverse primer binding sites (Fig. 1). This had a GC content similar to that of the HSV amplicon. The IC was produced in two rounds of amplifications using the LightCycler (Roche Diagnostics GmBH, Germany). Details of the primers and probes used for the production and detection of the IC are shown in Table 1 (all primers and probes used in this study
Results
The IC has a G + C content of 52%, identical to the HSV 1 amplicon, while the HSV 2 amplicon has a G + C content of 53%, due to A → G and T → C substitutions in the type 2 genome at base positions 2852 and 2859, respectively. Titrating the IC against a range of HSV 1and 2 tissue-culture positive controls showed that a 10−11 dilution of the purified final round PCR product was the most suitable, this equated to approximately 2 × 1010 copies μl−1. Typical melting curve peaks with melting temperatures (Tms)
Discussion
To improve our routine diagnostic service, we have developed a PCR IC for detecting inhibition in samples assayed for HSV 1 and 2 DNA by LightCycler PCR. The IC was produced by PCR amplification of plasmid DNA with HSV-plasmid chimeric primers and subsequent amplification of the resulting amplicons with HSV primers. This method has the advantage of requiring only two relatively straightforward sequential amplifications, compared with other reports (Müller et al., 1998, Rosenstraus et al., 1998,
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