Julian Hodgson1, Mark Zuckerman, Melvyn Smith. 1. HPA London, London South Specialist Virology Centre, Rayne Institute, 123 Coldharbour Lane, London SE5 9NU, UK.
Abstract
BACKGROUND: Detection of herpes simplex virus types 1 and 2 DNA by polymerase chain reaction (PCR) is the method of choice in many laboratories due to improved sensitivity, specificity and turnaround times compared with culture and antigen detection. However, internal controls need to be employed to ensure that inhibitors in samples do not produce false negative results. OBJECTIVE: We have developed an internal control for our routine diagnostic HSV 1 and 2 LightCycler PCR assay that has identical primer binding sites to the HSV target DNA but an internal sequence derived from plasmid DNA and detected by a different probe and fluorophore combination. METHODS: Production of the internal control was achieved using a straightforward two-step PCR technique in which plasmid DNA was amplified with HSV-plasmid chimeric primers, followed by amplification of the resulting amplicons with HSV primers and purification for subsequent use. RESULTS: Both the internal control and viral DNA were amplified in initial tests with 11 tissue-culture HSV 1 and 2 positives (22 in total), with little or no inhibition of the target sequences. A high level (98%) of concordant results were obtained with 272 clinical samples assayed in parallel with and without the internal control. CONCLUSION: These results are sufficient to justify the incorporation of the internal control into the routine LightCycler HSV DNA assay in our laboratory.
BACKGROUND: Detection of herpes simplex virus types 1 and 2 DNA by polymerase chain reaction (PCR) is the method of choice in many laboratories due to improved sensitivity, specificity and turnaround times compared with culture and antigen detection. However, internal controls need to be employed to ensure that inhibitors in samples do not produce false negative results. OBJECTIVE: We have developed an internal control for our routine diagnostic HSV 1 and 2 LightCycler PCR assay that has identical primer binding sites to the HSV target DNA but an internal sequence derived from plasmid DNA and detected by a different probe and fluorophore combination. METHODS: Production of the internal control was achieved using a straightforward two-step PCR technique in which plasmid DNA was amplified with HSV-plasmid chimeric primers, followed by amplification of the resulting amplicons with HSV primers and purification for subsequent use. RESULTS: Both the internal control and viral DNA were amplified in initial tests with 11 tissue-culture HSV 1 and 2 positives (22 in total), with little or no inhibition of the target sequences. A high level (98%) of concordant results were obtained with 272 clinical samples assayed in parallel with and without the internal control. CONCLUSION: These results are sufficient to justify the incorporation of the internal control into the routine LightCycler HSV DNA assay in our laboratory.
Authors: Thomas Köller; Daniel Kurze; Mirjam Lange; Martin Scherdin; Andreas Podbielski; Philipp Warnke Journal: PLoS One Date: 2016-04-19 Impact factor: 3.240