A simple approach to the generation of heterologous competitive internal controls for real-time PCR assays on the LightCycler.

BACKGROUND: The real-time PCR technology allows convenient detection and quantification of virus derived DNA. This approach is used in many PCR based assays in clinical laboratories. Detection and quantification of virus derived DNA is usually performed against external controls or external standards. Thus, adequacy within a clinical sample is not monitored for. This can be achieved using internal controls that are co-amplified with the specific target within the same reaction vessel. OBJECTIVES: We describe a convenient way to prepare heterologous internal controls as competitors for real-time PCR based assays. STUDY DESIGN: The internal controls were devised as competitors in real-time PCR, e.g. LightCycler-PCR. The bacterial neomycin phosphotransferase gene (neo) was used as source for heterologous DNA. Within the neo gene a box was chosen containing sequences for four differently spaced forward primers, one reverse primer, and a pair of neo specific hybridization probes. Pairs of primers were constructed to compose of virus-specific primer sequences and neo box specific primer sequences. Using those composite primers in conventional preparative PCR four types of internal controls were amplified from the neo box and subsequently cloned. RESULTS: A panel of the four differently sized internal controls was generated and tested by LightCycler PCR using their virus-specific primers. All four different PCR products were detected with the single pair of neo specific FRET-hybridization probes. CONCLUSION: The presented approach to generate competitive internal controls for use in LightCycler PCR assays proved convenient und rapid. The obtained internal controls match most PCR product sizes used in clinical routine molecular assays and will assist to discriminate true from false negative results.

Automated detection of five human herpes virus DNAs by a set of LightCycler PCRs complemented with a single multiple internal control.

BACKGROUND: Herpes viruses represent important causes of morbidity and mortality especially in immuno-compromised patients. To assist in rapid diagnosis real-time PCR assays have been developed for the detection of herpes virus DNA in patient specimens. A recently described set of real-time PCR assays using LightCycler technology enabled parallel detection of DNA from cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus type 1 and 2 (HSV-1/-2), and varicella-zoster virus (VZV) by using a single LightCycler program [J. Clin. Virol. 26 (2003) 85]. The set of assays lacked automation of DNA purification and of PCR mixture preparation, and was not furnished with measures to monitor for sample adequacy. OBJECTIVES: Development of a set of automated LightCycler-PCR assays for the detection CMV-, EBV-, HSV-1/-2- and VZV-DNA in plasma samples and complementation of the assays with internal amplification controls (ICs). STUDY DESIGN: The MagNA Pure LC instrument was used for automated DNA purification and automated preparation of PCR mixtures. A single multiple IC-DNA specific for all four herpes virus type-specific PCRs was generated and used in all four LightCycler assays. Detection limits were determined and clinical samples were evaluated. RESULTS: With quantified herpes virus type-specific reference DNA spiked into EDTA plasma, the detection limits were found at 250 copies/ml of CMV-, EBV-, HSV-1/-2-DNA and at 500 copies/ml of VZV-DNA. The novel set of assays was evaluated by testing 112 EDTA plasma samples. The use of the IC led to the detection of PCR-inhibited samples. CONCLUSION: The set of automated LightCycler assays was found rapid, markedly labour saving and suitable for the routine diagnostic laboratory. The use of the one internal control molecule simplified the assay protocol and allowed monitoring for sample adequacy.

Entirely automated quantification of human immunodeficiency virus type 1 (HIV-1) RNA in plasma by using the ultrasensitive COBAS AMPLICOR HIV-1 monitor test and RNA purification on the MagNA pure LC instrument.

The ultrasensitive COBAS AMPLICOR HIV-1 Monitor test was complemented with automated RNA purification on the MagNA Pure LC instrument. This enabled entirely automated ultrasensitive assessment of viral loads in human immunodeficiency virus type 1 (HIV-1)-infected individuals. The detection limit of the fully automated assay and the viral load measurements in 80 clinical samples were found to be in good agreement with those of the conventional ultrasensitive COBAS AMPLICOR HIV-1 Monitor test. The fully automated assay showed markedly reduced hands-on time and was found to be suitable for the routine assessment of HIV-1 viral loads in a clinical diagnostic laboratory.

Fully automated, internally controlled quantification of hepatitis B Virus DNA by real-time PCR by use of the MagNA Pure LC and LightCycler instruments.

We report on the development of a fully automated real-time PCR assay for the quantitative detection of hepatitis B virus (HBV) DNA in plasma with EDTA (EDTA plasma). The MagNA Pure LC instrument was used for automated DNA purification and automated preparation of PCR mixtures. Real-time PCR was performed on the LightCycler instrument. An internal amplification control was devised as a PCR competitor and was introduced into the assay at the stage of DNA purification to permit monitoring for sample adequacy. The detection limit of the assay was found to be 200 HBV DNA copies/ml, with a linear dynamic range of 8 orders of magnitude. When samples from the European Union Quality Control Concerted Action HBV Proficiency Panel 1999 were examined, the results were found to be in acceptable agreement with the HBV DNA concentrations of the panel members. In a clinical laboratory evaluation of 123 EDTA plasma samples, a significant correlation was found with the results obtained by the Roche HBV Monitor test on the Cobas Amplicor analyzer within the dynamic range of that system. In conclusion, the newly developed assay has a markedly reduced hands-on time, permits monitoring for sample adequacy, and is suitable for the quantitative detection of HBV DNA in plasma in a routine clinical laboratory.