Real-time PCR for rapid diagnosis of entero- and rhinovirus infections using LightCycler

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Abstract

Background: PCR techniques have proved to be more sensitive than traditional cell culture in the diagnosis of enterovirus and rhinovirus infections and are widely used in clinical virus laboratories. However, PCR assays are relatively time-consuming and labor intensive, particularly if separate hybridization steps are used to confirm the specificity of positive findings. Objectives: The aim of the present study was to develop fast and sensitive real-time PCR assay, which would allow simultaneous detection of entero- and rhinoviruses and their quantification in clinical and experimental samples. Study design: Two real-time RT-PCR protocols were developed using LightCycler (LC) technology; SYBRGreen and hybridization probe assays. The sensitivity of these assays to detect entero- and rhinoviruses was compared with that of a traditional reference RT-PCR-hybridization assay and cell culture. All PCR protocols used the same primers amplifying the 5′-non coding region (NCR) of entero- and rhinoviruses. The LC probe assay and the reference RT-PCR used almost identical detection probes, which bind to enterovirus specific amplicons. Results and conclusions: Both real-time PCR assays were equally sensitive as the reference RT-PCR-assay and all were more sensitive than cell culture. Both real-time assays quantified reliably the amount of the virus and took much shorter time than the reference RT-PCR. As the real-time SYBRGreen assay detects both entero- and rhinoviruses it can be used for primary screening of samples, which can be positive for either of these viruses. The real-time probe-assay can confirm the presence of enterovirus in SYBRGreen positive samples or it can be used for selective screening of enteroviruses e.g. from CSF samples.

Introduction

PCR has markedly improved the diagnosis of enterovirus and rhinovirus infections by increasing the sensitivity compared with traditional cell culture (Andreoletti et al., 1998, Pozo et al., 1998, Kessler et al., 1997, Rotbart et al., 1994). In most occasions PCR is also more rapid than cell culture but still many RT-PCR protocols are time-consuming and labor intensive, particularly if accompanied by separate hybridization step.

New real-time PCR techniques have enabled rapid virus PCR assays and quantification during the PCR reaction (Wittwer et al., 1997a, Wittwer et al., 1997b). These are important advantages in the diagnosis of enterovirus and rhinovirus infections allowing rapid diagnosis and right treatment. Quantification may become more important in future when treatments with new antiviral agents will be available.

The aim of the present study was to develop a rapid and sensitive real-time PCR method, which would simultaneously detect entero- and rhinoviruses, distinguish enteroviruses from rhinoviruses, quantify the viral load and be applicable for routine diagnostic laboratory work. We used the LightCycler (LC) (Roche Diagnostics) protocol, which is a fast real-time monitoring PCR system, where amplification and detection can be accomplished in one closed capillary.

Section snippets

Viral stocks and clinical specimen

Prototype enteroviruses and rhinoviruses were obtained from the American Type Culture Collection (ATCC, Manassas, VA). All clinical specimens originated from the diagnostic virus laboratory of the Tampere University Hospital. The samples were collected during the years 1999 and 2001, these included cerebrospinal fluids (CSF), nasopharyngeal aspirates (NPA) and stool samples. All samples were stored at −70 °C until analyzed. Vero cells and A549 cells were used for virus isolation from clinical

RT-reaction

LC allows one-tube RT-PCR reaction where both RT and PCR reactions can be carried out in the same capillary without any addition of reagents using the LC-RNA amplification kits developed for this purpose. We found that the results obtained using this type of one tube system were more variable than when RT-reaction was carried out prior PCR in a separate tube. In repeated tests the same clearly RNA positive sample gave sometimes negative and sometimes positive results. Therefore, we used

Discussion

PCR methods have significantly improved the diagnosis of enterovirus infections by increasing sensitivity and giving results in a shorter time than traditional cell culture. Better sensitivity is an important advantage when the amount of the virus is low leading to false negative findings in cell culture (like CSF samples in viral meningitis). Currently, PCR methods are widely used in clinical laboratories to diagnose enterovirus infections, but virus isolation is still important particularly

Acknowledgements

We thank Olfert Landt from TIBMolbiol for help in probe designing. We gratefully acknowledge Marja-Leena Vuorinen for technical assistance. The study was financially supported by grants from the Tampere University Hospital, the Academy of Finland and the Juvenile Diabetes Research Foundation.

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