Abstract 4978: Rapid and sensitive detection of oncoviruses using a simplified real-time RT-qPCR system

Abstract

Abstract For cancers caused by oncoviruses, patients at risk can be identified before the disease has progressed to the stage of tumor formation. Traditional immunological detection methods have several drawbacks. In the case of Human Papillomavirus (HPV) which can cause cervical cancer, detection is difficult due to low expression of early viral proteins and a lack of sensitive and specific high-quality antibodies that can discriminate HPV types. Test results can also be misleading or inconclusive when a previous infection causes antibody levels to remain elevated in a patient from months to years afterwards, as in the case of the Epstein-Barr Virus (EBV), which is associated with Hodgkin's lymphoma and other carcinomas. Polymerase Chain Reaction (PCR) based detection of viral nucleic acids are becoming more prevalent in recent years due to better sensitivity, specificity, and ease-of-use than traditional methods. Sequence-specific probe binding adds one more layer of specificity to real-time PCR and has the additional advantage of providing viral load information. We have developed a simplified real-time Reverse Transcription, quantitative Polymerase Chain Reaction (RT-qPCR) system for the rapid detection of RNA and DNA samples that minimizes operator hands-on time to reduce error and risk of cross-contamination. HPV, EBV, Hepatitis B and C Virus (HBV and HCV) nucleic acid test controls were purified using conventional spin column methods, and the purified samples were amplified in a dilution series using the 4x TaqMan® Fast Virus 1-Step Master Mix (FVMM) (Life Technologies, CA) to evaluate sensitivity based on Cq, linearity, and efficiency. Common PCR inhibitors such as heparin, EDTA, hematin, and humic acid were spiked into reactions to evaluate the tolerance of the system based on ΔCq between inhibitor-spiked samples and control samples. High inhibitor tolerance would mean there is no need for discarding precious samples, re-purification, or dilution of samples with low abundance targets. Results show that 0.5 IU of HBV, 1.3 IU of HCV, and ∼10 copies of EBV can be detected per reaction. ΔCq of ≤1 is observed for Adenovirus and exogenous Internal Positive Control (IPC) assays when reactions are spiked with up to 0.0625 U heparin, 7.2 μg EDTA, 10 μM hematin, or 100 ng humic acid per reaction. This simple RT-qPCR system can be easily incorporated into any existing workflow. PCR is completed in less than 1 hour, making it ideal for screening tests. Multiplex feasibility as shown by the incorporation of an exogenous IPC in our experiments means users may combine both type identification and quantification of an oncovirus in a single reaction, further saving time, cost, and samples. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4978. doi:10.1158/1538-7445.AM2011-4978