ORIGINAL ARTICLE Comparison of salivary collection and processing methods for quantitative HHV-8 detection DJ Speicher 1,2,3 , NW Johnson 2,3 1 School of Dentistry and Oral Health, Griffith University, Gold Coast, Qld; 2 Molecular Basis of Disease Research Program, Griffith Health Institute, Griffith University, Gold Coast, Qld; 3 Population & Social Health Research Program (Population Oral Health), Griffith Health Institute, Griffith University, Gold Coast, Qld, Australia OBJECTIVES: Saliva is a proved diagnostic fluid for the qualitative detection of infectious agents, but the accu- racy of viral load determinations is unknown. Stabilising fluids impede nucleic acid degradation, compared with collection onto ice and then freezing, and we have shown that the DNA Genotek P-021 prototype kit (P-021) can produce high-quality DNA after 14 months of storage at room temperature. Here we evaluate the quantitative capability of 10 collection/processing methods. METHODS: Unstimulated whole mouth fluid was spiked with a mixture of HHV-8 cloned constructs, 10-fold serial dilutions were produced, and samples were extracted and then examined with quantitative PCR (qPCR). Calibration curves were compared by linear regression and qPCR dynamics. RESULTS: All methods extracted with commercial spin columns produced linear calibration curves with large dynamic range and gave accurate viral loads. Ethanol precipitation of the P-021 does not produce a linear standard curve, and virus is lost in the cell pellet. DNA extractions from the P-021 using commercial spin col- umns produced linear standard curves with wide dynamic range and excellent limit of detection. CONCLUSION: When extracted with spin columns, the P-021 enables accurate viral loads down to 23 cop- ies ll À1 DNA. The quantitative and long-term storage capability of this system makes it ideal for study of sali- vary DNA viruses in resource-poor settings. Oral Diseases (2013) doi:10.1111/odi.12196 Keywords: salivary diagnostics; HHV-8; herpesvirus; quantitative; DNA Genotek Introduction The mouth is a mirror of the body’s health, and saliva is abundant, easy and minimally invasive to collect, thus makes a powerful diagnostic fluid for nucleic acid-based tests, especially when combined with a sensitive quantita- tive polymerase chain reaction (qPCR) assay (Streckfus and Bigler, 2002; Segal and Wong, 2008; Lee et al, 2009). This coupling gives oral fluids many advantages over blood and plasma for the detection and monitoring of infectious agents, such as viruses (Roy et al, 1999; Pauk et al, 2000; Biggar and Goedert, 2001) and bacteria (Nurkka et al, 2003), biomarkers for cancer and periodon- tal disease (Arellano-Garcia et al, 2008; Bigler et al, 2009; Giannobile et al, 2009), and drug testing (Mandel, 1993). Oral fluids are also easier and safer to use as they do not clot and represent a much lower risk of exposure of the healthcare worker to blood-borne infectious agents. Despite these benefits, the main problem with oral fluids is that biomarkers are present in lower concentration than in blood, and the presence of various nucleolytic enzymes, such as endonucleases, ribonucleases and bacterial prote- ases, is detrimental (Eichel et al, 1964; Bardon and Shu- gar, 1980; Kilian et al, 1996; Park et al, 2006a). To circumvent these problems, salivary diagnostics have fol- lowed two particular approaches: a focus on antibody assays, often point-of-care (POC) diagnostic tests, such as the OraQuick Advance Rapid HIV-1/2 and HCV tests; and stabilizing solutions that inhibit the enzymatic activity and allow samples to be stored longer before processing (Roberts et al, 2007). Traditionally, for the detection of infectious agents, saliva has been collected as whole mouth fluid (WMF) (Pauk et al, 2000; Al-Otaibi et al, 2009) or as throat gargles (TG) with phosphate-buffered saline (PBS) (Cook et al, 2002; Webster-Cyriaque et al, 2006), saline or the commercial mouthwash Listerine [essential oils in an alcoholic solution] (Marshall et al, 2007) in chilled cups and then either extracted immedi- ately for DNA or RNA, or frozen immediately to À80°C to slow enzymatic activity. While several commercial kits and PBS have aided in collection and stabilisation of oral fluids, there appears to Correspondence: David J. Speicher, Griffith Health Institute, Gold Coast Campus, Griffith University, Gold Coast, Qld 4222, Australia. Tel: +61 7 5678 0438, Fax: +61 7 5678 0708, E-mail: d.speicher@griffith. edu.au Received 21 August 2013; accepted 27 September 2013 Oral Diseases (2013) doi:10.1111/odi.12196 © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd All rights reserved www.wiley.com