Comparison of salivary collection and processing methods for quantitative HHV-8 detection.

OBJECTIVES: Saliva is a proved diagnostic fluid for the qualitative detection of infectious agents, but the accuracy 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 columns 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 copies µl(-1) DNA. The quantitative and long-term storage capability of this system makes it ideal for study of salivary DNA viruses in resource-poor settings.

Comparative performance analyses of commercially available products for salivary collection and nucleic acid processing in the newborn.

Analysis of saliva for clinical monitoring and biomarker detection holds great promise for improving health care. Commercially available assays are not intended for use with neonates, however, and collection and processing of saliva for subsequent transcriptomic analysis presents unique challenges in this population. We compared RNA yield, quality, stability and RT-qPCR performance for two commonly used commercial systems: the Qiagen RNeasy Protect Saliva Mini Kit(®) and the DNA Genotek Oragene•RNA(®) assay. Two 10 µl saliva samples were collected from ten newborns and stabilized for each assay. Total RNA was extracted following incubation for 3, 10, 15 or 20 days. Total RNA extracted from each assay was analyzed for integrity, quality and quantity using the Agilent BioAnalyzer 2100. RT-qPCR was performed for the reference gene, GAPDH, to assess subsequent performance of the extracted RNA. Although the DNA Genotek extraction protocol required nearly twice the time of the Qiagen protocol, RNA integrity did not differ between the kits. RNA concentration using the DNA Genotek assay, however, was 3,264 pg/µl (range: 262 - 10,336 pg/µl) compared to 822.4 pg/µl (range: 0 - 1,856 pg/µl) for the Qiagen protocol. Linear regression analysis showed a stronger correlation between the threshold cycle and RNA concentration using DNA Genotek (r(2) = 0.356) compared to Qiagen (r(2) = 0.0331). Our results suggest that although the Qiagen assay may reduce overall extraction time, RNA yield and performance in subsequent transcriptomic analysis is more robust using the DNA Genotek assay.

Detecting DNA viruses in oral fluids: evaluation of collection and storage methods.

Storing saliva for nucleic acid diagnostics is problematic in resource-constrained settings. DNA Genotek's OMNIgene™·DISCOVER kit aims to stabilise microbial DNA at room temperature. We evaluate this for long-term storage, determining DNA quantity/purity and human herpesvirus 8 (HHV-8) load as indicator. Viral loads and DNA degradation were assayed over 14months in HHV-8-negative saliva spiked with cell-associated and cell-free virus and saliva collected fresh frozen and into kits from 10 HIV-positive patients. Viral loads remained constant for 6-9months, yielding high quantities of DNA: subsequent losses were ≤48%. Patient samples, frozen or kit stored, produced pure DNA of comparable concentration. Higher HHV-8 detection in frozen saliva resulted from losses during ethanol precipitation using kits. After 14months, DNA degradation was significant in frozen saliva, but that in kits had integrity similar to fresh samples. Storing frozen saliva is detrimental. This kit is well suited for collection, long-term storage, and assay of viral DNA in resource-constrained settings.