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...

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Vydané v:	Oral diseases Ročník 20; číslo 7; s. 720 - 728
Hlavní autori:	Speicher, DJ, Johnson, NW
Médium:	Journal Article
Jazyk:	English
Vydavateľské údaje:	Denmark Blackwell Publishing Ltd 01.10.2014 Wiley Subscription Services, Inc
Predmet:	Body fluids Deoxyribonucleic acid DNA DNA Genotek herpesvirus Herpesvirus 8, Human - isolation & purification HHV-8 Human herpesvirus 8 Humans quantitative Saliva - virology salivary diagnostics Specimen Handling - methods Viral Load Virology DNA Genotek HHV-8 herpesvirus quantitative salivary diagnostics
ISSN:	1354-523X, 1601-0825, 1601-0825
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Abstract	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.
AbstractList	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. 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. 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. 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. 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. 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. 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. When extracted with spin columns, the P-021 enables accurate viral loads down to 23 copies mu l-1DNA. The quantitative and long-term storage capability of this system makes it ideal for study of salivary DNA viruses in resource-poor settings. 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.OBJECTIVESSaliva 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.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.METHODSUnstimulated 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.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.RESULTSAll 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.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.CONCLUSIONWhen 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. 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. [PUBLICATION ABSTRACT] 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.
Author	Speicher, DJ Johnson, NW
Author_xml	– sequence: 1 givenname: DJ surname: Speicher fullname: Speicher, DJ email: d.speicher@griffith.edu.au organization: School of Dentistry and Oral Health, Griffith University, Gold Coast, Qld, Australia – sequence: 2 givenname: NW surname: Johnson fullname: Johnson, NW organization: Molecular Basis of Disease Research Program, Griffith Health Institute, Griffith University, Gold Coast, Qld, Australia
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References_xml	– reference: Tholen D (2004). Evaluation of linearity using the newly approved NCCLS EP6-A protocol. Clin Lab News 30: 10-12. – reference: Roberts KJ, Grusky O, Swanson AN (2007). Outcomes of blood and oral fluid rapid HIV testing: a literature review, 2000-2006. AIDS Patient Care STDS 21: 621-637. – reference: Speicher DJ, Johnson NW (2012). Detection of human herpesvirus 8 by quantitative polymerase chain reaction: development and standardisation of methods. BMC Infect Dis 12: 210. – reference: Kilian M, Reinholdt J, Lomholt H, Poulsen K, Frandsen EV (1996). Biological significance of IgA1 proteases in bacterial colonization and pathogenesis: critical evaluation of experimental evidence. APMIS 104: 321-338. – reference: Lee YH, Wong DT (2009). Saliva: an emerging biofluid for early detection of diseases. Am J Dent 22: 241-248. – reference: Eichel HJ, Conger N, Chernick WS (1964). Acid and alkaline ribonucleases of human parotid, submaxillary, and whole saliva. Arch Biochem Biophys 107: 197-208. – reference: Bigler LR, Streckfus CF, Dubinsky WP (2009). Salivary biomarkers for the detection of malignant tumors that are remote from the oral cavity. Clin Lab Med 29: 71-85. – reference: Al-Otaibi LM, Al-Sulaiman MH, Teo CG, Porter SR (2009). Extensive oral shedding of human herpesvirus 8 in a renal allograft recipient. Oral Microbiol Immunol 24: 109-115. – reference: Cook RD, Hodgson TA, Waugh AC et al (2002). Mixed patterns of transmission of human herpesvirus-8 (Kaposi's sarcoma-associated herpesvirus) in Malawian families. J Gen Virol 83: 1613-1619. – reference: Marshall V, Parks T, Bagni R et al (2007). Conservation of virally encoded microRNAs in Kaposi sarcoma-associated herpesvirus in primary effusion lymphoma cell lines and in patients with Kaposi sarcoma or multicentric Castleman disease. J Infect Dis 195: 645-659. – reference: Arellano-Garcia ME, Hu S, Wang J et al (2008). 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Snippet	Objectives Saliva is a proved diagnostic fluid for the qualitative detection of infectious agents, but the accuracy of viral load determinations is unknown.... Saliva is a proved diagnostic fluid for the qualitative detection of infectious agents, but the accuracy of viral load determinations is unknown. Stabilising... Objectives Saliva is a proved diagnostic fluid for the qualitative detection of infectious agents, but the accuracy of viral load determinations is unknown....
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SubjectTerms	Body fluids Deoxyribonucleic acid DNA DNA Genotek herpesvirus Herpesvirus 8, Human - isolation & purification HHV-8 Human herpesvirus 8 Humans quantitative Saliva - virology salivary diagnostics Specimen Handling - methods Viral Load Virology
Title	Comparison of salivary collection and processing methods for quantitative HHV-8 detection
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