Comparison of HCMV Loads Using In-house Quantitative CMV PCR, ArtusCMV TM PCR and COBAS Amplicor CMV Monitor Test
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CMV real-time PCR quantitative
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Abstract
A real-time PCR assay based on highly conserved cytomegalovirus glycoprotein B (CMV-gB) gene was developed to quantify the CMV genome loads in human plasma obtained fromimmunocompromised patients. The sequence comparison from several strains resulted in the bestpossible set of primers and a probe that directed against the most highly conserved region. Thedetection limit of In-house TaqMan polymerase chain reaction assay for CMV-DNA was 2.3 log10copies/ml and the linear measure interval was 2.3 to 8.3 log10 copies/ml (R > 0.99). The In-houseTaqMan assay was compared to commercial quantitative PCR test, COBAS Amplicor CMV monitortest and Artus CMV TM PCR test, in the determination of CMV DNA load in plasma samples. Thecorrelation between In-house TaqMan and COBAS assays was statistically significant, R = 0.915 (P <0.05; n = 27). Also correlation between In-house TaqMan and Artus CMV assays was statisticallysignificant and higher (R = 0.998; P < 0.05; n = 8). When the In-house TaqMan system was evaluatedwith the Nucleospin® RNA virus kit and QIAamp blood minikit, no significant difference was found atthe 98.2% confidence interval. Preliminary results indicate that the In-house TaqMan real-time PCRwith the designed primer and probe set can be an alternative tool for quantification of CMV loads inclinical routine laboratories.
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Okada, P. A., Suksomboon, T., Peungpa, A., Pholampaisathit, S., & Sawanpanyalert, P. (2013). Comparison of HCMV Loads Using In-house Quantitative CMV PCR, ArtusCMV TM PCR and COBAS Amplicor CMV Monitor Test. Science, Engineering and Health Studies, 4(1), 24–35. https://doi.org/10.14456/sustj.2010.3
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Research Articles
References
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Zhao, J., Bai, Y., Zhang, Q., Wan, Y., Li, D., and Yan, X. (2005). Detection of hepatitis B virus DNA by real-time PCR using TaqMan-MGB probe technology. World Journal of Gastroenterology, 11: 508-510.
Boeckh, M. and Boivin, G. (1998). Quantitation of cytomegalovirus: methodologic aspects and clinical applications. Clinical Micro-biology Reviews, 11: 533-554.
Boeckh, M., Gallewez-Hawkins, M., Myerson, D., Zaia, J. A., and Bowden, R. A. (1997). Plasma polymerase chain reaction for cytomegalovirus DNA after allogeneic marrow transplantation: comparison with polymerase
chain reaction using peripheral blood leucocytes, pp.65 antigenemia, and viral culture. Transplantation, 64: 108-113.
Caliendo, A., St George, K., Kao, S., Allega, J., Tan, B-H., LaFontaine, R., Bui, L., and Rinaldo, C. R. (2000). Comparison of quantitative cytomegalovirus (CMV) PCR in plasma and CMV antigenemia assay: clinical utility of the prototype AMPLICOR CMV MONITOR test in transplant recipients. Journal of Clinical Microbiology, 38: 2122-2127.
Chou, S. W. and Dennison, K. M. (1991). Analysis of interstrain variation in cytomegalovirus glycoprotein B sequences encoding neutral-lization-related epitopes. Journal of Infectious Diseases, 163: 1229-1234.
Delgado, R., Lumbreras, C., Alba, C., Pedraza M. A., Otero, J. R., Gomez, R., Moreno, E., Noriega, A. R., and Paya, C. V. (1992). Low predictive value of polymerase chain reaction for diagnosis of cytomegalovirus disease in liver transplant recipients. Journal of Clinical Microbiology, 30: 1876-1878.
Fernandez, C., Boutolleau, D., Manichanh, C., Mangeney, N., Agut, H., and Gautheret-Dejean, A. (2002). Quantitation of HHV-7 genome by real-time polymerase chain reaction assay using MGB probe technology. Journal of Virological Methods, 106: 11-16.
Gouarin, S., Vabret, A., Scieux, C., Agbalika, F., Cherot, J., Mengelle, C., Deback, C., Petitjean, J., Dina, J., and Freymuth, F. (2007). Multicentric evaluation of a new commercial cytomegalo-virus real-time PCR quantitation assay. Journal of Virological Methods, 146: 147-154.
Herrmann, B., Larsson, V. C., Rubin, C., Sund, F., Eriksson, B., Arvidson, J., Yun, Z., Bondeson, K., and Blomberg, J. (2004). Comparison of a duplex quantitative real-time PCR assay and the COBAS amplicor CMV monitor test for detection of cytomegalovirus. Journal of Clinical Micro-biology, 42: 1909-1914.
Humar, A., Paya, C., Pescovitz, MD., Dominguez, E., Washburn, K., Blumberg, E., Alexander, B., Freeman, R., Heaton, N., and Mueller, B. (2004). Clinical Utility of cytomegalovirus viral loads testing for predicting CMV disease in D+/R- solid organ transplant recipients. American Journal of Transplantation, 4: 644-649.
Ikewaki, J., Ohtsuka, E., Kawano, R., Ogata, M., Kikuchi, H., and Nasu, M. (2003). Realtime PCR assay compared to nested PCR and antigenemia assays for detecting cytomegalovirus reactivation in adult T-cell leukemia-lymphoma patients. Journal of Clinical Microbiology, 41: 4382-4387.
Murphy, E., Yu, D., Grimwood, J., Schmutz, J., Dickson, M., Jarvis, M. A., Hahn, G., Nelson, J. A., Myers, R. M., and Shenk, T. E. (2003). Coding potential of laboratory and clinical strains of human cytomegalovirus. Proceedings of the National Academy of Sciences USA, 100: 14976-14981.
Piiparinen, H., Hockerstedt, K., Lappalainen, M., Suni, J., and Lautenschlager, I. (2002). Monitoring of viral loads by quantitative plasma PCR during active cytomegalovirus infection individual liver transplant patients. Journal of Clinical Microbiology, 40: 2945-2952.
Shepp, D. H., Match, M. E., Lipson, S. M., and Pergolizzi, R. G. (1998). A fifth human cytomegalovirus glycoprotein B genotype. Research in Virology, 149: 109-114.
Tiziano, A., Marco, E., Fabrizia, P., Silva, V., Alessandro, F., Giovanna, M., and Valeria, G. (2006). Quantitation of cytomegalo-virus DNA by real-time polymerase chain reaction in peripheral blood specimens of patients with solid organ transplants: comparison with end-point PCR and pp65 antigen test. Journal of Medical Virology, 78: 915-922.
Vincent, E., Gu, Z., Morgenstern, M., Gibson, C., Pan, J., and Hayden, RT. (2009). Detection of cytomegalovirus in whole blood using three different real-time PCR chemistries. Journal of Molecular diagnostics, 11: 54-59.
Wirgart, B. Z., Brytting, M., Linde, A., Wahren, B., and Grillner, L. (1998). Sequence variation within three important cytomegalovirus gene regions in isolates from four different patient populations. Journal of Clinical Microbiology, 36: 3662-3669.
Yun, Z., Lewensohn-Fuchs, I., Ljungman, P., and Vahlne, A. (2000). Real-time monitoring of cytomegalovirus infections after stem cell transplantation using the TaqMan polymerase chain reaction assays. Transplantation, 69: 1733-1736.
Yun, Z., Lewensohn-Fuchs, I., Ljungman, P., Ringholm, L., Jonsson, J., and Albert, J. (2003). A real-time TaqMan PCR for routine quantitation of cytomegalovirus DNA in crude leukocyte lysates from stem cell transplant patients. Journal of Virological Methods, 110: 73-79.
Zhao, J., Bai, Y., Zhang, Q., Wan, Y., Li, D., and Yan, X. (2005). Detection of hepatitis B virus DNA by real-time PCR using TaqMan-MGB probe technology. World Journal of Gastroenterology, 11: 508-510.