BACKGROUND: The polyomaviruses that infect humans, BK virus (BKV), JC virus (JCV), and simian virus 40 (SV40), typically establish subclinical persistent infections. However, reactivation of these viruses in immunocompromised hosts is associated with renal nephropathy and hemorrhagic cystitis (HC) caused by BKV and with progressive multifocal leukoencephalopathy (PML) caused by JCV. Additionally, SV40 is associated with several types of human cancers including primary brain and bone cancers, mesotheliomas, and non-Hodgkin's lymphoma. Advancements in detection of these viruses may contribute to improved diagnosis and treatment of affected patients. OBJECTIVE: To develop sensitive and specific real time quantitative polymerase chain reaction (RQ-PCR) assays for the detection of T-antigen DNA sequences of the human polyomaviruses BKV, JCV, and SV40 using the ABI Prism 7000 Sequence Detection System. STUDY DESIGN: Assays for absolute quantification of the viral T-ag sequences were designed and the sensitivity and specificity were evaluated. A quantitative assay to measure the single copy human RNAse P gene was also developed and evaluated in order to normalize viral gene copy numbers to cell numbers. RESULTS: Quantification of the target genes is sensitive and specific over a 7 log dynamic range. Ten copies each of the viral and cellular genes are reproducibly and accurately detected. The sensitivity of detection of the RQ-PCR assays is increased 10- to 100-fold compared to conventional PCR and agarose gel protocols. The primers and probes used to detect the viral genes are specific for each virus and there is no cross reactivity within the dynamic range of the standard dilutions. The sensitivity of detection for these assays is not reduced in human cellular extracts; however, different DNA extraction protocols may affect quantification. CONCLUSION: These assays provide a technique for rapid and specific quantification of polyomavirus genomes per cell in human samples.
BACKGROUND: The polyomaviruses that infect humans, BK virus (BKV), JC virus (JCV), and simian virus 40 (SV40), typically establish subclinical persistent infections. However, reactivation of these viruses in immunocompromised hosts is associated with renal nephropathy and hemorrhagic cystitis (HC) caused by BKV and with progressive multifocal leukoencephalopathy (PML) caused by JCV. Additionally, SV40 is associated with several types of humancancers including primary brain and bone cancers, mesotheliomas, and non-Hodgkin's lymphoma. Advancements in detection of these viruses may contribute to improved diagnosis and treatment of affected patients. OBJECTIVE: To develop sensitive and specific real time quantitative polymerase chain reaction (RQ-PCR) assays for the detection of T-antigen DNA sequences of the humanpolyomavirusesBKV, JCV, and SV40 using the ABI Prism 7000 Sequence Detection System. STUDY DESIGN: Assays for absolute quantification of the viral T-ag sequences were designed and the sensitivity and specificity were evaluated. A quantitative assay to measure the single copy human RNAse P gene was also developed and evaluated in order to normalize viral gene copy numbers to cell numbers. RESULTS: Quantification of the target genes is sensitive and specific over a 7 log dynamic range. Ten copies each of the viral and cellular genes are reproducibly and accurately detected. The sensitivity of detection of the RQ-PCR assays is increased 10- to 100-fold compared to conventional PCR and agarose gel protocols. The primers and probes used to detect the viral genes are specific for each virus and there is no cross reactivity within the dynamic range of the standard dilutions. The sensitivity of detection for these assays is not reduced in human cellular extracts; however, different DNA extraction protocols may affect quantification. CONCLUSION: These assays provide a technique for rapid and specific quantification of polyomavirus genomes per cell in human samples.
Authors: Sonia Toracchio; Claudia A Kozinetz; Deanna E Killen; Andrea M Sheehan; Eugenio I Banez; Michael M Ittmann; Vojtech Sroller; Janet S Butel Journal: J Clin Virol Date: 2009-07-23 Impact factor: 3.168
Authors: Lora D Thomas; Aaron P Milstone; Regis A Vilchez; Preeti Zanwar; Janet S Butel; J Stephen Dummer Journal: Transplantation Date: 2009-08-15 Impact factor: 4.939
Authors: Marc A Sze; Pedro A Dimitriu; Shizu Hayashi; W Mark Elliott; John E McDonough; John V Gosselink; Joel Cooper; Don D Sin; William W Mohn; James C Hogg Journal: Am J Respir Crit Care Med Date: 2012-03-15 Impact factor: 21.405
Authors: Shimon Kusne; Regis A Vilchez; Preeti Zanwar; Jorge Quiroz; Marek J Mazur; Raymond L Heilman; David Mulligan; Janet S Butel Journal: J Infect Dis Date: 2012-07-16 Impact factor: 5.226
Authors: Niraj C Patel; Steven J Halvorson; Vojtech Sroller; Amy S Arrington; Connie Wong; E O'Brian Smith; Regis A Vilchez; Janet S Butel Journal: Virology Date: 2009-01-31 Impact factor: 3.616
Authors: John A Vanchiere; Suhaib Abudayyeh; Christina M Copeland; Lee B Lu; David Y Graham; Janet S Butel Journal: J Clin Microbiol Date: 2009-06-03 Impact factor: 5.948