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Measuring Dengue Virus RNA in the Culture Supernatant of Infected Cells by Real-time Quantitative Polymerase Chain Reaction
Published on: November 1, 2018
Standardized detection of Simian virus 40 by real-time quantitative polymerase chain reaction in pediatric
Susanne Heinsohn1, Steffi Golta, Hartmut Kabisch
1Centre for Women's and Children's Health, Clinic and Policlinic for Paediatric Haematology and Oncology, University Hospital Hamburg-Eppendorf, Hamburg, Germany. heinsohn@uke.uni-hamburg.de
Background And Objectives:
Recent studies have detected Simian virus 40 (SV40) DNA in specific human tumors albeit with significant discrepancies in frequency. Possible inefficiency of DNA isolation and different detection methods do not allow comparable and precise quantification of SV40 in human samples. A standardized and common detection method is, therefore, essential for further routine SV40 analysis.
Design And Methods:
We established a real time quantitative (RQ-) polymerase chain reaction (PCR) based TaqMan assay on the LightCycler system for reproducible detection and quantification of SV40 DNA. We used 500 ng of the COS-1 cell line, containing one single integrated copy of SV40 DNA, as the quantification standard. Amplification of b-globin served as the quality control for DNA integrity. For the extraction of the episomal form of SV40 we compared a column and a precipitation-based DNA extraction method. DNA samples from 149 healthy controls, from 26 fresh frozen childhood cases of acute lymphoblastic leukemia (ALL) (B-, BCP- and T-ALL) and from 12 paraffin-embedded osteosarcomas were investigated.
Results:
The RQ-PCR assay had a linear amplification rate from 10 to 100,000 copies of SV40 in 500 ng genomic DNA. Very low copy numbers of SV40 DNA were detectable in 2/149 (1,3%) blood samples from healthy German controls. Various amounts of SV40 were detectable in 20/26 (77%) childhood ALL samples of German origin and, in part, high amounts were visible in 11/12 (92%) paraffin embedded Hungarian osteosarcomas. The column-based DNA isolation method allowed the detection of both, the integrated and the episomal forms of SV40.
Interpretation And Conclusions:
Our assay provides a standardized and reproducible quantification of SV40 DNA in a wide spectrum of specimens. Exact quantification strongly depends on the source, as well as on the quality, of the DNA used. Quantification of paraffin-embedded DNA generally leads to lower sensitivity of SV40 DNA detection. We strongly recommend this RQ-PCR assay for standardized detection of SV40.
