Evaluation of the Broad-Range PCR-Electrospray Ionization Mass Spectrometry PCR-ESI-MS System and Virus Microarrays for Virus DetectionReport as inadecuate




Evaluation of the Broad-Range PCR-Electrospray Ionization Mass Spectrometry PCR-ESI-MS System and Virus Microarrays for Virus Detection - Download this document for free, or read online. Document in PDF available to download.

Laboratory of Retroviruses, Division of Viral Products, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, 8800 Rockville Pike, HFM-454, Bethesda, MD 20892, USA



Current address: Division of Infectious Diseases and Vaccinology, School of Public Health, University of California Berkeley, 50 University Ave, Berkeley, CA 94720, USA





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Author to whom correspondence should be addressed.



Abstract Advanced nucleic acid-based technologies are powerful research tools for novel virus discovery but need to be standardized for broader applications such as virus detection in biological products and clinical samples. We have used well-characterized retrovirus stocks to evaluate the limit of detection LOD for broad-range PCR with electrospray ionization mass spectrometry PCR-ESI-MS or PLEX-ID, RT-PCR assays, and virus microarrays. The results indicated that in the absence of background cellular nucleic acids, PLEX-ID and RT-PCR had a similar LOD for xenotropic murine retrovirus-related virus XMRV; 3.12 particles per µL whereas sensitivity of virus detection was 10-fold greater using virus microarrays. When virus was spiked into a background of cellular nucleic acids, the LOD using PLEX-ID remained the same, whereas virus detection by RT-PCR was 10-fold less sensitive, and no virus could be detected by microarrays. Expected endogenous retrovirus ERV sequences were detected in cell lines tested and known species-specific viral sequences were detected in bovine serum and porcine trypsin. A follow-up strategy was developed using PCR amplification, nucleotide sequencing, and bioinformatics to demonstrate that an RD114-like retrovirus sequence that was detected by PLEX-ID in canine cell lines Madin-Darby canine kidney MDCK and Cf2Th canine thymus was due to defective, endogenous gammaretrovirus-related sequences. View Full-Text

Keywords: broad-range PCR amplification; electrospray ionization-mass spectrometry; PLEX-ID; virus microarray; RT-PCR; endogenous retroviruses broad-range PCR amplification; electrospray ionization-mass spectrometry; PLEX-ID; virus microarray; RT-PCR; endogenous retroviruses





Author: Lanyn P. Taliaferro, Teresa A. Galvin, Hailun Ma, Syed Shaheduzzaman, Dhanya K. Williams, Dustin R. Glasner † and Arifa S. Khan *

Source: http://mdpi.com/



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