Evaluation of the Broad-Range PCR-Electrospray Ionization Mass Spectrometry (PCR/ESI-MS) System and Virus Microarrays for Virus Detection

Viruses

Lanyn P. Taliaferro²  Teresa A. Galvin²  Hailun Ma²  Syed Shaheduzzaman²  Dhanya K. Williams²  Dustin R. Glasner¹ 

[1] 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;

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

DOI  :  10.3390/v6051876

来源: mdpi

【 摘 要 】

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.

【 授权许可】

CC BY   
© 2014 by the authors; licensee MDPI, Basel, Switzerland.