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Abbott's Plex-ID Outperforms RT-PCR and Microarrays in FDA Viral ID Study


NEW YORK (GenomeWeb) — Scientists at the US Food and Drug Administration Division of Viral Products have demonstrated the limits of detection of different virus discovery tools, and recommended that they be used in combination for hunting novel viruses lurking in cell lines and lab products used to develop vaccines.

In a study published in the journal Viruses, the group found that combined PCR/electrospray ionization mass spectrometry, or PCR/ESI-MS — sold commercially by Abbott as Plex-ID — was superior in detecting retroviral RNA on a background of cellular nucleic acids, as compared to RT-PCR and viral microarrays. However, when Plex-ID and microarrays were deployed to scan for novel viruses, the researchers concluded a combination approach was warranted, as different assays picked up different species.

Detecting the presence of new viruses could be particularly important in vaccine research. This is because vaccine development for current and re-emerging diseases, such as HIV and pandemic influenza, has sometimes required using cell lines and biological products from humans, dogs, birds, and insects.

Lab products, such as bovine serum or porcine trypsin, also often come from species that could potentially carry dangerous viruses.

Researchers must also address regulatory concerns that these viruses may go undetected by the routinely recommended cell line safety assays. The types of viruses could include latent DNA viruses and endogenous retroviruses, as well as occult and unknown viruses, the researchers said.

Thus, the rationale for rigorous and systematic evaluation, assay standardization, and determining the limits of detection of various detection platforms, was to attempt to definitively determine whether cell lines and lab products are free of viruses that could potentially harm people.

The task began with assay standardization. The study compared virus microarrays, RT-PCR assays, and Plex-ID with respect to the limit of detection of viral RNA in the presence or absence of background cellular nucleic acids.

The viral RNA was from well-characterized retroviral stocks of xenotropic murine retrovirus-related virus, or XMRV, and simian foamy virus serotype 1, or SFV-1.

The study used the Lawrence Livermore Microbial Detection Array, LLMDA v.2, and the ViroChip v.5, from the University of California, San Francisco, each of which can detect about 2,000 different viruses.

The study also used the Plex-ID system from Abbott, a platform that company has continued to develop since acquiring it when it bought Ibis Biosciences. The platform combines automated sample preparation, broad PCR amplification, and electrospray ionization mass spectrometry of DNA amplicons to identify base composition as determined by molecular weight.

More specifically, conserved regions of viral nucleic acids are used to design broad-range primers for detection of virus families. The weight of the amplicons is determined by mass spectrometry, and a base count composition is generated. Known or novel organisms are then detected and identified by comparing genomic signatures to a curated base count database.

For detecting XMRV in the absence of background nucleic acids, the study showed Plex-ID was as good as RT-PCR, and better in the presence of 100,000 cell equivalents of nucleic acids from an insect cell line.

Plex-ID, however, could not detect SFV-1 due to the absence of relevant primers.

Meanwhile, LLMDA was more sensitive in detecting XMRV and SFV-1, but detection was obscured completely by only 10,000 cell equivalents of background DNA and RNA. ViroChip was also unable to detect SFV-1, and samples with background nucleic acids were not tested on this microarray in the current study.

According to the Viruses study, current Plex-ID viral assays can detect 35 viral families and potentially more than 1,000 species, but approximately 96 families are known to exist. Thus, the assay does not detect all families, or any heretofore unknown families. The base count database does however contain over 850,000 entries, according to a study published in PLoS One in 2012.

Examining 11 different cell lines, the Viruses study detected a number of "unknown" base counts that were not in the Plex-ID curated database. The study then followed up on these hits of potential viral nucleic acids using a strategy involving PCR, sequencing, and bioinformatics.

This method of virus hunting is comparable to a method previously covered in PCR Insider. Developed by Eric Delwart at the Blood Systems Research Institute and University of California, San Francisco, the technique is intended to characterize the virome of the US blood supply. To do this, 3' ends are randomly amplified, followed by massively parallel sequencing using Illumina MiSeq, and BlastX of all possible protein translation products.

The Viruses study detected a number of different viruses in the various cell lines and ultimately concluded that "different viruses were detected by using different technologies," and using a combination strategy is important in evaluating safety of biological products.

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