PCR is a workhorse of biotech—whether it’s being used to prepare samples for another assay, or to assay samples from another preparation. From the search for point mutations to discovery of methylated DNA biomarkers, homebrew tests can be crafted to fill in where off-the-shelf kits fall short.
Real-time (q)PCR gives quantitative readings while end-point PCR affords digital answers, which can themselves be quantitative. Researchers at Select Sciences’ “qPCR Europe Conference”, held in Munich last month, powwowed about what uses they put it to in their search for more reliable, sensitive, automatable, and innovative diagnostics.
Ready-to-use kits are available for PCR-based diagnosis of many, if not most, of the viral infections that routinely find their way into the clinic. Yet when it comes to highly pathogenic viruses like pox viruses—which, because they can quickly kill off their hosts, tend to be far less common—“there’s definitely no market,” noted Andreas Nitsche, Ph.D., of the Center for Biological Safety 1 at the Robert Koch Institute in Berlin.
To identify such pathogens “you first need your own assays—your own assay design, your own controls, your own procedures how to establish such an assay,” he explained. “It’s not like a routine clinical lab.” The generic infectious agent needs to be found, and then the species or variant identified. For this, the pox expert says, bioinformatics plays an increasingly important role.
Viruses like the pox viruses have a very conserved genome, and their sequences can be compared by eye or with standard software tools. But in cases involving more heterogeneous viruses like the flaviviruses, dengue, or yellow fever, “we are talking about many, many thousands of sequences, and you have new sequence entries in GenBank almost every week,” he pointed out.
Assays need to be checked to make sure they’re still valid for all the known variants. And aligning and comparing thousands of sequences can take weeks or months. Dr. Nitsche is currently testing a plug-in for commercial software—to be released soon—that promises to streamline the process by providing automatic tools for sequence control and theoretical validation of assays.
Even identifying a particular virus type—for example, that the etiological agent of SARS is a coronavirus—can also be a challenge. This had been done with electron microscopy (EM), which is “the perfect open-view tool—you see everything that is in the sample,” Dr. Nitsche said.
But EM has its limitations: it will only identify the family of pathogen (it can’t discriminate between smallpox and vaccinia, for example), and it requires a relatively large pathogen concentration (in the range of 105-6) that may not be found in blood or other infected body parts.
Dr. Nitsche and other virus hunters have been exploiting techniques such as generic PCR—using generic primers that will amplify everything in the sample—as well as deep sequencing to find sequences that may not have been known before. Then, “you have a real need for bioinformatics because you have to handle a ton of data.”