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qPCR's Big Bag of Tricks

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The sweeping rationale behind the increased use of quantitative PCR in the clinic — that almost any classically trained lab technician can perform the assay in any standard molecular biology lab — might also be the technique's biggest downfall in establishing itself as a reputable diagnostic. While qPCR is now commonly used to detect infectious disease pathogens for diagnostic purposes, serious doubts remain as to whether it will ever replace conventional culturing or standby sequencing techniques.

But despite these concerns, researchers are relying on qPCR more and more as a detection and validation tool for various applications. From food- and water-borne pathogen detection, to epidemiological analyses of bacterial serovars, to enhancing molecular genetic tests for idiopathic short stature, qPCR has demonstrated its versatility in the past year. And with the prospects of automation, multiplexing, and standardization on the rise, researchers have more than enough reason to expect that the technique will continue to be embraced across disciplines.

"qPCR expression profiling has been widely applied in research as well as in diagnostic settings," says Barbara D'haene, a project manager at the Ghent University-affiliated qPCR design and analysis firm Bio-gazelle in Belgium. She adds, "[The] recent introduction of very precise automatic liquid handlers and high-throughput platforms holds the promise of taking this application to the next level."

Infectious agent detection

Medical microbiologists, such as Udo Reischl at University Hospital Regensburg in Germany, have been using qPCR to detect bacterial, fungal, and viral pathogens for some time — and for good reason, he says, especially when testing to discover the source of an unknown infection.

"If you don't know which organism you are looking for, you [can] just make a broad-range PCR which amplifies DNA segments of every bacterial species and not only, let's say, Staph aureus," he says. "You're looking with a general approach … contributing to a cheaper assay cost and to high-throughput testing."

Crystal Icenhour, the president and director of research at the Charlottesville, Va.-based firm Phthisis Diagnostics, says that real-time PCR diagnostics for infectious disease show promise because they are "more sensitive and specific than antibody technologies are," and they offer improved ease of use and time savings for the operator. Additionally, she says, "any technician who's familiar with real-time PCR instrumentation could use our product[s] with no additional training."

Working in collaboration with Phthisis co-founder Eric Houpt at the University of Virginia, Icenhour's team is developing a real-time PCR-based diagnostic for Cryptosporidium and Giardia infections — diarrheal illnesses associated with the intestinal parasites — which they hope to bring to market in July 2011. "The process that we've engineered for Cryptosporidium and Giardia diagnostics is easily transferrable to other infectious diseases," she says. "We can change a few components in the product and diagnose a completely different infectious agent."

In the same vein, researchers at the Food and Drug Administration are developing qPCR tests for infectious bacteria, such as Salmonella, E. coli, and other pathogens in fresh produce. Wen Lin, an interdisciplinary scientist at FDA, and his colleagues have recently developed a qPCR test that screens for conserved regions of the specific ipaH gene of Shigella species in 15 varieties of inoculated fruits and vegetables. "In FDA produce assignment, Shigella is one of the food pathogens [that has] been under intensive surveillance since 1999," Lin says. The standard Bacteriological Analytical Manual detection method employs culture techniques, while FDA has traditionally used conventional PCR, "which take days, and four to five hours, respectively. The real-time PCR I developed can finish the analysis from broth enriched overnight within an hour and at about 100 times the sensitivity," he says. Lin's results were published in the February issue of the Journal of Food Protection.

Epidemiological analyses

Matthew Stevens, a research officer at the Murdoch Children's Research Institute in Parkville, Australia, is using multiple qPCR assays to trace Chlamydia trachomatis infections by identifying serovars — or sub-strains — of the bacterium. By interrogating a targeted region of the ompA gene in C. trachomatis, Stevens and his team performed two to four qPCR screens to distinguish serovars, based upon serovar group complexity. "The first qPCR identifies which serovar group the particular strain belongs to," Stevens says. "From this, the necessary secondary set of qPCRs can then be used to characterize at the serovar level."

For example, C. trachomatis serovars in the F and G, or intermediate, groups are closely related and must be identified by separate qPCR runs, while C group serovars — of which there are seven — require four qPCR runs to be distinguishable from one another. In this way, Stevens says, qPCR can facilitate epidemiological studies that track C. trachomatis infections. And while "the assay could be readily adopted for use in other laboratories," he is quick to note that it is "not recommended [for] use as a primary diagnostic test" and is "for research use only."

Genetic detection

At the intersection between research and diagnostic applications of qPCR lies idiopathic short stature — a poorly understood condition that affects three out of every 100 children worldwide. Biogazelle's D'haene and her colleagues have developed a qPCR-based copy-number profiling screen for the SHOX gene and its associated regulatory regions. D'haene says that her group formerly used multiplex ligation-dependent probe amplification screens for SHOX and its neighbor PAR1 copy numbers, but switched to using qPCR. The MLPA assay involves a "lengthy ligation step" and "multiple post-PCR handling steps," she says, and the probe mix requires "extensive in-house modification." Real-time PCR, on the other hand, offers "relatively lowconsumable costs" and quicker turnaround. D'haene notes that while qPCR expression profiling is widely used in molecular labs, "the use of qPCR for copy-number profiling is less popular." The team reported its ISS qPCR assay in the Journal of Clinical Endocrinology & Metabolism in April.

Another boon for qPCR has been the detection of RNA targets in circulating cells. In a Bone paper in press, Ulrike Mödder, assistant professor of medicine at the Mayo Clinic, and her colleagues report that their combination of microarray and qPCR approaches allowed them to distinguish differential gene expression patterns of peripheral blood and bone marrow stromal osteogenic cells. "We thought it was fascinating that the peripheral blood cells are so much more quiescent but then they express, to a greater degree, markers to probably support osteoclastogenesis and, we also think, hematopoiesis," Mödder says. "It's very interesting that just because the cells [are] in different environments, they express different genes."

She says that her team chose to use a combination of microarray-qPCR techniques to effectively see the forest as well as the trees. Going into the study, the team had certain pathways of interest in mind. "But the microarray up front gives you a broader overview and it might lead you, actually, into new directions that you want to follow up," Mödder says. "We followed [our study] with qPCR, and I think certainly it's the best way to look into gene expression patterns right now."

Mödder echoes the meme of simplicity, sensitivity, and specificity surrounding qPCR. When examining gene expression patterns, she says, "you need a system that is very specific and also very sensitive. I think we are past the idea that we just want to see if a gene is there or not — we really want to see the levels of RNA. … [qPCR] gives you the possibility to detect differences, probably down to 15 or 20 percent — and it's significant, and probably also biologically meaningful in humans." Though Mödder's group began designing its own primers three years ago, she says that the osteoblast marker qPCR array can be easily performed in other labs. "I think by now in a good molecular biology laboratory, most of them can do it," she says.

Environmental effects

Alexey Fedulov, a research scientist in environmental health at the Harvard School of Public Health, and his collaborators are also using the microarray-qPCR combination — only their work, published in the Journal of Toxicology and Environmental Health in January, interrogates the inflammatory response pathway triggered by environmental exposures in pregnant mice.

Fedulov's lab happened upon the "unusual ability of a negative control substance — titanium dioxide particles — to induce inflammatory response in the lungs during pregnancy," he says. "The finding sparked the proposal to evaluate the entire responseof the lung," via microarray gene-expression experiments followed by real-time PCR in a hypothesis-driven approach. The team tested its PCR array results by pathway analysis and "found that, in combination, the genes [we] identified via 'blind' genome-wide approach, and those found through [our] hypothesis-driven insight are combined in several known pathways relevant to the biology of the study," Fedulov says.

While Fedulov adds that his team's microarray-qPCR method suggests the potential for predicting outcomes of titanium dioxide exposure in pregnant mice as well as the specific deleterious actions of environmental pollutants, he "would be skeptical if the implication was that gene expression data — PCR or other — could be diagnostic to identify the type of environmental toxin or pollutant."

Assuring quality

But as qPCR has been gaining attention for its applications that have identified new targets, it hasn't been kept properly in check, says Stephen Bustin, a professor of molecular science at the Queen Mary University of London. "This attention is accompanied by a reluctance to question the reliability and relevance of the qPCR data," Bustin says. "Unlike many diagnostic assays it is threatening to replace," he continues, "qPCR is not a mature technology — there are serious disagreements on how best to perform the assay, how to obtain copy numbers or relative quantification data from raw quantification cycles, and whether linear regression or non-linear regression algorithms are most suitable for data analysis."

In a 2009 Clinical Chemistry paper, Bustin and other PCR experts suggested the minimum information for publication of quantitative real-time experiments, or MIQE. Because Bustin et al. note that "full disclosure of all reagents, sequences, and analysis methods is necessary to enable other investigators to reproduce results," they argue that MIQE details should accompany every peer-reviewed qPCR paper, whether in an abbreviated form or as a supplement.

Bustin predicts that there will be "continued publication of contradictory results, persistence of uncertainty, and, consequently, lack of confidence by clinicians in PCR data as the basis for their diagnostic and prognostic decision-making" until there is a community-wide agreement on how to best standardize the technology.

Ghent's D'haene says a "big challenge remains [in the] adherence of qPCR-based scientific articles to the recently published MIQE guidelines." In abiding by standardized practices, she says, studies will become "much more transparent, reproducibly in other labs, and simply lead to higher-quality and trustworthy conclusions."

Challenges and concerns

Beyond the issues of standardization, qPCR is far from a perfect fit for certain clinical scenarios. In the microbiology realm, the University of Regensburg's Reischl says that there are a number of unresolved challenges, not the least of which is clinical sample complexity. "You cannot quantify organisms in a swab because a swab is not a standardized material," he says. "If you take 10 swabs from the same patient, you may observe 10 different quantitative results."

Many patient samples, like stool, he says, are especially complex in and of themselves. For their part, Phthisis Diagnostic's Icenhour says her team's DNA stool extraction kit — set to become available in July — will allow users to "extract DNA from patient stool samples," a "highly variable sample type," for use with any real-time PCR downstream diagnostic.

Reischl also says that distinguishing between living and dead pathogenic microorganisms — infectious versus no longer a threat — will likely continue to be an issue. "Because DNA is very stable, [even] after initiation of an antibiotic treatment, for example, you may detect DNA in the lung of a tuberculosis patient for six months or almost a year," he says. The PCR test will continue to produce a positive result, even if the patient has been sufficiently treated and is no longer infectious.

Furthermore, Reischl adds, qPCR may never be able to replace traditional culture methods for detecting the complete spectrum of antibiotic resistance in bacteria. For any given antibiotic, a bacterium could employ "20 different pathways" and a variety of genetic mechanisms to escape the antibiotic pressure, PCR just can't keep up, he says. "I think PCR can compete with culture for detection, but not for comprehensive resistance testing," he says. "And as long as PCR does not provide reliable results for antibiotic susceptibility testing speculations — mainly the resistance patterns of individual organisms — and as long as molecular methods cannot reliably analyze complete resistance patterns, they cannot substitute diagnostic culture. They have to be performed in parallel."

Michele Van Dyke at the University of Waterloo, whose team used qPCR to screen for Yersinia in surface water, says that sample concentration issues and difficulties associated with removing PCR inhibitors are just two reasons why "it may be advantageous to do concurrent analysis using both PCR and culture methods," for waterborne pathogen detection.

Bustin is also concerned that increased qPCR automation — and therefore the hype surrounding the technique's ease of use in the clinic — could hinder the technology. His "considerations go hand-in-hand with the prevailing tendency to rely on pre-mixed reagents that de-skill qPCR to such an extent that serious trouble-shooting for the complex procedure is no longer possible because the operators do not understand the basics of the assay," he says.

Bustin does have confidence, however, that "well-designed" qPCR assays can reliably identify DNA sequence variants down to even single base-pair deletions. "I am also excited about the potential of immuno-qPCR, which combines the detection specificity of an antibody with the amplification power of qPCR," he says. The "focus on expressed proteins makes it an especially effective diagnostic tool to test for the presence of toxins and biomarkers, which can be more informative than simply detecting the presence of a nucleic acid."

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