By Ben Butkus
Life Technologies scientists, in collaboration with an epidemiologist from George Washington University, have developed a nucleic acid extraction method that enables PCR-based detection of pathogens in blood culture more quickly and with higher sensitivity than commonly used techniques.
The protocol uses products from Life Technologies marketed for use in forensics, food safety, and pharmaceutical quality control, and as such cannot be employed in the clinical diagnostics space without appropriate regulatory approval.
Nevertheless, the method could be a boon for Life Tech in the future should it tackle the sample prep space as part of its burgeoning molecular diagnostics business.
The Life Tech researchers, along with Jeanne Jordan, a professor of epidemiology and biostatistics at GWU and director of the International Institute of Public Health, detailed the proof of principle for the sample prep method in a paper published online last week in the Journal of Molecular Diagnostics.
In the study, which was sponsored in large part by Life Tech, the scientists compared three mechanistically different sample extraction methods — silica spin columns, phenol-chloroform, and automated magnetic capture of polymer-complexed DNA using a Life Tech AutoMate Express instrument — for their abilities to purify nucleic acids from clinical blood culture samples for downstream PCR-based testing.
Although PCR-based testing of blood culture to identify blood-borne pathogens is rapidly becoming a preferred alternative to culture-based methods, PCR assay sensitivity still suffers due to inhibitors that are native to blood; and in particular to an inhibitor called sodium polyanetholsulfonate, or SPS, which is routinely added to blood culture media to inhibit bacterial growth, prevent blood coagulation, and interfere with some antibiotics.
The phenol-chloroform extraction method, which the researchers deemed the gold standard, is a sufficiently sensitive method usually requiring minimal sample dilutions, but uses hazardous organic chemicals and is labor-intensive, the study's authors explained. Meantime, silica-based extraction is easier to perform but has been reported to carry PCR inhibitors into nucleic acid extracts, and thus requires increased sample dilution, thus increasing labor and time to result.
In PCR- or sequencing-based molecular testing, "there are a lot of [PCR]-inhibiting compounds depending on what type of [sample] matrix you're dealing with," Manohar Furtado, vice president of R&D at Life Technologies and a co-author on the study, told PCR Insider this week.
"In most culture, if you want to do genetic analysis, you have to have a system that removes SPS efficiently," Furtado said. "The idea was to come up with a system that would very efficiently bind DNA to magnetic beads … and then optimize the chemistry with certain additives … so we could wash off all the inhibitors, and then change the composition to get the DNA off the beads.
"We have magnetic beads that are … a different chemistry from the routine silica beads that you see in other systems," he added. "We thought, 'Let's try this system to see how it works.'"
Life Tech sells its magnetic bead-based sample prep chemistry under the brand names PrepFiler for the forensics market and PrepSEQ for the food and environmental testing and pharma QC markets. In addition, the company markets the PrepFiler assays along with the AutoMate Express benchtop instrument system to forensics customers.
"Essentially the system was developed so it could deal with difficult matrices — anything that has a lot of [PCR] inhibitors," Furtado said. "We have optimized it … so we don't lose DNA during extraction but still get rid of the maximum amount of inhibitors."
Furtado added that a few other research papers in the forensics arena have demonstrated the efficiency of the Life Tech platform for purifying DNA from various blood samples, but noted that the Journal of Molecular Diagnostics study was the first to test clinical samples.
In their study, Furtado, Jordan, and colleagues used the three methods to purify DNA from 60 clinical blood samples collected into standard Bactec blood culture bottles; then analyzed the extracts for the presence of DNA from Staphylococcus aureus and methicillin-resistant S. aureus using TaqMan assays on an ABI 7500 real-time PCR system.
The extracts from silica columns required 100- to 1,000-fold dilutions to sufficiently reduce the inhibitory effects of SPS. In contrast, samples extracted using either phenol-chloroform or the Automate Express platform required little to no dilution, resulting in an approximate 100-fold improvement in assay sensitivity and the ability to detect organisms two hours earlier than with the silica column-based method. Of the three methodologies, Automate Express had the shortest time to result, requiring approximately 80 minutes to process 12 samples, the authors reported.
In addition, the Automate Express platform was the only technology of the three that was capable of purifying nucleic acids from 50-µL blood culture samples without requiring dilution of the extracts prior to PCR; and the system "effectively processed blood culture samples as large as 256 uL in volume, which may further enhance the probability of detecting low numbers of blood-borne pathogens," the researchers wrote in their paper.
Overall, the researchers estimated that the ability of the Automate Express to adequately purify nucleic acids from blood culture samples effectively cut the cost of analysis in half because it was not necessary to screen multiple dilutions.
"The Automate Express platform not only reduced hands-on time, but the DNA extraction workflow reduced the time to result to a greater extent than was possible when either silica columns or phenol-chloroform was used to extract the samples," the authors concluded. However, they included the caveat that their findings "support a recent report that different extraction technologies have advantages in certain applications and that no one methodology is ideally suited for all applications."
Life Tech has also tested the chemistry to extract nucleic acids from bird stool samples in order to detect infectious agents such as influenza virus, Furtado said. "We don't have direct data on human stool," he added. "The reason we haven't used it on the human diagnostic side is we haven't put this through the [US Food and Drug Administration]. It's a research product only." The company currently has no approved products for clinical diagnostic sample prep.
As for Life Tech potentially using the Automate Express platform and associated chemistry in clinical molecular diagnostics, Furtado said that the company "has been thinking about it, as right now in diagnostics we have a PCR instrument … and some sequencing instruments out there that are CE-IVD marked in Europe. At some point in time I'm sure we'll start thinking about playing in the sample prep market, as well, but I can't speak to that right now."
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