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UC-Denver Researchers Use RT-qPCR to Detect Bacteria in Cystic Fibrosis Airway Specimens

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By Ben Butkus

Real-time quantitative PCR is equal to or better than more time-consuming culture-based methods for identifying several bacterial species associated with lung disease and chronic airway infections in cystic fibrosis patients, according to a recent study published by University of Colorado-Denver clinical researchers.

As such, the researchers believe that qPCR may be a useful tool for understanding polymicrobial CF lung infections, lung disease progression, and eventually developing antimicrobial therapies.

In particular, the technique can more readily identify certain anaerobic bacterial species that are believed to play as important a role in CF-related pulmonary infections as more common aerobic bacteria, but have thus far been difficult to study because of the complexity of culturing them.

"We know that CF patients have chronic lung infections," Edith Zemanick, assistant professor of pediatrics at UCD and lead author on the study, told PCR Insider this week.

"Right now we use culture to identify bacteria that are known to be associated with CF," such as Pseudomonas aeruginosa and Staphylococcus aureus, Zemanick said. "But there's a lot of thought that anaerobes probably also play a role in CF lung disease. The problem is that those are very difficult to capture by culture. Clinical labs in general are not doing anaerobic cultures from CF patients."

Real-time qPCR provides "an easier way to study anaerobes that doesn't rely on culture," she added.

In their study, published Nov. 30 in PLoS One, Zemanick and colleagues used RT-qPCR to test 84 sputa, 47 oropharyngeal, and 27 salivary specimens from 16 pediatric CF patients.

In most cases, they used qPCR assays to measure DNA extracts for total bacteria present, also known as a bacterial load assay; for three of the most typical CF pathogens, P. aeruginosa, S. aureus, and Haemophilus influenzae; and for five anaerobic bacteria, Prevotella melaninogenica, Prevotella oris, Prevotella denticola, Fusobacterium species, and Peptostreptococcus micros.

All of the primers and assays used had been previously described in the literature, except for the H. influenza primers, which the researchers designed from an in-house database of sequences obtained from CF airway specimens for the bacterium.

For bacterial load assays, Zemanick and colleagues measured total ribosomal RNA gene copy number using a qPCR TaqMan assay from Roche Molecular Systems; and for species-specific assays, they used Power SYBR Green PCR Master Mix from Life Technologies' Applied Biosystems business.

All of the assays were run in duplicate on Eppendorf's Mastercycler EP Realplex system. "We didn't do an extensive comparison on other [PCR] platforms, but I doubt there would be major issues," study co-author Kirk Harris told PCR Insider. "Moving forward, if we were going to try and validate these more thoroughly, then that would be something we'd have to look at. But at this point we're really just looking at the reliability of taking a sample and processing it twice, and seeing if you get the same answer."

Overall, the qPCR assays detected bacterial DNA in more than 97 percent of specimens; were highly reproducible at quantities greater than 102 ribosomal RNA gene copies per reaction; and showed excellent agreement when processed in duplicate, the researchers reported.

The assays also revealed that anaerobic bacteria were highly prevalent and were detectable in mean quantities similar to that of typical CF pathogens.

However, their study also showed that qPCR and culture had variable sensitivities when compared to a composite gold standard of culture with qPCR for detecting the three common pathogenic aerobic bacteria, P. aeruginosa, S. aureus, and H. influenza, in CF airway samples.

The researchers chalked this result up to several factors, including the relatively small amounts of bacterial DNA amidst human DNA background in CF sputum samples; and the possibility of insufficient cell lysis. They also claimed that more sensitive primers for these organisms may be available in the literature.

However, Zemanick said that the ability to detect several different types of anaerobic and aerobic bacteria using qPCR was one of the most notable aspects of the study.

"Our focus has really been more on the anaerobes, because culture is reasonably good for things like Pseudomonas and Staphylococcus," Zemanick said. "We're not really looking to replace culture. This is something that would supplement culture. The biggest advantage here is the ability to identify any type of bacteria, to be able to get all of those … that are difficult to grow in culture or that you can grow in culture."

Nevertheless, she added that the study also shows how qPCR may be useful in quickly identifying common pathogenic bacteria in CF patients so that physicians could more quickly and accurately prescribe appropriate therapies.

"When you have CF patients admitted for pulmonary exacerbations, and they get started on [intravenous] antibiotics, it would be helpful if you knew early on what you were treating," Zemanick said.

The researchers said that they will now focus on refining their qPCR assays, including using alternative primers, to help increase their sensitivity in detecting some of the common CF-associated pathogenic bacteria.


Have topics you'd like to see covered in PCR Insider? Contact the editor at bbutkus [at] genomeweb [.] com.

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