New drug-resistance test uses molecular beacons
New drug-resistance test uses molecular beacons
Advantages include speed, high specificity
In the past year, a handful of molecular-based tests have been developed for drug susceptibility to tuberculosis. Researchers at Montefiore Medical Center report on an innovative yet relatively simple test using a genetic approach that is highly specific to rifampin and is being tested on several other TB drugs as well.
The test takes advantage of the fact that the TB genome is highly conserved, meaning that a mutation in a drug-resistant strain almost always indicates the strain is drug-resistant and that wild-type strains are always drug-sensitive, says David Alland, MD, assistant professor of medicine at Albert Einstein College of Medicine in Philadelphia and attending physician at Montefiore Medical Center in Bronx, NY.
Alland and his colleagues have developed the test using molecular beacons or fluorescent probes, which are highly specific to the DNA strain for which they are made. The results of the test on rifampin-resistant strains were published in the most recent issue of Nature Biotechnology.
"We can use them to probe areas of the TB genome in a PCR assay and see whether that area is wild-type or not because if there are any mutations, they will not fluoresce," he explains.
One advantage of a test based on molecular beacons is they are relatively simple yet robust. A sample is placed in several tubes, the beacons are put inside the tubes, the assay is performed, and then the fluorescence is analyzed. The procedure takes only three hours. But equally important, the tubes never have to be opened, thus exposing them to contamination, which is a major concern in high-volume labs.
"Other nucleic acid-based amplification techniques all require that the tubes be opened and analyzed in some way," he says. "We believe it is something that is not going to work either in labs that do high through-put or certainly in developing countries where tubes have to stay closed."
Currently, the test requires five tubes, but Alland expects the number can be reduced to one for labs that have the appropriate technology. Although an expensive PCR machine is required, it is possible to read the fluorescence simply by placing the tubes in a UV box. "We are getting close but not there yet to having a very simple assay for drug-resistance testing. You just do a simple PCR assay and take the tubes and see if they glow, and that is about as simple as you can get with an amplification-based technique," he adds.
So far, the test has been run on 75 isolates cultured on solid media L-J slants and correctly identified all of the drug-resistant strains. The next step is to use the test on clinical samples. Alland expects the test also could be used on positive smears. Currently, the assay is being tested on other TB drugs, including isoniazid, but the results have not been published.
As with other nucleic acid-based diagnostics, the test requires expensive technology. The goal, Alland says, is to develop an inexpensive kit that can be used commercially. He knows making that leap can prove difficult.
"There are a number of tests being developed, and all have their shortcomings," he says. "Possibly ours has not been around long enough to find a shortcoming."
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