NEW YORK (GenomeWeb) – Detecting point mutations that confer drug resistance in HIV's RNA-based genome is routine in resource-rich settings. In low-resource locations, however, such personalized medical treatment is a challenge.
Now, using K103N mutant HIV-1 as a test case, researchers at Brown University have developed a method that allows RNA point mutations to be detected in a single step. They have also begun adapting the technique to a microfluidic device that may some day be suitable for point-of-care use in global settings without high-tech labs.
As described in The Journal of Molecular Diagnostics, the ligation on RNA, or LRA, method essentially eliminates the need to create a cDNA library from RNA, and merges ligation and amplification steps to detect single point mutations.
The work was the result of collaboration between the biomedical engineering lab of Anubhav Tripathi at Brown and the HIV drug resistance lab of Rami Kantor in the division of infectious diseases at Brown's Warren Alpert Medical School.
HIV point mutations are very common in patients who fail antiretroviral therapy, Kantor told GenomeWeb.
An estimated 15 to 20 percent of patients fail initial, first-line regimens, and up to 40 percent fail more advanced, second-line regimens, he said. "Of those who fail therapy, about 80 to 90 percent have drug-resistant point mutations."
For the detection method, Tripathi said direct ligation and amplification on DNA is well known, and it has been performed on RNA in multiple separate steps, but "nobody to our knowledge has tried this technique directly on RNA, in a single step," he noted in an interview.
In LRA, the ligase joins a so-called common probe, which is fully complementary to the RNA target, with a detector probe that has a 3' end nucleotide only complementary to the variant to be detected, according to the JMD study.
These are hybridized adjacent to the RNA, and if both probes match up perfectly they will be joined by T4 DNA ligase, with the ligation rate slowed if there is a single-nucleotide mismatch.
In the next stage, the ligase is heat inactivated and a hot-start DNA polymerase becomes active. For amplification, the researchers used the Thermo Fisher Scientific PikoReal Real-Time PCR System, as well as a custom droplet-based real-time PCR device previously reported in an Analytical Chemistry study and covered by GenomeWeb.
The study also documented various steps the researchers took to make the method work efficiently, including chemically modifying the probes, adjusting probe lengths, and optimizing buffer pH so that it could accommodate both enzymatic reactions.
While it required about a year to get the assay running, it is now "very good," and has made the detection of point mutations in RNA about ten times faster than previous methods, Tripathi said.
During the time it took the team to perfect the technique it was supported by seed money from the Center for Aids Research at the National Institutes of Health, and the group has now been invited to present its work at an upcoming CFAR event.
The team plans to next direct the method toward clinical samples. Kantor noted that patient samples are available in his laboratory from around the world.
"At the end of the day the real usefulness of this technique is the application to patient samples, and that's what we care most about," Tripathi said.