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Analytical Chemistry

Mobile Test Could Detect Viral DNA Without PCR

Bioengineering: Simple test strip removes background DNA, allowing researchers to use PCR alternative to detect HIV

by Louisa Dalton
January 26, 2015

ENRICHED BLOOD
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Credit: Anal. Chem.
Bioengineers concentrate the DNA they seek on a strip of nitrocellulose paper. As a blood sample travels up the strip, immobilized nucleotides (red) capture the target sequence (green). Other DNA (yellow) is washed away, enriching a portion (pink) for detection by recombinase polymerase amplification.
20150126lnp2-enrich.jpg
Credit: Anal. Chem.
Bioengineers concentrate the DNA they seek on a strip of nitrocellulose paper. As a blood sample travels up the strip, immobilized nucleotides (red) capture the target sequence (green). Other DNA (yellow) is washed away, enriching a portion (pink) for detection by recombinase polymerase amplification.

Researchers want to develop viral detection tests that don’t depend on the polymerase chain reaction. PCR amplifies nucleic acids nicely, yet it requires equipment that isn’t always handy, especially in developing nations. A mobile test that doesn’t depend on laboratory access would be better.

One of the most promising alternatives to PCR is recombinase polymerase amplification (RPA), which amplifies DNA in less than 15 minutes. Unfortunately, the reaction doesn’t work in whole blood. Now bioengineers have developed a simple pretreatment step that isolates viral DNA from blood samples, moving an RPA-based test closer to being practical (Anal. Chem. 2015, DOI: 10.1021/ac504365v).

A baby born to a mother with HIV should be tested for the disease straightaway, says Brittany Rohrman, a bioengineering graduate student at Rice University. Currently, doctors use a PCR test, which seeks out HIV DNA that incorporates itself into the host chromosome.

During PCR, the sample first must be heated to separate DNA into single strands, then cooled so that primers bind the strands, and finally warmed to a third temperature to amplify strands with polymerase. The steps are then repeated. Such temperature finagling requires a thermal cycler.

To avoid the need for such specialized lab equipment, many research groups, including Rebecca Richards-Kortum’s laboratory where Rohrman works, have turned to RPA, a method for copying nucleic acids that works quickly at one temperature. Instead of temperature cycling, recombinase enzymes bind primers to the target DNA sequences so a polymerase can then extend the strands. This simplicity could allow for fast-turnaround diagnostics, providing a result at the mother’s side before she treks back home with her new baby.

Rohrman wanted to figure out why RPA seems to stop working when run in whole-blood samples. She thought the high concentration of nontarget DNA in the samples could be the culprit: She noticed that diluting samples allowed the test to work. So she titrated background DNA to find out exactly how much RPA could tolerate before the method stopped working.

Then she designed a simple method to strip away background DNA from whole blood. She adds a sample to the end of a nitrocellulose strip, much like the ones found in pregnancy tests. The sample travels up the strip, passing a section of immobilized DNA that grabs target HIV sequences. She washes the strip to rid it of unbound DNA, leaving a concentrated sample of her target DNA that is ready for RPA.

Rohrman tested the assay on a solution that simulated blood: It contained a plasmid of HIV DNA to serve as the target and salmon sperm DNA to serve as background noise. It worked, she says, but it still takes too long and has too many steps: The nitrocellulose enrichment step takes 50 minutes, and the RPA test takes 15. She would like to combine both tests into an all-in-one microfluidic assay to save time.

Manfred Weidmann of the University of Stirling, in the U.K., thinks that a closed device would be an improvement, adding that it also has the advantage of decreasing the likelihood of contamination from external DNA. David S. Boyle of PATH, a nonprofit global health group, would like to see testing performed on whole blood spiked with HIV-infected cells, but he likes the simple approach for capturing target DNA.

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Comments
Matthew Forrest (January 26, 2015 12:31 PM)
I think that it's important to note that RPA can work with whole blood, it's just that this particular assay doesn't work very well with it. It all depends on the primer and probe sequences being used.

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