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Scientists at Penn State have developed an assay that lets them to directly measure HIV viral load in a drop of blood. The technology is also faster and less expensive than current approaches. At present, RT-PCR is typically used to assess HIV levels in a patient’s blood, requiring genetic material to be amplified before it can be measured. This is time-consuming and does not provide a direct measurement of viral loads, but rather a close estimate. This new technology, called Self-digitization Through Automated Membrane-based Partitioning (STAMP), aims to directly measure viral levels in just a drop of blood. It involves mixing viral RNA with the Cas13 protein, which is part of the CRISPR-Cas system. When activated by the presence of HIV RNA, the Cas13 protein will cleave a reporter molecule, resulting in a measurable signal. The assay is faster, less expensive, and requires less blood than RT-PCR.

Measuring HIV viral loads is an important step in monitoring patient progress during treatment. It can vary wildly, from 20 viral particles to over 500,000 per drop of blood, depending on the stage of infection and the patient’s treatment history. It is necessary to measure the viral load several times during treatment to ensure that things are progressing as required. However, current tests to achieve this, which typically involve RT-PCR, are time consuming and expensive to run, and do not provide a direct measurement of viral load, but rather a proxy estimate.

To create a more direct diagnostic technology, and one that is faster and less expensive to run, these researchers have turned to the CRISPR-Cas system. The CRISPR-Cas system is becoming well-known as a gene editing tool, but it also has a role in diagnostic technologies because of its ability to highly specifically identify and manipulate genetic material.

Penn State’s STAMP assay involves mixing HIV RNA with the Cas13 protein. Then the researchers place a polycarbonate membrane containing nanopores over the sample. The pores are so small that they only allow a tiny droplet to enter, which contains one RNA molecule with a Cas13 protein attached. In the presence of HIV RNA, the Cas13 protein is activated, cleaving a reporter molecule, and creating a measurable signal that can be viewed inside the nanopore-enclosed droplets.

“By counting the number of droplets showing this signal, we can determine the amount of HIV in the person’s blood,” said Weihua Guan, a researcher involved in the project. “The more droplets with the signal, the higher the viral load. While further improvements are needed to enhance its detection limit and automate the setup, the STAMP-based digital CRISPR method shows great potential for advancing HIV viral load monitoring.”  

Top image: In the new quantification method developed by Penn State researchers, a molecular signal shines green when HIV is detected in an RNA molecule. By counting the signals, researchers can quantify the viral load in a sample. Credit: Provided by Weihua Guan.

Study in journal ACS Nano: STAMP-Based Digital CRISPR-Cas13a for Amplification-Free Quantification of HIV-1 Plasma Viral Loads

Via: Penn State





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