Rapid and sensitive paper-based nucleic acid assay for SARS-CoV-2


In healthcare, paper-based diagnostics have revolutionized point-of-care approaches by providing inexpensive, disposable tools that can be used remotely. Developed by the World Health Organization (WHO), paper-based diagnostics meet ASSURED criteria (Affordable, Sensitive, Specific, User-Friendly, Rapid and Robust, Equipment-Free, and Deliverable to End Users). Paper-based diagnostics have the advantage of being affordable, equipment-free, and easily accessible.

Study: Sensitive detection of SARS-CoV-2 on paper.  Image <a class=credit: NIAID” class=”rounded-img enlarge-image-child” src=”https://d2jx2rerrg6sh3.cloudfront.net/images/news/ImageForNews_723147_16613030023453581.jpg” srcset=”https://d2jx2rerrg6sh3.cloudfront.net/image-handler/ts/20220823090329/ri/2000/src/images/news/ImageForNews_723147_16613030023453581.jpg 2000w, https://d2jx2rerrg6sh3.cloudfront.net/image-handler/ts/20220823090329/ri/1950/src/images/news/ImageForNews_723147_16613030023453581.jpg 1950w, https://d2jx2rerrg6sh3.cloudfront.net/image-handler/ts/20220823090329/ri/1750/src/images/news/ImageForNews_723147_16613030023453581.jpg 1750w, https://d2jx2rerrg6sh3.cloudfront.net/image-handler/ts/20220823090329/ri/1550/src/images/news/ImageForNews_723147_16613030023453581.jpg 1550w, https://d2jx2rerrg6sh3.cloudfront.net/image-handler/ts/20220823090329/ri/1350/src/images/news/ImageForNews_723147_16613030023453581.jpg 1350w, https://d2jx2rerrg6sh3.cloudfront.net/image-handler/ts/20220823090329/ri/1150/src/images/news/ImageForNews_723147_16613030023453581.jpg 1150w, https://d2jx2rerrg6sh3.cloudfront.net/image-handler/ts/20220823090329/ri/950/src/images/news/ImageForNews_723147_16613030023453581.jpg 950w, https://d2jx2rerrg6sh3.cloudfront.net/image-handler/ts/20220823090329/ri/750/src/images/news/ImageForNews_723147_16613030023453581.jpg 750w, https://d2jx2rerrg6sh3.cloudfront.net/image-handler/ts/20220823090329/ri/550/src/images/news/ImageForNews_723147_16613030023453581.jpg 550w, https://d2jx2rerrg6sh3.cloudfront.net/image-handler/ts/20220823090329/ri/450/src/images/news/ImageForNews_723147_16613030023453581.jpg 450w” sizes=”(min-width: 1200px) 673px, (min-width: 1090px) 667px, (min-width: 992px) calc(66.6vw – 60px), (min-width: 480px) calc(100vw – 40px), calc(100vw – 30px)” style=”width: 2000px; height: 1658px;” width=”2000″ height=”1658″/>Study: Sensitive detection of SARS-CoV-2 on paper. Image credit: NIAID

A paper-based diagnosis was developed by Jinghong Li, Weimin Li, Ruijin Deng, and colleagues at Boston University for the identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with high sensitivity. Developed in just 18 days, the device can detect 400 copies of viral ribonucleic acid (RNA) per microliter and is capable of identifying three coronaviruses (SARS-CoV, Middle East respiratory syndrome coronavirus (MERS-CoV) and SARS-CoV-2), as well as seven influenza subtypes. The test is inexpensive and does not require trained technicians for evaluation and reporting. The research is published in the journal Nature Biomedical Engineering.

Assay of nucleic acids on paper: the mechanism

The researchers developed a test called MARVE (Multiplexed, Nucleic Acid-Free, Single Nucleotide-Resolved Viral Evolution) that uses a pH indicator that changes color (from red to yellow) as the pH decreases. In their investigation, the authors ingeniously used silver (Ag) ions to interact with two components – a deoxyribonucleic acid (DNA) probe and the urease enzyme.

Ag(I) ions interact with DNA to form C–Ag–C complexes. A free Ag(I) ion inhibits the enzyme urease, which hydrolyzes urea to a weak base, ammonia.

In order to detect viral RNA, the authors designed the DNA probe to release Ag(I) after it binds to a sequence of SARS-CoV-2. The released Ag(I) suppresses urease activity, resulting in a lower pH and a yellow reading. When the probe is not accompanied by the viral RNA, it retains the Ag(I) and the active urease degrades the urea to produce ammonia, which makes the pH conditions higher and emits a Red color.

These reactions can be carried out in solution or on paper media resulting in colorimetric test results that can be observed with the naked eye and can be analyzed with a smartphone.

DNA-toehold: The crucial element

The DNA-foot exchange probe used in Li and co-authors’ test is critical to its specificity. A DNA-tip exchange probe consists of two complementary DNA strands that are hybridized together due to their complementary nucleotide sequences. Two exchange reactions are initiated by toeholds, short sequences at the ends of the probe strand complementary to the target sequence or the protective strand.

A DNA-foot exchange probe binds to the viral RNA target, releasing Ag(I) ions – to prevent urease hydrolysis – and establishing the low pH conditions that lead to color change – from red to yellow (positive reaction) . Invalid viral RNA with a single point mutation is unable to activate the probe. These prevent the release of Ag(I) and lead to active hydrolysis of urease and high pH conditions which produce a red reading.

Embedding assay reactions in origami paper

The reaction steps proceed sequentially as the layers of paper are folded over each other, allowing the reaction intermediates to move between the layers. A smartphone can be used to assess the reading.

Test Highlights

The MARVE System integrates the specificity of nucleic acid testing with analysis times and costs – such as those associated with lateral flow testing. To achieve these characteristics, only one enzyme and one DNA probe are required, and no nucleic acid amplification step is necessary. This helps reduce costs and provides a less complex procedure that takes less time.

Since MARVE reactions occur at room temperature (except for the thermolysis step), there is no need for a heating block. Additionally, MARVE assays are quick to develop since only one probe is needed. MARVE’s ease of use and accuracy indicate that the tests are suitable for home testing.

In fact, MARVE can be modified to eliminate the need for origami folding steps and can be made more user friendly, such as lateral flow trials.

Source link