Instead of PCR
Scientists have developed a DNA machine to detect pathogens
Scientists at ITMO University and the University of Central Florida have developed a DNA machine for detecting pathogens - viruses, bacteria and other microorganisms that are dangerous to health. The new method, unlike PCR tests, does not require the use of expensive equipment. The development allows testing even at room temperature. With the help of a color signal, the result can be seen with the naked eye. The article was published in the journal Chemical Communications (Gorbenko et al., DNA nanomachine for visual detection of structured RNA and double stranded DNA).
PCR is one of the most accurate and sensitive methods of molecular diagnostics of infectious diseases. However, it also has its limitations: laboratory conditions, trained personnel and expensive equipment are needed to analyze samples for the presence of pathogen genetic material. For example, devices for increasing copies of fragments of nucleic acids, RNA and DNA, are thermocyclers that change the temperature at different stages. As an alternative, methods of isothermal amplification are being developed, in which copying (amplification) RNA and DNA occur in the same temperature regime. They are close in accuracy to the PCR indicators.
Biochemists from ITMO managed to advance in this direction and create a convenient tool for verifying and visualizing the results of isothermal amplification.
"RNA or DNA is usually in a folded state and unfolds only at high temperatures, which complicates the detection of target fragments. We have developed DNA machines that are capable of detecting the necessary sites - analytes – of single–stranded RNAs belonging to certain pathogens without special devices and heating. DNA machines "get close to the target", grab it and force the nucleic acid to turn around," explains Daria Gorbenko, an employee of the SCAMT International Research Center at ITMO University.
The proposed DNA machine is a small platform made of double-stranded nucleic acid. Special sections are connected to it, figuratively speaking, "hands" — there may be two, three or even four of them. For each pathogen, its own platform is created, it is enough to pick it up once. It should be arranged in such a way that there is no possibility of cross-complementarity, that is, undesirable crossing of the structural elements of the DNA machine with the RNA analyte.
"If the desired pathogen is present in the sample, the machine will connect with the analyte and form a four—stranded DNA - G-quadruplex. The properties of this structure open up space for creating a simple and visual test system: when adding special reagents, we will get a color signal. In our case, the sample is colored brown," explains Daria Gorbenko.
The whole process of testing using a DNA machine - from taking the material and isolating nucleic acid to forming a G—quadruplex and receiving a color signal - takes about two to three hours, but the authors of the project are confident that this time can be shortened. At this stage of the study, experiments were carried out on synthetic samples and on copies of RNA and DNA: the article describes the results of the detection of cytomegalovirus (a type of herpes), hemophilic bacillus that causes respiratory damage, and listeria monocytogenes (acute food poisoning).
Visual detection of target fragments of listeria monocytogenes and cytomegalovirus RNA.
An unexpected discovery was that the DNA machine in some cases coped with the detection of not only single-stranded RNA, but also double—stranded DNA - and all this at room temperature. Thanks to this result, the article got into the top 10 urgent publications of this year.
Scientists plan to focus on detecting copies of DNA to explain this phenomenon, as well as increase the selectivity of the DNA machine to search for a specific nucleic acid in a solution of several others. According to the authors of the project, in the future the proposed method will be "packaged" in a compact device that can be used outside the laboratory.
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