The accurate direct detection of dengue virus and Zika virus in samples from patients without any need for preparations or laboratory equipment has been made possible by a combination of Sherlock and Hudson – yes, this time Watson has been left out.
Developed at the Broad Institute of MIT and Harvard in the US, the diagnostic platform SHERLOCK (short for Specific High Sensitivity Reporter unLOCKing) detects nucleic acids (RNA and DNA) in various types of samples in a highly specific manner via an enzymatic reaction that can be performed in a test tube or on a strip of paper. A laboratory is not required.
To achieve this goal, the scientists adapted an enzyme called CRISPR-Cas13, which recognizes nucleic acids, by adding reporter molecules that indicate the presence of a genetic target such as a virus.
Until now, to process samples from patients on this platform, it was necessary to extract and isolate the nucleic acids they contained, requiring laboratory infrastructure and trained personnel. This requirement hindered its use in the field.
To facilitate the process and lower its cost, a team led by Pardis Sabeti at the Broad Institute created HUDSON (Heating Unextracted Diagnostic Samples to Obliterate Nucleases), which consists of a rapid chemical and heat treatment used on samples to inactivate certain enzymes that would otherwise degrade the genetic targets.
This new method enables the enzyme to detect its target directly in body fluids such as saliva, urine and blood. The processed clinical samples are then run through SHERLOCK, and the final positive or negative result is visible to the naked eye on a paper readout.
“Rapid and sensitive tools are critical for diagnosing, surveilling and characterizing an infection,” Sabeti said in a press release from the Broad Institute. “This system is putting us even closer to a fast and user-friendly diagnostic that can be easily deployed anywhere.”
The new system was validated using samples provided by Brazilian patients as part of a project supported by FAPESP and led by Maurício Lacerda Nogueira, a professor at the São José do Rio Preto Medical School (FAMERP) in São Paulo State.
“We’ve done epidemiological studies focusing on dengue for 15 years now, and more recently also on Zika. This has enabled us to build up a very large collection of well-characterized samples,” Nogueira told Agência FAPESP.
The samples used in the study were collected in 2015 and 2016, a period when both Zika and dengue were circulating abundantly in the São José do Rio Preto region. SHERLOCK proved capable of processing the samples and presenting results in under two hours.
“We selected a set of samples that represented a challenge to any novel method,” Nogueira said. “Dengue and Zika are very similar viruses, and test results often suffer from cross-reactivity. SHERLOCK diagnosed them with a 100% success rate even for mixed samples, meaning positive for more than one virus.”
He added that he has been collaborating with researchers at MIT for over ten years in search of faster and cheaper diagnostic technologies that can be used in the field for the real-time surveillance of epidemics.
“One of the advantages of this type of technology is the ease with which the test can be adapted to the needs of the moment. In the case of an epidemic involving a new virus, a kit with reagents can be developed quickly and taken to the area. However, we’re still a few years away from commercial applications of the method,” Nogueira said.
The article by Sabeti’s group shows that adapted tests can be rapidly developed to allow the use of SHERLOCK to distinguish the four dengue virus serotypes and region-specific strains of Zika virus from the 2015-16 pandemic.
The researchers also developed assays capable of identifying clinically relevant genetic variants (polymorphisms) of the viruses. In the case of Zika, the platform detected patient samples with a mutation in a viral protein called prM, which could contribute to the development of fetal microcephaly, according to research published in Science in 2017.
Evolution of PCR
As the authors note, existing methods of viral nucleic acid detection in samples, such as real-time polymerase chain reaction (PCR), are very sensitive and rapidly adaptable, but most require extensive sample manipulation and expensive machinery.
Antigen-based rapid diagnostic tests require minimal equipment but have lower sensitivity and specificity.
“An ideal diagnostic would combine the sensitivity, specificity and flexibility of nucleic acid diagnostics with the speed and ease-of-use of antigen-based tests. Such a diagnostic could be rapidly developed and deployed in the face of emerging viral outbreaks and would be suitable for disease surveillance or routine clinical use in any context”, the authors say in the article.
Two other articles on the same topic were published in the April 26 issue of Science. One of them describes the detection of human papilloma virus (HPV) in patient samples using CRISPR-based technology.
Source : By Karina Toledo | Agência FAPESP