Now, in a new study, researchers from Weiss Bioinspired Engineering Institute of Harvard University, Massachusetts Institute of Technology and several hospitals in Boston have developed a cheap diagnostic test based on CRISPR, which allows users to test novel coronavirus and its various virus variants at home without additional instruments. The related research results were published in the journal ScienceAdvances on August 6th, 200212002, and the title of the paper was "Minimal Instrumental Sherlock Diagnosis of bedside novel coronavirus and mergingvariables Based on Crispr".
This diagnostic device, called MinimallyInstrumentedSHERLOCK, is easy to use and provides results that can be read and verified by supported smartphone applications within one hour. It successfully distinguished three different novel coronavirus variants in the experiment, and can be quickly reconfigured to detect more variants, such as Delta variants. The equipment can be assembled with three-dimensional printers and common parts, and the price is about 15 USD. Reusing these components can reduce the cost of a single test to $6.
HelenadePuig, the first author of the paper and a postdoctoral researcher at Harvard University's Weiss Institute of Bioinspired Engineering and Massachusetts Institute of Technology, said, "miSHERLOCK eliminates the need to transport patient samples to centralized testing sites, greatly simplifying the sample preparation steps, so that patients and doctors can know the health status of individuals and communities more quickly and accurately, which is very important in the growing epidemic. "
From supply chain to Sherlock
As pediatricians specializing in infectious diseases in Boston Children's Hospital, the paper * * * and the first author, Dr. RoseLee, have been working in the front line of coronavirus pneumonia-19 epidemic for more than a year. Her experience in the clinic provided inspiration for the final development of the Mi Sherlock project.
Lee said, "Some simple things that used to be seen everywhere in hospitals, such as nasopharyngeal swabs, suddenly became difficult to obtain, so the routine sample processing procedures were disrupted, which was a big problem in the context of the epidemic. The motivation of our team to carry out this project is to eliminate these bottlenecks, provide accurate diagnosis methods for coronavirus pneumonia-19 without much dependence on the global supply chain, and also accurately detect emerging virus variants. "
For the novel coronavirus detection part of their diagnostic equipment, these authors chose the technology based on CRISPR, which was built by Dr. JimCollins, the author of the paper and the core teacher of the Weiss Institute of Bioinspired Engineering at Harvard University, and named Shylock. Shylock uses CRISPR's "molecular scissors" to cut DNA or RNA at specific sites, which has an additional benefit: this specific type of molecular scissors will also cut other DNA fragments in the surrounding area, enabling it to cut single-stranded nucleic acid probe molecules carrying fluorescent signals, thus generating fluorescent signals, indicating that the target has been successfully cut.
The diagnostic program only requires the user to spit saliva into the sample preparation room, then transfer the collection tray to the reaction room and press the plunger, thus activating the reaction and minimizing the risk of cross-contamination. Image from ScienceAdvances, 202 1, doi:10.1kloc-0/26/sciadv.abh2944.
These authors created a Shylock test to cut a specific region of novel coronavirus nucleoprotein coding gene, which is conserved in many variants of the virus. When this molecular scissor named Cas 12a successfully binds and cleaves NP gene, the nearby single-stranded DNA probe is also cleaved, thus generating a fluorescent signal. They also created an additional Shylock test to target a group of virus mutations in the spike protein sequence, which represent three novel coronavirus virus variants: α, β and γ.
Through a detection method that can reliably detect viral RNA within the acceptable concentration range of diagnostic tests authorized by the US Food and Drug Administration, these authors then focused on solving the most difficult challenge in diagnosis: sample preparation.
Spit, wait, scan
The paper and the first author, Dr. Tan Xiao, a clinical researcher at Harvard University's Weiss Institute of Bioinspired Engineering, said, "When you test nucleic acids in a sample, you need to do many steps to prepare the sample so that you can really extract and amplify these nucleic acids. When transporting the sample to the testing institution, you must protect the sample. If you are dealing with infectious diseases, please ensure that it is not contagious. In order to make it a truly easy-to-use diagnostic test, we must simplify it as much as possible. "
These authors choose to use saliva instead of nasopharyngeal swab samples as their collection method, because it is easier for users to collect saliva, and research shows that novel coronavirus can be detected in saliva for more days after infection. However, untreated saliva also brings its own challenges: it contains enzymes that degrade various molecules, resulting in a high false positive rate.
These authors have developed a new technology to solve this problem. First, they added two chemicals DTT and EGTA to saliva and heated the sample to 95 C3 3 minutes, which eliminated the false positive signal in untreated saliva and cracked any virus particles. Then they added a porous membrane to capture RNA on its surface, and finally they could directly add it to Shylock reaction to produce test results.
In order to integrate saliva sample preparation and Shylock reaction into the diagnostic device, these authors designed a simple battery-powered device with two chambers: a heated sample preparation chamber and an unheated reaction chamber. Users spit into the sample preparation room, heat it, wait for three to six minutes, and saliva will enter the filter. The user takes out the filter and transfers it to the column of the reaction chamber, then presses the plunger, puts the filter into the reaction chamber and pierces the reservoir to activate the Shylock reaction. After 55 minutes, the user observed the reaction chamber through the colored transparent window to confirm whether there was a fluorescent signal. They can also use a matching smartphone application to analyze the pixels recorded by the smartphone camera, thus providing a clear positive or negative diagnosis result.
These authors tested their diagnostic equipment with clinical saliva samples from 27 patients with coronavirus pneumonia-19 and 2 1 healthy patients, and found that miSHERLOCK can correctly identify patients with coronavirus pneumonia-19 and patients without disease 95% of the time. They also tested the performance of this diagnostic device in detecting α, β and γ-CoV-2 variants of SARS-CoV-2 by adding full-length synthetic viral RNA containing mutations representing each virus variant to saliva of healthy people, and found that the device was effective at various viral RNA concentrations.
Together with DevoraNajjar, the first author and research assistant of Collins Lab, the paper said, "One advantage of miSHERLOCK is that it is completely modular. The device itself is separate from the detection method, so you can integrate different detection methods to detect the specific RNA or DNA sequence you want to detect. The cost of this equipment is about 15 US dollars, but it will be reduced to about 3 US dollars after mass production. The detection method of the new target can be established in about two weeks, so that a new variant detection method for diseases such as coronavirus pneumonia-19 can be quickly developed. "
Prepare for practical application
These authors considered the low-resource environment when building their diagnostic equipment, because the epidemic situation made people see the great inequality of medical services between different parts of the world. The components of the equipment can be made by anyone who can use a three-dimensional printer, and relevant data files and circuit designs can be publicly obtained on the Internet. Adding smart phone applications is also aimed at the environment with limited resources, because mobile phone services can be used almost anywhere in the world, even in areas that are difficult to reach on foot. They are eager to cooperate with manufacturers who are interested in mass production of miSHERLOCK for global distribution.
Collins said, "When the miSHERLOCK project started, there was almost no monitoring of the novel coronavirus variety. We know that it will be very important to track this virus variant when evaluating the long-term impact of coronavirus pneumonia-19 on local and global communities, so we push ourselves to establish a truly decentralized, flexible and user-friendly diagnosis platform. By solving the problem of sample preparation, we have ensured that this equipment can actually be used by consumers, and we are very happy to cooperate with industrial partners to commercialize it. "
"By combining cutting-edge biotechnology with low-cost materials, this research team has built a powerful diagnostic device that can be manufactured and used locally by people without advanced medical degrees," said Dr. Doningbo, founding director of the Weiss Institute of Bioinspired Engineering at Harvard University. This is a perfect example of the mission of the Weiss Institute of Bioengineering at Harvard University: to give life-changing innovations to those who need them. "
References:
Puyg Tal, Leonard. Diagnosis of SARS-CoV-2 at the point of care based on CRISPR and minimal instrumental Sherlock describing the variant. Scientific progress, 202 1, doi:10.1126/sciadv.abh2944.