As part of ongoing efforts to try to reduce the spread of COVID-19, researchers around the world are working hard to develop novel diagnostic technologies. Rapid and simple identification of people infected with SARS-CoV-2 plays a crucial role in reducing transmission of the virus, by enabling appropriate isolation measures and contact tracing to take place.The lab of Dr Changchun Liu at the University of Connecticut Health Center has recently reported progress in this area, with the development of a simple, low-cost, CRISPR-based method which can detect infectious diseases such as COVID-19.1 Technology Networks spoke to Dr Liu to learn more about the method, the advantages it offers over PCR-based methods of detection, and the next steps to be taken before a point-of-care diagnostic test using this method could become widely available.Anna MacDonald (AM): Many of the current diagnostic tests for infectious diseases such as COVID-19 are PCR-based. Why is this method currently the gold standard and what are some of the limitations associated with it?

Changchun Liu (CL): PCR/RT-PCR, particularly real-time PCR/RT-PCR, provides a highly sensitive and specific method for detection of infectious diseases (e.g., COVID-19). In addition, PCR/RT-PCR-based methods can enable early diagnosis of COVID-19 because it detects nucleic acid (e.g., RNA) of novel coronavirus SARS-CoV-2. PCR/RT-PCR-based methods are typically restricted in a centralized clinical laboratory due to the requirement for sophisticated equipment and well-trained personnel, which are not suitable for simple, rapid, point of care diagnostic applications. AM: What advantages could CRISPR-based detectionoffer? What is limiting greater use of these methods for the detection of infectious diseases so far? CL: CRISPR-based nucleic acid detection provides a highly sensitive, specific and reliable testing approach for nucleic acid-based molecular diagnostics. Unlike PCR-based methods, CRISPR-based nucleic acid detection methods can work at a constant temperature (e.g. 37oC) without the need for an expensive thermal cycler (e.g. PCR machine). However, currently available CRISPR-based nucleic acid detection methods typically require: i) separate nucleic acid amplification in different reaction systems, and ii) multiple manual operations, which undoubtedly complicates the testing procedures and potentially increases the risk of carry-over contaminations due to amplification products transferring.

AM: Can you tell us more about the CRISPR-based method you have developed and give us an overview of your studys results? CL: We developed an All-In-One Dual CRISPR-Cas12a (termed "AIOD-CRISPR") assay method for rapid, ultrasensitive, specific and visual detection of nucleic acid. To improve detection sensitivity, we have proposed a dual CRISPR-Cas12a detection strategy. In addition, unlike previous CRISPR-based nucleic acid detections, all reagents for nucleic acid detection in our AIOD-CRISPR assay can be incubated in one-pot, enabling simple, rapid, sensitive and specific nucleic acid detection. Our AIOD-CRISPR assay method has successfully been utilized to detect nucleic acids (DNA and RNA) of the SARS-CoV-2 and HIV with a sensitivity of a few copies. Also, it was evaluated by detecting HIV-1 RNA extracted from human plasma samples, achieving a comparable sensitivity with real-time RT-PCR, but within a shorter time (less than 20 minutes). AM: How does AIOD-CRISPR compare to other CRISPR-based detection methods?

CL: As mentioned above, we developed a dual CRISPR-Cas12a detection strategy to improve detection sensitivity in our AIOD-CRISPR assay. In addition, unlike previously reported CRISPR-based nucleic acid detections, all reagents of our AIOD-CRISPR assay can be incubated in one-pot, eliminating need for multiple manual operations and enabling simple, rapid point of care diagnostics. In summary, our AIOD-CRISPR assay provides a simple, rapid (typically 5-20 minutes), ultrasensitive (few copies) and highly specific method for nucleic acid-based molecular diagnostics at the point-of-care. AM: Why is a one-pot reaction system so important? CL: As mentioned above, previously reported CRISPR-based nucleic acid detections typically require separate nucleic acid amplification and multiple manual operations, which undoubtedly complicates the testing procedures and is not ideal for point of care diagnostics. In our AIOD-CRISPR assay, all components for isothermal amplification and CRISPR-based detection are prepared in a one-pot format, which greatly simplifies the detection procedures and eliminates the risk of carry-over contaminations. Thus, our AIOD-CRISPR assay method has a great potential for developing next-generation point-of-care molecular diagnostics. AM: What are the next steps before a point-of-care diagnostic test using this method could become widely available? CL: We are integrating our AIOD-CRISPR assay into our microfluidic diagnostic chip to develop a simple, rapid, affordable, point-of-care diagnostic platform for SARS-CoV-2 detection at home or small clinics. We have long focused on developing simple, low-cost, point-of-care diagnostic technologies for rapid detection of infectious diseases. For instance, during the 2015-2016 Zika outbreak, we developed an instrument-free point-of-care molecular diagnostic technology for Zika virus detection.2 AM: What difference could a rapid, affordable, point-of-care test such as the one you are developing make to the global response to the COVID-19 pandemic?

CL: Rapid and early detection of the SARS-CoV-2 virus plays a crucial role in facilitating early intervention and treatment (e.g. home isolation, social distancing) and preventing COVID-19 disease spread. We envision that such a simple, rapid, affordable, and point-of-care diagnostics technology can be widely used for detection of the SARS-CoV-2 at home or in small clinics, preventing or slowing the rapid spread of COVID-19. AM: Your method was used to detect SARs-CoV-2 and HIV could it be easily adapted in the future for other possible infectious disease outbreaks? CL: Yes, as a platform technology, our AIOD-CRISPR assay method can be easily adapted to detect other infectious disease in the future.

Changchun Liu, Associate Professor, Department of Biomedical Engineering, University of Connecticut Health Center, was speaking to Anna MacDonald, Science Writer, Technology Networks. References:

1: Ding, X., Yin, K., Li, Z., & Liu, C. (2020). All-in-One Dual CRISPR-Cas12a (AIOD-CRISPR) Assay: A Case for Rapid, Ultrasensitive and Visual Detection of Novel Coronavirus SARS-CoV-2 and HIV virus. https://doi.org/10.1101/2020.03.19.998724

2: Song, J., Mauk, M. G., Hackett, B. A., Cherry, S., Bau, H. H., & Liu, C. (2016). Instrument-Free Point-of-Care Molecular Detection of Zika Virus. Analytical Chemistry, 88(14), 72897294. https://doi.org/10.1021/acs.analchem.6b01632

Read more here:
A Point-of-Care CRISPR-based COVID-19 Diagnostic on the Horizon - Technology Networks

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