Development of a Rapid Point-of-Care Diagnostic Test for Zika Virus Infection Based on CRISPR-Cas12a Technology

Coralie Reimerink^*
*Correspondence: Coralie Reimerink, Department of Internal Medicine, University Medical Center Utrecht, 3584 CX Utrecht, The Netherlands, Email:

Keywords

Zika virus • Point-of-care diagnostics • CRISPR-Cas12a • Rapid detection • Infectious diseases

Introduction

Zika Virus (ZIKV) is an emerging pathogen that has caused several outbreaks worldwide, particularly in tropical and subtropical regions. The virus is primarily transmitted through Aedes mosquitoes and can lead to severe neurological complications, including microcephaly in newborns and Guillain-Barré syndrome in adults. Rapid and accurate diagnosis of ZIKV infection is crucial for managing outbreaks and preventing further transmission. Current diagnostic methods, such as RT-PCR, while highly accurate, require specialized laboratory equipment and trained personnel, limiting their accessibility in resource-poor areas. To address this gap, we have developed a point-of-care diagnostic test based on CRISPR-Cas12a technology, which combines the specificity of CRISPR-based nucleic acid detection with the simplicity and speed necessary for field deployment [1].

Literature Review

The diagnostic test we developed utilizes the CRISPR-Cas12a system, a powerful and precise molecular tool that can target and cleave specific sequences of the Zika virus (ZIKV) genome. The test design revolves around three core components: guide RNA (gRNA), the CRISPR-Cas12a enzyme, and a fluorescent reporter molecule. The gRNA is meticulously designed to bind exclusively to a conserved region of the ZIKV RNA, ensuring high specificity. Upon binding of the gRNA to the target ZIKV RNA, the CRISPR-Cas12a enzyme is activated. This activation triggers collateral cleavage of a fluorescent reporter, which is a short single-stranded DNA oligonucleotide tagged with both a fluorophore and a quencher. In its intact state, the reporter remains non-fluorescent due to the proximity of the quencher. However, once the Cas12a enzyme cleaves the reporter, the fluorophore is separated from the quencher, resulting in a detectable fluorescence signal [2].

The procedure for the test is straightforward and designed for ease of use in various settings. First, the patient sample is mixed with the CRISPR-Cas12a complex and the fluorescent reporter. This mixture is then incubated for a short period, typically between 30 to 60 minutes. During this time, if ZIKV RNA is present in the sample, the CRISPR-Cas12a enzyme will be activated, leading to cleavage of the reporter and emission of a fluorescent signal. The presence of this fluorescence indicates a positive test result, confirming ZIKV infection [3].

This design allows for direct detection of ZIKV RNA from patient samples without the need for extensive sample preparation or complex laboratory equipment. The simplicity and rapidity of the assay make it highly suitable for point-of-care settings, especially in resource-limited environments where traditional diagnostic infrastructure may be lacking. Furthermore, the high sensitivity of the test ensures that even low viral loads can be detected, providing reliable diagnostic capability during early stages of infection or in asymptomatic cases. The portability and minimal equipment requirements of this CRISPR-Cas12a-based diagnostic test thus represent a significant advancement in the field of infectious disease diagnostics, offering a practical and efficient solution for real-time ZIKV detection [4].

Discussion

The CRISPR-Cas12a-based diagnostic test offers several advantages over traditional methods. Its high specificity is ensured by the precise base-pairing between the gRNA and the target ZIKV RNA, reducing the likelihood of cross-reactivity with other viruses. The sensitivity of the assay, demonstrated to be on par with RT-PCR, allows for the detection of low viral loads. The rapid turnaround time, typically within 30-60 minutes, facilitates timely decision-making and intervention [5]. Furthermore, the minimal equipment requirement makes it suitable for use in field settings and resource-limited environments. Challenges to the implementation of this technology include ensuring the stability of the CRISPR-Cas12a components under varying environmental conditions and the need for user-friendly platforms to read the fluorescence signal. Future work should focus on optimizing the assay for robustness in diverse settings and integrating it into portable devices for broader application [6].

Conclusion

The development of a CRISPR-Cas12a-based point-of-care diagnostic test for Zika virus infection represents a significant advancement in the field of molecular diagnostics. This assay provides a rapid, sensitive, and specific method for detecting ZIKV, with the potential to greatly enhance the management of outbreaks, particularly in areas lacking advanced laboratory infrastructure. Continued development and field testing will be crucial in realizing the full potential of this technology in global health applications.

Acknowledgement

None.

Conflict of Interest

None.

References

1. Yalcin, Ahmet, Mustafa Ceylan, Omer Faruk Bayraktutan and Ali Rıza Sonkaya, et al. "Age and gender related prevalence of intracranial calcifications in CT imaging; Data from 12,000 healthy subjects." J Chem Neuroanat 78 (2016): 20-24.

Google Scholar, Crossref, Indexed at

2. Donzuso, Giulia, Giovanni Mostile, Alessandra Nicoletti and Mario Zappia. "Basal ganglia calcifications (Fahr’s syndrome): Related conditions and clinical features." Neurol Sci 40 (2019): 2251-2263.

Google Scholar, Crossref, Indexed at

3. Saleem, Shafaq, Hafiz Muhammad Aslam, Maheen Anwar and Shahzad Anwar, et al. "Fahr’s syndrome: Literature review of current evidence." Orphanet J Rare Dis 8 (2013): 1-9.

Google Scholar, Crossref, Indexed at

4. Carecchio, Miryam, Michele Mainardi and Giulia Bonato. "The clinical and genetic spectrum of primary familial brain calcification." J Neurol 270 (2023): 3270-3277.

Google Scholar, Crossref, Indexed at

5. Patel, Dushyant V., Ellen M. Holfels, Nicolas P. Vogel and Kenneth M. Boyer, et al. "Resolution of intracranial calcifications in infants with treated congenital toxoplasmosis." Radiol 199 (1996): 433-440.

Google Scholar, Crossref, Indexed at

6. Manyam, Bala V. "What is and what is not ‘Fahr's disease’." Parkinsonism Relat Disord 11 (2005): 73-80.

Google Scholar, Crossref, Indexed at