Development and characterization of a digital CRISPR/Cas13a based assay for rapid and sensitive diagnosis of Severe Fever with Thrombocytopenia Syndrome Virus

Following the first reports of Severe fever with thrombocytopenia syndrome virus (SFTSV) during 2009 in Central China [1], there have been outbreaks reported in over 20 Chinese provinces in subsequent years [2], as well as SFTSV cases reported in Japan [3] and Korea [4]. The early, non-specific clinical symptoms of Severe fever with thrombocytopenia syndrome (SFTS) can be clinically misdiagnosed and confused with other tick-borne viral diseases, such as anaplasmosis or scrub typhus [5]. This can result in a delayed admission of potentially severe SFTS cases. Due to rapid clinical progression to multiple organ failure within 8 days [6], [7] as well as a lack of approved medical countermeasures including vaccines and therapeutics [8], the case fatality rate of SFTS patients ranges from 6 – 30% [9].

Effective methods of SFTSV detection are urgently needed for specifically identifying infections at the earliest opportunity. Current epidemiological surveillance and clinical diagnostic methods for SFTSV infections are mainly based on serological screening for anti-SFTSV total antibodies by enzyme-linked immunosorbent assay (ELISA) [10] or a commercially nucleic acid testing (comNAT) assay based on one-step reverse transcription polymerase chain reaction (RT-PCR), which is approved for SFTSV testing in China [11]. Since the viral load in the blood of early SFTS patients may not reach the lower limit of quantitative detection (LOQD) of 10 TCID50/mL, or a cut-off Ct value of 35 cycles, as stipulated by the comNAT assay, it is estimated that over 30% of patients with suspected SFTS could not be confirmed by current laboratory and clinical diagnostic methods [12]. In addition, SFTSV infection with ticks carrying the virus primarily occurs in rural areas, which are typically medically under-resourced [13]. There is a need to establish a highly sensitive and rapid detection method for SFTSV suitable for point-of-care testing (POCT) in these regions.

In recent years, the discovery of the bacterial clustered regularly interspaced short palindromic repeats (CRISPR) and associated endonuclease (CRISPR/Cas) immune system has provided opportunities for diagnostics with isothermal preamplification methods [13]. These diagnostic assays can be performed without the need for sophisticated laboratory equipment, such as PCR thermocyclers. For CRISPR-based detection, Cas13a or Cas12a possess unique collateral trans-cleavage mechanisms that can be activated by recognition with the specific target RNA or DNA sequence, respectively, to efficiently cleave nearby reporter probes and produce a detection signal [14]. The CRISPR/Cas detection system is often coupled with nucleic acid amplification and is therefore usually divided into two separate steps. After a separate isothermal preamplification step, such as reverse-transcription recombinase polymerase amplification (RT-RPA), recombinase polymerase amplification (RPA), and Loop-mediated isothermal amplification (LAMP), Cas13a-based SHERLOCK (Specific High-sensitivity Enzymatic Reporter unlocking) [15] and Cas12a-based DETECTR (DNA endonuclease-targeted CRISPR trans reporter) assays [16] enables highly sensitive detection of targets with a limit of detection (LOD) at the level of ~103 genome copies per μL. The two-step CRISPR/Cas detection system primarily uses real-time fluorescence, end-point fluorescence, or strip tests to visualize a diagnostic result, which does not provide a quantitative measurement of viral genome samples. This can be resolved by combining the nucleic acid preamplification step and CRISPR/Cas detection into a one-pot method, in order to more accurately quantify the amount of the original viral genome.

Recently, the SHINE (Streamlined Highlighting of Infections to Navigate Epidemics) assay has been developed to detect SARS-CoV-2 RNA using a paper-based colorimetric readout or an in-tube fluorescent readout, combining RT-RPA based amplification and Cas13-based detection in a single step [17]. However, this assay introduces RT-RPA, T7 RNA polymerase transcription, and the CRISPR/Cas13a detection systems into one tube, which in the past has led to repeatability issues in sensitivity testing, due to macromolecular crowding and enzymatic incompatibility [18]. Therefore, for the CRISPR/Cas13a system, this can be resolved by isolating the RT step and combining the remaining three steps of RPA, T7 RNA polymerase transcription, and the CRISPR/Cas13a detection systems into a one-pot method, in order to ensure the stability of the quantitative reaction system. Compared with conventional tube-based (bulk) assays, the digital based CRISPR/Cas assay can evenly distribute the reaction system with the sample into nanoliter-sized droplets, thus leading to higher sensitivity and improved repeatability [19]. In addition to viral genome detection, the digital based CRISPR/Cas assay enables the absolute quantification of the target nucleic acid in an isothermal environment (37 °C).

Using this information, we developed a digital CRISPR/Cas13a based assay to detect and quantitate SFTSV nucleic acids. First, we developed a two-step CRISPR/Cas13a based assay as a platform for optimization and compared the sensitivity to previously published work. On this base platform, we then developed and tested an optimized one-pot bulk assay to achieve sensitive and robust quantification of the target nucleic acids, by directly reverse transcribing SFTSV RNA into cDNA as a template, and combining into one step the RPA, T7 RNA polymerase transcription and CRISPR/Cas13a detection systems. To optimize the bulk reaction system, we systematically investigated the effects of different components in this assay, such as reaction buffer, pH, enzymes, and other additives. We then used real-time fluorescence monitoring, end-point colorimetric strips and end-point fluorescence readout to test the sensitivity, specificity, and LOD of this one-pot bulk reaction system for SFTSV detection. Afterwards, we converted the bulk reaction system into a digital droplet reaction system, and showed that this assay achieved qualitative results within 10 minutes and quantitative detection within 30 minutes. Last, we used this system to quantitate the amount of viral RNA in simulated SFTSV samples, in which the digital CRISPR/Cas13a based assay demonstrated consistent results with those from cDNA results by quantitative PCR (qPCR).

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