ABSTRACT

Nucleic acids-based molecular diagnostic tools incorporating the CRISPR/Cas system are being developed as rapid and sensitive methods for pathogen detection. However, most CRISPR/Cas-based diagnostics lack quantitative detection ability.

Here, we report Warm Start RApid DIgital Crispr Approach (WS-RADICA), which uses commercially available digital chips for the rapid, sensitive, and quantitative detection of nucleic acids. In this method, digital detection is realized by dividing a one-pot warm-start RT-LAMP and CRISPR/Cas12b-based detection assay into thousands of nanoliter or subnanoliter reaction wells, and the quantitative result is obtained by counting the ratio of fluorescence-positive wells to all reaction wells. The warmstart activity of RT-LAMP prevents amplification at room temperature before sample partitioning and enables accurate digital quantification. A proof of concept assay was developed by targeting the nucleocapsid (N) gene of SARS-CoV-2. This method allowed qualitative detection in 40 min and quantitative results in 60 min in a 60 ºC water bath and could detect as little as 1 copy/uL of SARS-CoV-2 RNA. Moreover, WS-RADICA can be easily adapted to various digital devices : two digital devices were evaluated for both DNA and RNA quantification, with linear dynamic ranges of 0.8-12777 copies/µL for DNA and 1.2-18391 copies/µL for RNA (both R2 values > 0.99). The performances were comparable for WS-RADICA, RT-qPCR, and RT-dPCR reactions. WSRADICA showed better sensitivity and inhibitor tolerance than the bulk RT-LAMP-Cas12b method. WS-RADICA demonstrates enhanced speed and sensitivity compared to other methods and tolerates inhibitors; therefore, it offers an attractive option for nucleic acid quantification.

Materials

The sequences of primers, crRNA, and FQ reporters in this study, listed in Supplementary   Table 1, were synthesised by Integrated DNA Technologies. Plasmids containing the Ngene from each virus genomes (SARS-CoV-2, SARS-CoV, and MERS-CoV) were purchased from Integrated DNA Technologies. The synthetic RNA covering 99.9% of the bases of the SARS-CoV-2 viral genome was purchased from Twist Bioscience (Genbank ID:MN908947.3). CRISPR -Cas12b was purchased form Magigen Biotechnology. The DNA and RNA concentrations were measured by dPCR or RT-dPCRassay.

Fig. 1. Schematic illustration of WS-RADICA.

(A) Overview of the WS-RADICA process.

Nucleic acids (DNA and RNA) are extracted from different types of samples, then mixed with RT-LAMP reagent and Cas12b/crRNA/FQ reporter mix. The reaction mixtures can be subdivided into thousands of partitions by digital chips, followed by incubation at 60ºC for 1 h. The partitions containing the target yield a much higher fluorescent signal than the partitions without targets, and the end-point results are detected by a fluorescent detector to calculate the proportions of positive partitions.

(B) Reactions in the positive partitions.

The DNA/RNA, RT-LAMP reagents, and Cas12b/crRNA/FQ reporter are mixed in a one-pot format in each partition. The target DNA/RNA can be amplified by RT-LAMP mixtures into looped structures. Because the amplified targets are complementary with crRNA, they bind to a Cas12b/crRNA complex, triggering the trans cleavage of Cas12b protein to cut the FQ reporters, which in turn results in a fluorescent signal.

(C) Schematic

concept of the bulk RT-LAMP-Cas12b assay.

Source file

A Warm-start Digital CRISPR-based Method for the Quantitative Detection of Nucleic Acids

Critical Analytics for Manufacturing Personalized Medicine Interdisciplinary Research Group, Singapore-MIT Alliance for Research and Technology, Singapore 138602, Singapore

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