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Abstract
<p xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="first" dir="auto"
id="d7196866e117">Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated
(Cas) proteins are an innovative tool in molecular diagnostics owing to their high
specificity and modularity for target nucleic acid sequences. However, the sequence-indiscriminate
trans-cleavage activity of the Cas protein renders multiplex detection challenging.
In this study, we developed a Cas12a-based multiplex detection system by designing
blocker DNA complementary to reporter DNA, which enables the simultaneous detection
of two genes with a single Cas protein in a single reaction. As a proof of concept,
we chose high-risk human papillomavirus (HPV) 16 and 18 as the model targets and incorporated
recombinase polymerase amplification (RPA) and transcription reactions to achieve
high accuracy and sensitivity. Using the proposed system, we detected the genes of
both HPV 16 and 18 down to 1 aM within 80 min under isothermal conditions. We validated
the performance of the system in detecting genomic DNA from various cell lines and
clinical samples from cervical cancer patients with high specificity. The proposed
system facilitated rapid multiplex detection of high-risk HPVs in a single reaction
tube with only Cas12a, thus representing a more user-friendly and economical alternative
to previous Cas protein-based multiplex detection assays. The proposed system has
considerable potential for point-of-care testing and could be expanded to detect various
nucleic acid biomarkers.
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