%0 Journal Article
%T Ultrasensitive detection of Salmonella typhi using a PAM-free Cas14a-based biosensor.
%A Wei Y
%A Hu Y
%A Wang L
%A Liu C
%A Abdullaewich YS
%A Yang Z
%A Mao H
%A Wan Y
%J Biosens Bioelectron
%V 259
%N 0
%D 2024 Sep 1
%M 38781698
%F 12.545
%R 10.1016/j.bios.2024.116408
%X The effectiveness of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas14a1, widely utilized for pathogenic microorganism detection, has been limited by the requirement of a protospacer adjacent motif (PAM) on the target DNA strands. To overcome this limitation, this study developed a Single Primer isothermal amplification integrated-Cas14a1 biosensor (SPCas) for detecting Salmonella typhi that does not rely on a PAM sequence. The SPCas biosensor utilizes a novel primer design featuring an RNA-DNA primer and a 3'-biotin-modified primer capable of binding to the same single-stranded DNA (ssDNA) in the presence of the target gene. The RNA-DNA primer undergoes amplification and is blocked at the biotin-modified end. Subsequently, strand replacement is initiated to generate ssDNA assisted by RNase H and Bst enzymes, which activate the trans-cleavage activity of Cas14a1 even in the absence of a PAM sequence. Leveraging both cyclic chain replacement reaction amplification and Cas14a1 trans-cleavage activity, the SPCas biosensor exhibits a remarkable diagnostic sensitivity of 5 CFU/mL. Additionally, in the assessment of 20 milk samples, the SPCas platform demonstrated 100% diagnostic accuracy, which is consistent with the gold standard qPCR. This platform introduces a novel approach for developing innovative CRISPR-Cas-dependent biosensors without a PAM sequence.