Ultrasensitive detection of Salmonella typhi using a PAM-free Cas14a-based biosensor.

Publisher: Elsevier Advanced Technology Country of Publication: England NLM ID: 9001289 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1873-4235 (Electronic) Linking ISSN: 09565663 NLM ISO Abbreviation: Biosens Bioelectron Subsets: MEDLINE

Publication: Oxford : Elsevier Advanced Technology
Original Publication: [Barking, Essex, England] : Elsevier Applied Science, 1989-

Biosensing Techniques*/methods ; Salmonella typhi*/isolation & purification ; Salmonella typhi*/genetics ; CRISPR-Cas Systems* ; Milk*/microbiology; Animals ; Nucleic Acid Amplification Techniques/methods ; DNA, Single-Stranded/chemistry ; Limit of Detection ; Humans ; Typhoid Fever/diagnosis ; Typhoid Fever/microbiology ; DNA, Bacterial/genetics ; DNA, Bacterial/analysis ; DNA, Bacterial/isolation & purification

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.
Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
(Copyright © 2024 Elsevier B.V. All rights reserved.)

Keywords: CRISPR-Cas14a1; Salmonella typhi; Single primer amplification

0 (DNA, Single-Stranded)
0 (DNA, Bacterial)

Date Created: 20240523 Date Completed: 20240601 Latest Revision: 20240610

20240610

10.1016/j.bios.2024.116408

38781698