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

• Published in: Biosensors & bioelectronics Vol. 259; p. 116408
• Main Authors: Wei, Yangdao, Hu, Yuanzhao, Wang, Luchao, Liu, Chunsheng, Abdullaewich, Yuldoshov Sherzod, Yang, Zhiqing, Mao, Haimei, Wan, Yi
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 01-09-2024
• Subjects: Animals; Biosensing Techniques - methods; CRISPR-Cas Systems; CRISPR-Cas14a1; DNA, Bacterial - analysis; DNA, Bacterial - genetics; DNA, Bacterial - isolation & purification; DNA, Single-Stranded - chemistry; Humans; Limit of Detection; Milk - microbiology; Nucleic Acid Amplification Techniques - methods; Salmonella typhi; Salmonella typhi - genetics; Salmonella typhi - isolation & purification; Single primer amplification; Typhoid Fever - diagnosis; Typhoid Fever - microbiology; Salmonella typhi; Single primer amplification; CRISPR-Cas14a1
• ISSN: 0956-5663; 1873-4235; 1873-4235
• DOI: 10.1016/j.bios.2024.116408

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.