Cleavable hairpin beacon-enhanced fluorescence detection of nucleic acid isothermal amplification and smartphone-based readout

Abstract

Fluorescence detection of nucleic acid isothermal amplification utilizing energy-transfer-tagged oligonucleotide probes provides a highly sensitive and specific method for pathogen detection. However, currently available probes suffer from relatively weak fluorescence signals and are not suitable for simple, affordable smartphone-based detection at the point of care. Here, we present a cleavable hairpin beacon (CHB)-enhanced fluorescence detection for isothermal amplification assay. The CHB probe is a single fluorophore-tagged hairpin oligonucleotide with five continuous ribonucleotides which can be cleaved by the ribonuclease to specifically initiate DNA amplification and generate strong fluorescence signals. By coupling with loop-mediated isothermal amplification (LAMP), the CHB probe could detect Borrelia burgdorferi (B. burgdorferi) recA gene with a sensitivity of 100 copies within 25 min and generated stronger specific fluorescence signals which were easily read and analysed by our programmed smartphone. Also, this CHB-enhanced LAMP (CHB-LAMP) assay was successfully demonstrated to detect B. burgdorferi DNA extracted from tick species, showing comparable results to real-time PCR assay. In addition, our CHB probe was compatible with other isothermal amplifications, such as isothermal multiple-self-matching-initiated amplification (IMSA). Therefore, CHB-enhanced fluorescence detection is anticipated to facilitate the development of simple, sensitive smartphone-based point-of-care pathogen diagnostics in resource-limited settings.

Recent trends in smartphone‐based optical imaging biosensors for genetic testing: A review

“…In contrast to traditional genetic testing devices that rely on expensive and complex equipment requiring technical expertise, 68–71 smartphones with the advantages of numerous different sensors, high‐resolution cameras, small size, data processing ability, and connectivity have become dominant in resource‐limited settings and found their way into the field of POCT 41,45,47,52,72 . Various smartphone‐based optical imaging detection platforms have been applied in many genetic testing scenarios 46,73–77 . According to the different principles of genetic testing applications, the major applications of smartphone‐based optical imaging biosensors can be divided into the following three types: infectious disease, hereditary disease, and cancer.…”

“…41,45,47,52,72 Various smartphone-based optical imaging detection platforms have been applied in many genetic testing scenarios. 46,[73][74][75][76][77] According to the different principles of genetic testing applications, the major applications of smartphone-based optical imaging biosensors can be divided into the follow-ing three types: infectious disease, hereditary disease, and cancer.…”

Principles and Recent Advances in Biosensors for Pathogens Detection

“…Nevertheless, this approach is expensive and necessitates a prolonged time of analysis. Nowadays, this technique is extensively employed for recognizing S. epidermidis, [146] S. aureus, [146] Borrelia burgdorferi recA, [147] Salmonella typhimurium, human enteroviruses, papilloma viruses, hepatitis viruses, Salmonella species, M. tuberculosis, Karlodinium armiger, Karlodinium veneficum, and Staphylococcus aureus. [145]…”

DNA compaction enhances the sensitivity of fluorescence-based nucleic acid assays: a game changer in point of care sensors?

“…By integrating LAMP, Ding et al presented a cleavable, energy-transfer-tagged probe for rapid, sensitive, and accurate nucleic acid detection. 18 The developed Cleavable Hairpin Beacon (CHB) probe is a single fluorophore-tagged oligo comprising 5 consecutive ribonucleotides that are cleaved by ribonuclease to begin DNA amplification and generate enhanced fluorescence signals. 18 A ribonuclease-dependent cleavable beacon primer (CBP) enabling the fluorescence LAMP recognition of single nucleotide mutation was earlier developed by Ding et al 18 The aforementioned approaches are expensive, tedious, labour intensive and demand long assay time, and they are also prone to failures due to non-specific oligo preparations, primer dimer formation, stability issues, etc .…”