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.

Figure 1

CHB probe design and CHB-LAMP assay. (A) Structure design of the CHB probe. (B) The CHB probe sequence to amplify B. burgdorferi recA gene by the CHB-LAMP and its minimum free energy (MFE) analysis using the software NUPACK (Caltech) with concentrations of 0.8 μM CHB, 70 mM Na⁺ and 6 mM Mg²⁺. (C) Schematic of the target sequence recognition and enhanced fluoresce detection of the CHB probe. (D) Principle of the CHB-LAMP assay. In the assay, LAMP’s loop backward (LB) primer was used to design the CHB probe.

Figure 2

Real-time fluorescence quantitative detection of B. burgdorferi DNA by (A) CHB-LAMP and (B) MB-LAMP assays. Left, the real-time fluorescence curves; right, the linear relationship between the threshold time and the log₁₀ of template’s copy number. The plasmids (recA gene sequence included) with the copy number (cps) ranging from 10⁶ to 10¹ were used as the templates. NTC non-template control. Each error bar represents the standard deviation for three replicates.

Figure 3

Endpoint fluorescence detection of the EvaGreen-based LAMP, CHB-LAMP with RNase H2, CHB-LAMP without RNase H2, and MB-LAMP after 60-min amplification of various copies of plasmid DNA targets. (A, B) The fluorescence image and fluorescence intensity (FI) comparison of different methods. Each error bar represents the standard deviation for six replicates. (C, D) The fluorescence image and FI comparison of the CHB-LAMP and CBP-LAMP assay. Each error bar represents the standard deviation for four replicates. Positive, the CHB-LAMP or CBP-LAMP reaction with 10⁵ copies of plasmids (300-bp recA gene inserted). NTC non-template control. The images were captured by using the Bio-Rad Gel Imaging System. The FI was calculated using the Image J software. The delta FI was defined as the FI change between positive and the NTC (Delta FI = FI_Positive–FI_NTC).

Figure 4

Smartphone-based fluorescence readout of LAMP products of different methods. (A) Image of the LAMP products taken by smartphone. (B) Screenshot of the app interface for imaging/parameter setting. (C) Screenshot of the app interface for fluorescence quantitative readout. Well #1–#5, the reactions with 10⁵ to 10¹ copies of the plasmid templates. Well #6, non-template control (NTC).

Figure 5

Detection of B. burgdorferi DNA extracted from ten tick samples by both the CHB-LAMP and PCR methods. (A) Real-time fluorescence CHB-LAMP assay. (B) Endpoint CHB-LAMP detection in a 96-well plate. Images were taken on the Bio-Rad Gel Imaging System. (C) EvaGreen-based real-time PCR assay. (D) Melting curves of the PCR products from (C). PC positive control with 10⁶ copies plasmid templates, NTC non-template control.

Figure 6

CHB-IMSA assay of the tenfold serial dilution of plasmid bearing EV71 VP1 gene. (A) Real-time fluorescence curves of the CHB-IMSA assay to detect the plasmids (300-bp VP1 gene included) with the copy number (cps) ranging from 10⁷ to 10¹ copies per reaction. (B) The linear relationship between the threshold time and the log₁₀ of DNA copy number. NTC non-template control. Each error bar represents the standard deviation for three replicates.