Simultaneous and rapid colorimetric detection of distinct miRNAs using Split-LAMP

Abstract

Introduction: Aberrant microRNA (miRNA) expressions are often discovered in many life threatening diseases such as cancer. In particular, recent studies show combinations of miRNA levels have greater diagnostic accuracy as opposed to single miRNA levels. For point-of-care applications, rapid and sensitive isothermal amplification with loop-mediated isothermal amplification (LAMP) has gained significant interest. Method: We developed a cost-effective point-of-care testing (POCT) device for multiple miRNAs that can integrate miRNA signals into a single output. Results and Discussion: We demonstrate that the loop primers for LAMP can be broken and be used for miRNA detection. This split-LAMP approach provides a logic AND-gate output for two distinct miRNA inputs. We then show that this is potentially useable in point-of-care testing using pH-sensitive dye to give a rapid, colorimetric endpoint readout within 30 min. This novel logic gate approach can potentially be extended to multiple miRNAs such that there can be a powerful diagnostic concept for multiple short RNAs in a point-of-care rapid test.

Keywords: LAMP; POCT; multiplex miRNA; rapid colorimetric detection; split-LAMP.

FIGURE 1

Proof of Split-LAMP concept. (A) Illustration of Split-LAMP design versus Traditional-LAMP design. (B) Replacement of BIP with B2 and B1c. (C–F) Decreasing concentrations of B2, F2, B1c and B2/F2 respectively. Curves shows fluorescence (Rn) measured over time (minutes). Reactions were performed without replicates.

FIGURE 2

Evaluating the effect of different LB and LF concentrations on miRNA detection. Concentration of LB primer is used as (A) 0.1µM, (B) 0.01 µM and (C) 0 µM. Concentration of LF primer is used as (D) 0.1µM, (E) 0.01 µM and (F) 0 µM. Curves show fluorescence (Rn) measured over time (minutes). All reactions were performed in triplicates.

FIGURE 3

Quantification of target DNAs. (A) Decreasing concentrations of F2. (B) Decreasing concentrations of B2. Curves show the normalized fluorescence over time (minutes). (C) Table showing changes in TA values (ΔTA) measured with different combinations of B2 and F2 concentrations used. The intensity of the colour in the box is a visual representation of the TA value (white = lowest TA; dark gray = highest TA). All reactions were conducted in triplicates.

FIGURE 4

Evaluating the effect of miR-21 and miR-34 concentrations on miRNA detection. Concentration of miR-21 is used as (A) 300 nM and concentration of miR-34 is used as (B) 300 nM and (C) 30 nM. Curves show fluorescence (Rn) measured over time (minutes). (D) Table shows change in TA values (ΔTA) measured with different combinations of miR-34 and miR-21 concentrations. The intensity of the colour in the box is a visual representation of the TA value (white = lowest TA; dark gray = highest TA). Additionally, different cutoffs will result in different combinations of the miRNAs that would result in a positive (white box) signal. Gray boxes represent a negative output. Reactions were performed without replicates.

FIGURE 5

Test for selectivity using (A) Commercial buffer and (B) pH-responsive buffer with reactions containing miR-34a and/or miR-21 or negative control. (C) Images taken at 0 and 30-min timepoint showing colour change of the reaction mixtures. Curves show fluorescence (Rn) measured over time (minutes). All reactions were conducted in duplicates.