Strand Displacement Probes Combined with Isothermal Nucleic Acid Amplification for Instrument-Free Detection from Complex Samples

Abstract

Sensitive and specific detection of pathogens via nucleic acid amplification is currently constrained to laboratory settings and portable equipment with costly fluorescent detectors. Nucleic acid-detecting lateral flow immunoassay strips (LFIAs) offer a low-cost visual transduction strategy at points of need. Unfortunately, these LFIAs frequently detect amplification byproducts that can yield spurious results which can only be deciphered through statistical analysis. We integrated customizable strand displacement probes into standard loop mediated isothermal amplification (LAMP) assays to prevent byproduct capture on commercial LFIAs. We find that combining strand displacement with LAMP (SD-LAMP) yields LFIA test band intensities that can be unequivocally interpreted by human subjects without additional instrumentation, thereby alleviating the need for a portable reader's analysis. Using SD-LAMP, we capture target amplicons on commercially available LFIAs from as few as 3.5 Vibrio cholerae and 2 750 Escherichia coli bacteria without false positive or false negative interpretation. Moreover, we demonstrate that LFIA capture of SD-LAMP products remain specific even in the presence of complex sample matrixes, providing a significant step toward reliable instrument-free pathogen detection outside of laboratories.

Figure 1

Schematic of standard LAMP and SD-LAMP reactions and their subsequent LFIA detection. (A) The noncyclic step of both reactions in which F3, B3, and inner primers bind to a double-stranded target sequence and polymerase generates dumbbell-shaped products. (B) Dumbbell-shaped products enter the cyclic amplification step during which loop primers accelerate amplification. Products can be labeled by either standard tagging of each loop primer or by SD-LAMP, which uses one labeled loop primer along with a tagged strand displacement probe that hybridizes to the amplicons’ loop region. (C) Labeled amplicons visualized on a standard LFIA strip.

Figure 2

Detection of standard LAMP and SD-LAMP reactions in pure water. Electrophoresis gels verifying amplification (top), LFIA test results (middle), and LFIA test line quantification (bottom). (A) Standard LAMP reaction products for E. coli labeled with primers (LF-FAM and LB-Biotin) yield LFIA results too faint for visual interpretation. (B) LAMP products labeled with reconfigured primers (LF-Biotin and LB-FAM) yield false positive LFIA results in low concentration samples and no template control (NTC) reactions. (C) Probed strand displacement LAMP reactions yield no false positive LFIA results for E. coli. n = 4, replicates indicated by each circle. **** indicates p ≤ 0.0001; ** indicates p ≤ 0.01.

Figure 3

A total of 98 percent of test strips with a background subtracted test intensity of 0.020 are interpreted as positive by unaided human subjects.

Figure 4

Detection of standard LAMP and SD-LAMP reactions in pure water. Electrophoresis gels verifying amplification (top), LFIA test results (middle), and LFIA test line quantification (bottom). (A) Standard LAMP reaction products for V. cholerae labeled with primers yield false positive LFIA results in low concentration samples and no template control (NTC) reactions. (B) SD-LAMP reactions yield no false positive LFIA results for V. cholerae. n = 4, replicates indicated by each circle. *** indicates p ≤ 0.001; ** indicates p ≤ 0.01; * indicates p ≤ 0.05.

Figure 5

Detection of SD-LAMP reactions in complex matrixes. Electrophoresis gels verifying amplification (top), LFIA test results (middle), and LFIA test line quantification (bottom). SD-LAMP reactions yield no false positive LFIA results for E. coli diluted in (A) pond water and (B) human plasma. SD-LAMP reactions yield no false positive LFIA results for (C) V. cholerae diluted in pond water. n = 4, replicates indicated by each circle. **** indicates p ≤ 0.0001; *** indicates p ≤ 0.001; ** indicates p ≤ 0.01; * indicates p ≤ 0.05.