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.
Highlights
Isothermal nucleic acid amplification techniques, such as loop mediated amplification (LAMP), are promising methods for point-of-need pathogen identification because they can efficiently amplify targets without the expensive thermal cycling equipment of conventional PCR.[1−3] LAMP can amplify 10 ng of nucleic acids within 5−10 min[4] and from complex patient,[5,6] environmental,[7] and food sample matrixes.[8]
We further evaluated the unaided reading of lateral flow immunoassay strips (LFIAs) results by human subjects, determining LFIA test band intensities required for definitive interpretation
Rather than redesign the entire primer sets, we found the use of strand displacement probes greatly improved interpretability of LFIAs by increasing the intensity of positive test bands and preventing primer dimerization that yielded false positive test bands
Summary
The swapped primer tag configuration still yielded a limit of detection in agarose gels of 275 E. coli cells but resulted in positive test bands in all LFIAs, including negative controls. Adding the strand displacement probes to the LAMP protocol is a simpler modification than redesigning primer sets or painstakingly optimizing reaction times This essential modification mitigates spurious amplification and nonspecific detection in easy-to-use LFIAs, improving LAMP’s utility outside of a laboratory. We found LFIAs yielded positive test bands from SD-LAMP reactions performed with 50% pond water (the maximum possible ratio given that the reagents are prehydrated) and as few as 27 500 E. coli and 350 V. cholerae cells. SD-LAMP reactions yield no false positive LFIA results for E. coli diluted in (A) pond water and (B) human plasma. ■ AUTHOR INFORMATION strand displacement probes or capture efficiency of the LFIAs
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