2 research outputs found
Strand Displacement Probes Combined with Isothermal Nucleic Acid Amplification for Instrument-Free Detection from Complex Samples
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 <i>Vibrio cholerae</i> and 2 750 <i>Escherichia coli</i> 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
An Evolved Mxe GyrA Intein for Enhanced Production of Fusion Proteins
Expressing
antibodies as fusions to the non-self-cleaving Mxe GyrA
intein enables site-specific, carboxy-terminal chemical modification
of the antibodies by expressed protein ligation (EPL). Bacterial antibody-intein
fusion protein expression platforms typically yield insoluble inclusion
bodies that require refolding to obtain active antibody-intein fusion
proteins. Previously, we demonstrated that it was possible to employ
yeast surface display to express properly folded single-chain antibody
(scFv)-intein fusions, therefore permitting the direct small-scale
chemical functionalization of scFvs. Here, directed evolution of the
Mxe GyrA intein was performed to improve both the display and secretion
levels of scFv-intein fusion proteins from yeast. The engineered intein
was shown to increase the yeast display levels of eight different
scFvs by up to 3-fold. Additionally, scFv- and green fluorescent protein
(GFP)-intein fusion proteins can be secreted from yeast, and while
fusion of the scFvs to the wild-type intein resulted in low expression
levels, the engineered intein increased scFv-intein production levels
by up to 30-fold. The secreted scFv- and GFP-intein fusion proteins
retained their respective binding and fluorescent activities, and
upon intein release, EPL resulted in carboxy-terminal azide functionalization
of the target proteins. The azide-functionalized scFvs and GFP were
subsequently employed in a copper-free, strain-promoted click reaction
to site-specifically immobilize the proteins on surfaces, and it was
demonstrated that the functionalized, immobilized scFvs retained their
antigen binding specificity. Taken together, the evolved yeast intein
platform provides a robust alternative to bacterial intein expression
systems