Fig 3 -

(A) Visual detection of the dilution series of purified RPA products run on commercial gold nanoparticle-based LFA strips. (B) Smartphone images of the dilution series of purified RPA products run on in-house-made LFA strips with SBMSO nanophosphor reporters. (C) Normalized TL/CL intensity ratio of SBMSO reporters against the concentration of purified DNA amplicons. Three trials were run for each concentration, then the average was calculated. The red line signifies the detection limit cutoff, taken as the mean plus three times the standard deviation (μ+3σ) of the no-analyte control LFAs.</p

dsDNA standard curve obtained from the QuantiFluor dsDNA system.

The inset shows the fluorescence obtained with 4 μL of 5X diluted purified and unpurified RPA products (in red) and their respective dsDNA concentrations. According to the standard curve, the dsDNA amount of purified and unpurified samples is 16.23 and 9.37 ng/well, respectively. Therefore, the dsDNA concentration of the undiluted purified and unpurified amplicons is 20.3 and 11.7 ng/μL, respectively. (TIF)</p

A 3-D printed phone accessory with minimal optical hardware, containing a lens and a bundle of inexpensive plastic optical fibers but no electronic components, was used as a dark imaging compartment which was designed to hold a universal LFA cartridge (MICA-125; DCN Diagnostics) such that the result window of the cartridge is aligned with the rear camera of the iPhone 5S and occupies most of the field of view when the cartridge is fully inserted into the attachment.

A proprietary software application, “Luminostics”, controls the flash and the rear camera of the iPhone. The flash excites the nanophosphors for ~3 s, and, after switching off the flash, the camera captures the images after a ~100 ms time delay. The camera captures four images and generates the average result. We have described the iPhone reader in more detail in our previous publications [17,24]. (TIF)</p

Fig 4 -

(A) Visual detection of the dilution series of unpurified RPA products run on commercial gold nanoparticle-based LFA strips. (B) Smartphone images of the dilution series of unpurified RPA products run on in-house-made LFA strips with SBMSO nanophosphor reporters. (C) Normalized TL/CL intensity ratio of SBMSO reporters against the concentration of unpurified DNA amplicons. Three trials were run for each concentration, then the average was calculated. The red line signifies the detection limit cutoff, taken as the mean plus three times the standard deviation (μ+3σ) of the no-analyte control LFAs.</p

Fig 5 -

(A) Visual detection of RPA-amplified Leishmania parasite DNA dilution series (unpurified RPA products), run on commercial gold nanoparticle-based LFA strips. (B) Smartphone images of the RPA-amplified Leishmania parasite DNA dilution series (unpurified RPA products), run on in-house-made LFA strips with SBMSO nanophosphor reporters. (C) Normalized TL/CL intensity ratio of SBMSO reporters against the number of parasites added per RPA reaction. Three trials were run for each concentration, and the average was calculated. The red line signifies the detection limit cutoff, taken as the mean plus three times the standard deviation (μ+3σ) of the no-analyte control LFAs.</p

LFA architecture used to detect RPA products.

(A) DNA amplicons labeled with biotin and fluorescein (FAM) during the RPA reaction first bind to reporter particles conjugated with monoclonal mouse anti-FITC antibodies in the conjugate pad. (b) The complexes then travel along the membrane and are captured by anti-biotin antibodies immobilized at the test line. Anti-mouse antibodies capture the excess reporters at the control line. Positive samples show both the test and the control lines; negative samples show only a control line.</p

Agarose gel electrophoresis of RPA products: 10 μL of each RPA reaction was loaded on the gel.

Lane: (1) DNA ladder (100–1517 base pairs; New England BioLabs Inc.), (2) QIAquick-purified DNA amplicons, (3) Unpurified DNA amplicons sample 1, (4) Unpurified DNA amplicons sample 2, (5) Unpurified negative control RPA reaction mixture with Vero cell DNA.</p

Isolation and Barcoding of Trace Pollen-free DNA for Authentication of Honey

Adulteration and mislabeling of honey to mask its true origin have become a global concern. Pollen microscopy, the current gold standard for identifying honey’s geographical and plant origins, is laborious, requires extensive training, and fails to identify filtered honey and honey spiked with pollen from a more favorable plant to disguise its origins. We successfully isolated pollen-free DNA from filtered honey using three types of adsorbents: (i) anti-dsDNA antibodies coupled to magnetic microspheres; (ii) anion-exchange adsorbent; and (iii) ceramic hydroxyapatite. The internal transcribed spacer 2 region of the captured pollen-free DNA was polymerase chain reaction-amplified and subjected to next-generation sequencing. Using an in-house bioinformatics pipeline, initial experiments showed that anion exchange had the greatest capacity to capture trace pollen-free DNA, and it was successfully applied to isolate DNA from five honey samples. Enrichment of trace pollen-free DNA from filtered honey samples opens a new approach for identifying the true origins of honey