Fig 1.
Methods for tagging ink (OPACODE S-1-17823 black) with DNA then applying to acetaminophen capsules in August 2014.
(A) two ink-labeled acetaminophen capsules are displayed. Tagging was performed in 2014. The capsule on the left was DNA tagged in this pilot (with both an “L and a “5”) using standard high-speed capsule pad printing. The matched capsule on the right was tagged with the same “L” and “5” but without DNA in the black ink. Both the “+DNA” and “–DNA” capsules appear identical to the eye. (B) It can be seen that the DNA-tagged ink can be swabbed from the surface using ethanol as a wetting solvent. Also note, there is no marring of the surface of the capsule in the process. (C) The swab tip after swabbing of the capsule, with the [DNA+ink] complex positioned at the tip. (D) 0.2mL tubes for PCR amplification each contain the tip of a cotton swab (with DNA containing ink on it) to be analyzed via PCR/CE analysis (Fig 2), or via qPCR amplification and detection (Fig 3), or isothermal amplification and detection (Fig 4).
Fig 2.
PCR and CE analysis–Confirmatory testing for field-deployable nucleic acid analysis.
This is a display of the final output of the analysis by PCR/CE, with clear differentiation between DNA tagged (+DNA) and untagged (-DNA) capsules. The DNA of interest is less than 200 base pairs long and appears as a single discrete peak. The PCR-CE data also confirms that there is only one discreet DNA of interest in the capsule. In the (-DNA) capsule, where the “L “and “5” have both been swabbed off for analysis denoted as “-DNA (L+5)” there is no DNA peak observed at the appropriate area of the CE trace. On the other hand, for the DNA tagged capsules (+DNA) where the “L,” “5” or both “L + 5” have been sampled, DNA is detected at the correct size as indicated by the blue peaks without spurious amplification nor additional peaks. These specimens were collected by swabbing and analyzed in 2016, more than 2 years after the DNA-tagged capsules were manufactured.
Table 1.
The composition of the reagent Mix for the Polymerase Chain Reaction Assay.
Fig 3.
Field-deployable nucleic acid analysis—Real time, qPCR.
This is a graphical representation a molecular probe qPCR assay. This can be accessed and viewed on a laptop or PC. As depicted here, there is a clear differentiation in the amplification curves between the DNA tagged (+DNA) capsules versus the non-DNA tagged (-DNA) capsules with minor sample to sample variation. In all cases both the “L” and “5” symbols have been swabbed. As seen here, clear differentiation between tagged and untagged could be obtained by 30 cycles (25 minutes) but since we wanted to show a plateau, the reaction has been taken to completion (45 cycles). The software generates a table from such data where a human interpreter is required to call the sample as containing DNA. Work is in progress to introduce a user interpretation-free algorithm producing a simple yes/no output. These specimens were collected by swabbing and analyzed by qPCR in March 2017, more than 2 years after the DNA-tagged capsules were manufactured.
Table 2.
The composition of the Master Mix for the GE illustra PuReTaq Ready-To-Go PCR Beads (Panel A) and Thermo Fisher’s FastTaq Ready Mix (Panel B).
Fig 4.
Field-deployable nucleic acid analysis—Isothermal DNA amplification and detection.
Isothermal amplification block and detector was deployed using RPA real time quantitative isothermal amplification detection chemistry. (A) A representative real-time amplification data derived from the assay as deployed for direct analysis of a swab-DNA complex. As shown, there is a clear differentiation in the amplification curves of the DNA tagged (+DNA) capsules (“L+5” sampling) versus the untagged (-DNA) capsules (“L+5” sampling), with only minor sample to sample variation. As seen here, the differentiation between tagged and untagged could be called and stopped within 10 minutes, but for this assay (since we wanted to show a plateau), it has taken 15 minutes to achieve for completion. (B) Positive and negative results shown graphically as a Positive (+) or a Negative (–) on the machine display. This allows the removal of the user interpretation, and allows the machine to have learned a pattern and call a result as is. These specimens were collected by swabbing and analyzed by isothermal methods in 2016, more than 2 years after the DNA-tagged capsules were manufactured.
Table 3.
The composition of the master mix for the Customized TwistAmp exo assay as equivalent to the manual23.