Who Packed the Drugs? Application of Bayesian Networks to Address Questions of DNA Transfer, Persistence, and Recovery from Plastic Bags and Tape

Number of Individuals

20

Replicates per Individual and Condition

Varies by experiment: Direct zip-lock handling = 3 handlings per participant. Indirect personal-bag experiment = 1 exposure for 24 h. Tape experiment = participants contributed twice, swapping A/B roles, producing 20 wrapped cardboard samples. Shedder-status experiment = 3 × 10-s tube-holding replicates per participant separated by ≥3 h.

Depositor Characteristics

Shedder status was assessed: 4 high, 11 medium and 5 low shedders.

Criteria for Shedder Status

High: ≥2/3 samples above mean total RFU of 62,520 and ≥2/3 samples with 20/24 full loci. Low: all samples < 8,000 RFUtot and ≥2/3 negative or partial. Remaining participants classified as medium.

Previous Activities

Participants waited at least 1 h after washing their hands/using antibacterial liquid before deposition. During the 24-h Indirect personal-bag experiment, participants were instructed to carry/move their bags between work and home.

Contact Scenario

Direct zip-lock handling: after ≥1 h following hand washing, participant opened/closed and touched the upper surface of a zip-lock bag for ~30 s. Indirect personal-bag experiment: a DNA-free zip-lock bag containing a weighted tube was placed in the participant's own personal bag for 24 h while the bag was used/carried. Tape experiment: Participant B handled tape for 1 min by passing it hand-to-hand and stroking its sides toward the palms and a piece was torn off. After ≥10 days of storage, participant A used the tape to wrap cardboard. Shedder-status experiment: Participants held a plastic tube for 10s in their dominant hand.

Primary Substrate Type

Direct zip-lock handling: Zip-lock bag. Indirect personal-bag experiment: personal bag. Tape experiment: adhesive tape roll and cardboard. Shedder-status experiment: plastic tube.

Deposit

Direct zip-lock handling: 30 s contact × 3 Indirect personal-bag experiment: normal day to day life. Tape experiment: Person B - 1 min handling, including hand-to-hand passing/stroking and simulated use by tearing tape. Person A - wrap a cardboard box with tape. Shedder-status experiment: 10 s holding of plastic tube

Delay

Direct zip-lock handling: none. Indirect personal-bag experiment: 24 h between placement of zip-lock bag in personal bag and recovery. Tape experiment: minimum 10 days between tape handling by participant B and subsequent use by participant A. no delay after participant A handled the cardboard. Shedder-status experiment: none.

Secondary Substrate Type

Indirect personal-bag experiment: Zip-lock bag. Tape experiment: adhesive tape.

Secondary Substrate Contact

Indirect personal-bag experiment: zip-lock bag remained inside previously used personal bag for 24 h while the bag was moved/carried. Tape experiment: previously handled tape was left for at least 10 days in storage, before subsequently used by participant A to wrap cardboard.

Further Transfer

n.s.

Sampling Time

Generally immediately after completion of the experimental transfer scenario; indirect bag samples were collected after the 24-h storage period and tape/cardboard samples after the ≥10-day tape storage followed by wrapping. Exact elapsed time between final contact and swabbing is n.s.

Persistence

24 h in participants' previously used personal bags, with bags being carried/moved between locations. ≥10 days storage of previously handled tape before reuse. Storage environmental conditions not specified.

Sampling Method

Moistened swab

Sampling Area

Zip-lock bag: top 3 cm of outside and inside, including lock. Wrapped cardboard: full outside and tape edges around cardboard. Tube: full body excluding lid

Extraction

5% Chelex, 200 µL Chelex added to swabs, without prior incubation with water.

DNA Quantification

PowerQuant, Promega, using a 7500 Real-Time PCR system (Thermo Fisher Scientific)

Input for Profiling

1 ng template, 25 µL reaction volume and 29 amplification cycles. Where concentration was below the recommended input, maximum template volume of 15 µL was used.

Profiling

PowerPlex Fusion 6C System (Promega), Veriti 96-well thermal cycler, Applied Biosystems 3500 xL Genetic Analyzer, GeneMapper ID-X v1.6 with analytical threshold 100 RFU.

Reference Samples

n.s.

Profile Interpretation and Mixture Analysis

EuroForMix v3.2.0 was used to calculate likelihood ratios and mixture proportions. Unknown contributors were included as necessary to explain observed alleles. Average RFU was calculated over 23 loci excluding amelogenin. Mixture proportions were used to partition RFU between contributors. Bayesian networks were then used for activity-level evaluation.

RNA Data Interpretation

n.a.

DNA Quantity

Direct transfer experiment: 0,0002 ng/ul - 0,0428 ng/ul. Indirect transfer experiment: 0 ng/ul - 0,0333 ng/ul.

Profile Quality

Highly variable. Direct zip-lock transfer: 4 full + 7 partial profiles, 6 samples with ≤6 alleles and 4 with no result. Indirect transfer: 3 full + 2 partial POI profiles, 4 with ≤5 alleles and 10 with no result. Tape/cardboard: 3 no results, 3 with only four alleles. 7 full/partial C1 profiles, 5 C1+C2 mixtures and 2 partial C2 profiles.

Parameter Used for Comparison

DNA concentration. Number of alleles matching the POI. Average RFU/peak height. Total RFU. Shedder status. Profile completeness. Presence/absence of unknown contributors. Likelihood ratios.

Cautionary Remarks

N/A