Introduction

It is likely that organisms in patient samples will eventually decrease in potency as the days go by after collection. The sample stability depends on many factors such as the transport medium it is stored in, the type of analyte of concern, and many more factors. The importance of knowing the lifespan of your sample is relevant to standard testing.

Testing for diagnostic concerns or for R&D purposes, a stability study should be done to study the trend a target of interest will experience as time passes. It is inevitable that a sample will want to be retested so studying the effects of time is important to be able to make proper conclusions.

This experiment will review a general sample stability study specifically for fungal targets. The fungal targets will be Epidermophyton floccosum (ATCC 9646) and Trichophyton rubrum (ATCC 28188). The reference material was cultured before as instructed on the ATCC website. The cultured media was extracted by lysing the strong fungal cell walls to expose the genomic information for studying. The experiment was carried out across 5 days after being spiked into a transport media matrix that is of low saline concentration. The fungal targets presented little to no change from Day 1 vs Day 5 allowing me to make conclusions about fungal targets from that time range.

Method

• Instrument: Biorad CFX96 C1000 Touch
• Extraction Kit: Magnetic Bead based extraction using Universal DNA/RNA Extraction Kit (Filtrous, EXK-UN-0096)
• Centrifuge capable of doing 2400 RCF.
• Incubator (with shaking preferred) that can go up to 95°C
• Reference Material: Cultured Epidermophyton floccosum (ATCC 9646) and Trichophyton rubrum (ATCC 28188)
• 2X Buffer NLY 
• PCR Kit: Nail Fungal Infections qPCR Detection Kit (Filtrous, OSK-NF-0100)

  • PCR Protocol:

    • Sample Volume: 20µL
    • Initial Denaturation: 95°C for 2 minutes
    • 40 Cycles Total
      • Denaturation: 95°C for 10 seconds
      • Annealing: 60°C for 30 seconds with data capture at the annealing step.

  • Detection Channels

    • Trichophyton Rubrum: Channel 1 (FAM)
    • Epidermophyton floccosum: Channel 3 (ROX, CFR 610)

Sample Preparation

Extraction Procedure

Cultured reference was spiked into a neutralizing VTM (low saline concentration) for the purposes of this study. 10uL of cultured reference material was spiked into 2mL of neutralizing transport media.

200 µL of 2X Buffer NLY was pipetted into a 2 mL nuclease-free tube, followed by the addition of 200 µL of dry nail sample and 10 µL of lytic enzyme mix. The mixture was shaken at 100 RCF at 45 °C for 15 minutes, then allowed to cool to room temperature. After cooling, the sample was shaken again at 100 RCF at 90 °C for 15 minutes. The sample was allowed to rest at room temperature for 10 minutes following heating. It was then centrifuged at 2,400 RCF for 2 minutes, and the resulting supernatant was used for nucleic acid extraction.

The extraction used the prepared fungal sample as a sample template, with the preparation of a 96-well sample plate. 10 µL Proteinase K, 2 µL L Solution, 250 µL Buffer MYE, and 500 µL isopropanol were added to each well. 5 samples worth of this solution was prepared initially and a total of 762uL was added into each well.

250 µL of patient sample was then added to its designated well and mixed thoroughly by pipetting. The plate was incubated at room temperature for 5 minutes.

Next, 10 µL MagPure beads were added to each well. The bead container had been vortexed beforehand to ensure thorough resuspension. After mixing the beads into the wells, the plate was incubated again at room temperature for 5 minutes.

The sample plate was then placed on a magnetic rack to magnetize the beads. Once the beads had settled at the bottom of the wells and the solution became clear, the supernatant was carefully removed and discarded.

To wash the beads, the plate was removed from the magnetic rack and the beads were resuspended in 500 µL Wash Buffer Solution (prepared by adding 48 mL ethanol to 12 mL Wash Buffer). The plate was returned to the magnetic rack, and the supernatant was removed once the beads had fully cleared from the solution.

The samples were air-dried for 10 minutes, ensuring complete evaporation of the residual wash buffer.

Finally, the beads were resuspended in 50 µL DEPC-treated water, and the plate was placed back on the magnetic rack. Once the beads had cleared, the eluted nucleic acids were transferred to a clean elution plate. The elutions were left in the fridge until ready to be used for PCR reactions. The sample was left at room temperature to study the effects of its stability on day 5.

PCR Procedure

Referencing Nail Fungal Infections qPCR Detection Kit (Filtrous, OSK-NF-0100),  15uL of each of the mixes was added into an individual well on a 0.2mL 96-Well Clear plate compatible with the CFX96 Machine. 5uL of extracted samples were added to each unique mix.

Stability Procedure

The same exact process was repeated on Day 1 and Day 5. With Day 5 exception being that the sample was not recreated (contriving the VTM with organism). The sample samples made on day 1, were reused on day 5.

Results and Discussion

Day 1 Results

Epidermophyton floccosum Day 1: CT average at 25.3 RFU Max: ~130,000

Trichophyton Rubrum Day 1: CT average at 19.4 RFU Max: ~390,000 (2 Data points used)

Day 5 Results

Epidermophyton floccosum Day 5: CT average at 25.8 RFU Max: ~115,000

Epidermophyton floccosum Day 5: CT average at 20.3 RFU Max: ~340,000

A very nominal change between the sample being stored at low saline concentrations from Day 1 and Day 5.

Conclusion

The results of the stability study represent a nominal change of sample potency from Day 1 to Day 5 when stored in low saline concentration. The data from a stability study allows more conclusions to be made after a sample is being extensively tested. Fungi samples have strong cell walls and are low activity species that it is expected to remain stable. Limited activity of a sample and exposure of genomic information makes fungal stable to be studied by PCR.

Although more parameters exist that would be necessary to do a full extensive stability study. These studies include, freeze thaws, stand alone sample matrix stability, heating, and many more. Stability studies should be done as applicable for a lab. If a lab is frequently using a lot of a reagent that needs to be stored at freezing conditions, freeze thaw stability is an effort to look towards. A study such as the one that occurred in the focused experiment is ideal for laboratories that receive samples to be tested.