Introduction

The detection of bacterial DNA in urine samples is a common application of PCR in clinical laboratories, particularly for the diagnosis of urinary tract infections (UTIs). RT-qPCR is often used for its rapid turnaround time and high specificity through probe-based detection, allowing for the identification of both bacterial pathogens and relevant resistance genes directly from clinical samples.

Urine, while a convenient and non-invasive sample type, contains several PCR inhibitors that can interfere with DNA amplification. These include urea, salts (such as NaCl and phosphates), cellular debris, and proteins. Urea in particular can denature enzymes and destabilize nucleic acids, reducing amplification efficiency or causing false negatives.

To overcome these challenges, a nucleic acid extraction step is critical. This process not only concentrates the bacterial DNA but also removes inhibitory substances, ensuring that the target sequences are accessible and compatible with downstream PCR amplification. Without proper extraction, the sensitivity and accuracy of DNA-based detection for UTI pathogens in urine samples may be significantly compromised, particularly for organisms present at low abundance or for targets such as antibiotic resistance genes.

This experiment examined Klebsiella pneumoniae (KP) and its associated KPC (Klebsiella pneumoniae carbapenemase) resistance gene, using urine samples collected from human patients. The results demonstrated that detection of both the bacterial genome and the antibiotic resistance marker required prior DNA extraction. KP was partially detectable without extraction although it did not have consistent results. The resistance gene KPC was consistently not detectable without extraction.

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.
• Reference Material: Cultured ATCC BAA-1898 (Klebsiella pneumoniae (Schroeter) Trevisan)
• PBS adjusted to 7.4pH
• PCR Kit: Urinary Tract Infection qPCR Detection Kit (Filtrous, OSK-UT-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

    • Klebsiella Pneumoniae: Mix 1, Channel 2 (HEX, VIC, CIV-550)
    • KPC: Channel 2 (HEX, VIC, CIV-550)

• PCR Kit: Antibiotic Resistance qPCR Detection Kit (Filtrous, OSK-TR-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

    • KPC: Mix 2, Channel 2 (HEX, VIC, CIV-550)
      

Sample Preparation

Extraction Procedure

Cultured ATCC BAA-1898 (Klebsiella pneumoniae (Schroeter) Trevisan) is sourced from human urine. 50uL of the reagent was spiked into 1450uL of negative urine to be used as a sample.

A total of 1.5 mL of urine sample was transferred to a 2 mL microcentrifuge tube and centrifuged at 2400RCF for 5 minutes. After centrifugation, the supernatant was carefully removed, leaving 50 µL of urine in the tube without disturbing the pellet. 200 µL of PBS and 20 µL of lytic enzyme mix were then added to the pellet. The sample was mixed thoroughly by pipetting or vortexing and incubated at room temperature for 5 minutes.

The extraction used the prepared urine 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.

PCR Procedure

Referencing Universal Urinary Tract InfectionsqPCR Detection Kit (Filtrous, OSK-UT-0100) and Antibiotic Resistance qPCR Detection Kit (OSK-TR-0100), UTI Mix 1 and ABR Mix 2 were used.  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. The following samples were run using the 5uL for template delivery.

• Cultured ATCC BAA-1898 (Klebsiella pneumoniae (Schroeter) Trevisan)
• Cultured and extracted ATCC BAA-1898 (Klebsiella pneumoniae (Schroeter) Trevisan)
• Contrived Urine Sample using ATCC BAA-1898 not extracted
• Contrived Urine Sample using ATCC BAA-1898 extracted
• UTI Positive Control
• ABR Positive Control
• Nuclease Free Water (Negative Control)

Results and Discussion

Klebsiella Pneumoniae (KP)

The blue line represents the sample extracted, while the red line represents the sample not extracted. The urine extract sample resulted in a higher RFU which represents that there was less interference in the extracted sample.

Klebsiella Pneumoniae (KPC)

The blue line represents the sample extracted, while the red line represents the sample not extracted. The urine extract sample resulted in a higher RFU which represents that there was less interference in the extracted sample.

Conclusion

Both organisms were detectable without the need of extraction. The urine interference was not sufficient to prevent the detection of the sample. Although, it is notable the difference between extraction and no extraction. Amplification efficiency was much improved as a result of extraction. This removed PCR inhibitors in urine, such as urea and β-hCG, allowing the PCR reaction to be more readily reactable.

In identifying those with UTI, it is possible that the organisms exist at really low concentrations. An extraction is supposed to help concentration and clean up a sample to make sure DNA/RNA is readily detectable to allow for proper identification and detection. Diets also affect individuals and the potency of the organism in urine. Cleaning up a sample by ridding most of the urine and reconstituting it into a stable buffer for extraction allows the clean up of inhibitors.