Introduction

The detection of bacterial and viral targets is a routine procedure in laboratories, commonly performed using PCR. RT-qPCR is often preferred due to its rapid turnaround time and the use of probes for highly specific detection. However, sample preparation before PCR is critical, as various inhibitors present in biological samples can significantly impact results.

These inhibitors include heme (from blood), urea (from urine), polysaccharides (from plant and fungal samples), lipids (from tissues and stool), proteins (such as nucleases and collagen), salts (such as excess NaCl or EDTA from storage buffers), and humic acids (from environmental samples like soil and water). To ensure accurate detection, an extraction step is typically required to isolate DNA or RNA, making the target sequences readily available for amplification.

This experiment examined Moraxella catarrhalis (a bacterial target) and Human Metapneumovirus (a viral target) using nasopharyngeal swab samples from human patients. The results showed that Moraxella catarrhalis was undetectable without prior DNA extraction, whereas Human Metapneumovirus could be detected with and without an extraction step.

Method

Instrument: BioRad CFX-96 w/ C1000 Touch ThermalCycler
Extraction Kit: Magnetic Bead based extraction using Universal DNA/RNA Extraction Kit (Filtrous, EXK-UN-0096)
PCR Kit: Universal Respiratory qPCR Detection Kit (Filtrous, OSK-RP-0100)

• PCR Protocol:

  • Sample Volume: 20µL
  • Reverse Transcription: 50°C for 5 minutes
  • 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

  • Human Metapneumovirus: Channel 3 (Cal Fluor 610, ROX)
  • Moraxella catarrhalis: Channel 2 (HEX, VIC, CIV-550)

Sample Preparation

Extraction Procedure

The extraction process began 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 Respiratory qPCR Detection Kit (Filtrous, OSK-RP-0100), RPP PCR Mix 2, and RPP PCR Mix 4 were used. RPP PCR Mix 2 was used by creating 20 samples worth of a mixture of 14uL of PCR Mix 2 and 1uL of RT-Enzyme Mix per sample, resulting in 280uL of PCR Mix 2 + 20uL of RT-Enzyme Mix. 15uL of the Master Mix was added into a well on a 0.2mL 96-Well Clear plate compatible with the CFX96 Machine. RPP PCR Mix 4 added 15uL directly to the 96-well plate to 18 total wells. The following samples were run in triplicate and 5uL of template were added to a unique well for each mix.

• Human Metapneumovirus from patient sample
• Human Metapneumovirus extracted
• Moraxella catarrhalis from patient sample
• Moraxella catarrhalis extracted
• RPP Positive Control
• Nuclease Free Water (Negative Control)

Results and Discussion

Moraxella catarrhalis (Bacteria)

Unextracted (Direct) Sample: minimal amplification, expected of an unextracted bacterial sample, as target DNA is not readily available in unlysed cells. CT = 27.12; RFU = 258. 

Extracted Sample: Improved amplification efficiency evident by the increase of slope of RFU. Precise between technical triplicates. CT = 25.04; RFU = 1684.

This patient sample of moraxella catarhhalis was partially detectable without extraction. When compared to the negative control, the unextracted result would come out as positive. Although in comparison the amplification efficiency is much more significant with extraction as there are less PCR inhibitors making it clearer about the target analyte.

Human Metapneumovirus (Viral)

Unextracted (Direct) Sample: Very good initial signal, not surprising of a viral RNA based assay. Extraction efficiency should be gauged off of preservation/improvement of signal relative to this baseline. CT = 19.85; RFU = 2234. 

Extracted Sample: Increased RFU relative to direct sample, indicating successful extraction of RNA. CT = 20.19; RFU = 2528. 

The patient sample of Human Metapneumovirus was detectable with and without extraction. This means that the patient sample transport medium is not going to cause interference with PCR. The genomic information from the viral target is readily available as compared to bacteria. Although evident in the extraction, the extraction did improve amplification efficiency due to the increase of RFU.

Conclusion

The bacterial analyte was detectable without extraction. Although the amplification efficiency was very poor. Once the sample was extracted, the amplification efficiency greatly increased. With reference to the viral data, this is more reflective of the readily available genomic information that is free floating in solution that occurs before extraction. The bacterial genomic information is inhibited and not readily available without lysing. The only lysing occurring before extraction is during the denaturation steps of PCR. This allows for some genomic information to be available for amplification reflecting the small amounts of amplification occurring for the bacterial target. Although some bacterial walls may be more difficult to lyse than by just applying heat. This is evident of a need for extraction when concerned with targets of interest that are bacteria. 

Viral, however, was detectable without extraction and with extraction at similar amplification efficiencies. Viral analytes tend to have genomic information readily available. These targets are smaller and are generally less inhibited by the inhibitors in patient samples. The characteristic of amplifying RNA is single stranded which is readily more reactable than a DNA double strand.

When working with bacteria targets, it is important to do a nucleic extraction to guarantee results and readiness of reaction for PCR. Cell walls are tough and a heat lysis might not be sufficient to get nucleic information.