Why do the Tickborne and Gut Zoomer tests use real time polymerase chain reaction (RT-PCR) and not next generation sequencing (NGS)?

1. Targeting Specificity and Sensitivity

• RT-PCR is designed to detect specific nucleic acid sequences with high sensitivity and specificity.
• For known gut pathogens, validated primers and probes allow fast, accurate identification even at low pathogen loads.
• NGS, on the other hand, sequences all nucleic acids in a sample. While this offers a broad, unbiased look, it may dilute signals from pathogens if they are present at low concentrations.

2. Speed and Turnaround Time

• RT-PCR assays typically yield results in 2–4 hours.
• NGS workflows require library preparation, sequencing (which can take 12–48 hours), and bioinformatics analysis, leading to total turnaround times of 24–72+ hours.

3. Cost and Complexity

• RT-PCR is significantly more cost-effective and technically accessible for routine testing.
• NGS requires expensive instruments, high reagent costs, and advanced computational infrastructure and personnel.

🔄 Number of Cycles: RT-PCR vs NGS

RT-PCR:

• RT-PCR typically runs for 40–45 cycles.
• Each cycle amplifies the target sequence, and Ct (Cycle threshold) values reflect pathogen load.
• The signal from fluorescent probes is detected in real-time, enabling quantitative output.

NGS:

• NGS does not operate using amplification cycles in the same way as PCR.
• It involves:
  • Library preparation, including PCR amplification (usually 12–18 PCR cycles) for adapter ligation and enrichment.
  • Sequencing cycles: Modern sequencers can perform 50–300 sequencing cycles, depending on read length (e.g., 2 × 150 bp).
• These cycles do not selectively amplify specific pathogens and instead sequence everything in the sample.

🧬 Probe Usage: RT-PCR vs NGS

RT-PCR:

• Uses sequence-specific primers and fluorescent probes, such as TaqMan probes, for each target pathogen.
• Multiplex RT-PCR panels can detect up to 20–30 pathogens in a single run.
• The number of probes used is equal to the number of targets, plus internal controls. Each probe is uniquely labeled to avoid spectral overlap.

NGS:

• No specific probes are required for pathogen detection.
• Detection relies on computational alignment of sequencing reads to reference databases.
• This makes it more flexible for discovering novel pathogens (e.g., COVID-19) but less targeted and efficient for detecting known organisms.

✅ Summary: When RT-PCR Is Preferable

RT-PCR is ideal when the goal is rapid, sensitive, and cost-effective detection of known gut pathogens. NGS has advantages when detecting rare, unknown, or co-infecting organisms in complex samples, but its time, cost, and complexity make it less practical for routine clinical diagnosis of well-characterized gut infections.