Use of amplicon sequencing to improve sensitivity in PCR-based detection of microbial pathogen in environmental...

Area of Science:

• Environmental microbiology
• Molecular diagnostics
• Next-generation sequencing

Background:

• Current DNA-based pathogen detection methods face challenges in sensitivity, specificity, and robustness.
• These limitations are often linked to the requirement for specific and adequate amplification (SAA) in Polymerase Chain Reaction (PCR).

Purpose of the Study:

• To investigate the use of next-generation sequencing (NGS) to detect target DNA sequences directly from false-negative PCR reactions (PCR-NGS).
• To eliminate the need for SAA in PCR by leveraging NGS for enhanced detection.
• To improve the sensitivity and accuracy of microbial detection in complex environmental matrices.

Main Methods:

• False-negative PCR and quantitative PCR (qPCR) reactions were generated using serial dilutions of Salmonella genomic DNA.
• These reactions were directly analyzed by NGS to detect target invA sequences.
• The PCR-NGS approach was validated using Salmonella-spiked stream water and sediment samples.
• The method was applied to urban stream water samples for real-world performance assessment.

Main Results:

• Target invA sequences were successfully detected in all false-negative PCR and qPCR reactions, significantly lowering detection limits.
• The PCR-NGS approach demonstrated effectiveness in correcting false-negative results in environmentally relevant samples.
• Eight out of ten urban stream water samples, initially deemed Salmonella-negative by conventional methods, tested positive using PCR-NGS.
• Analysis of non-target sequences identified primer dimer-like sequences as a primary cause of false negativity.

Conclusions:

• The PCR-NGS approach offers a significant improvement in method sensitivity for microbial pathogen detection.
• This method can effectively correct false-negative detections, providing more accurate results.
• PCR-NGS enables sequence-based failure diagnostics, aiding in understanding limitations of PCR in complex environmental samples.