Improving sensitivity of single tube nested PCR to detect fastidious microorganisms

Area of Science:

• Microbiology
• Molecular Biology
• Clinical Diagnostics

Background:

• Single Tube Nested PCR (ST-nPCR) is valuable for detecting fastidious microorganisms in resource-limited settings.
• Optimizing outer primer performance is key to enhancing ST-nPCR sensitivity.
• Fastidious intracellular bacteria like *C. abortus* and *C. psittaci* are difficult to diagnose.

Purpose of the Study:

• To investigate methods for optimizing outer primer performance in ST-nPCR.
• To improve the detection sensitivity of ST-nPCR for fastidious pathogens.
• To evaluate the efficacy of Q5 Taq polymerase in ST-nPCR.

Main Methods:

• Investigated decreasing outer primer concentrations, using antisense oligonucleotides, chemically modified inner primers, and Q5 Taq polymerase.
• Tested optimization approaches on *C. abortus* and *C. psittaci*.
• Validated the optimized assay using 70 veterinary clinical samples.

Main Results:

• Q5 Taq polymerase, lacking 5'-3' exonuclease activity, yielded the best results.
• Achieved a detection limit of 0.1-1 ag (0.2-2 plasmid copies), comparable to real-time PCR.
• 10% of veterinary clinical samples tested positive.

Conclusions:

• Taq polymerases lacking 5'-3' exonuclease improve ST-nPCR sensitivity for fastidious microorganisms.
• The optimized ST-nPCR assay is affordable, applicable to various pathogens, and suitable for limited-resource laboratories.
• This method offers a sensitive and practical approach for diagnosing difficult-to-detect bacterial infections.