Targeting the 16S rRNA Gene by Reverse Complement PCR Next-Generation Sequencing: Specific and Sensitive Detection...

Area of Science:

• Clinical microbiology
• Molecular diagnostics
• Next-generation sequencing (NGS) applications

Background:

• Accurate bacterial identification is critical for effective antibiotic treatment.
• 16S rRNA gene sequencing is a common molecular method when cultures fail, but its sensitivity varies.
• Novel, sensitive, and clinically applicable methods for bacterial detection are needed.

Purpose of the Study:

• To evaluate the clinical utility and performance of 16S rRNA reverse complement PCR (16S RC-PCR) for bacterial species identification.
• To compare 16S RC-PCR with traditional culture methods and Sanger sequencing.

Main Methods:

• Developed and tested 16S RC-PCR, a next-generation sequencing-based method.
• Applied the method to 11 bacterial isolates, 2 polymicrobial samples, and 59 clinical samples.
• Compared results against culture and 16S Sanger sequencing.

Main Results:

• 16S RC-PCR accurately identified all bacterial isolates to the species level.
• In culture-negative samples, 16S RC-PCR increased identification rates from 17.1% (Sanger) to 46.3%.
• The method demonstrated increased sensitivity for detecting bacterial pathogens.

Conclusions:

• 16S RC-PCR enhances bacterial detection sensitivity in clinical samples.
• This method can increase the number of diagnosed bacterial infections, improving patient care.
• 16S RC-PCR is automatable and suitable for clinical diagnostic laboratories.