Species-specific identification of Pseudomonas based on 16S–23S rRNA gene internal transcribed spacer (ITS) and its combined application with next-generation sequencing

Abstract

Background Pseudomonas species are widely distributed in the human body, animals, plants, soil, fresh water, seawater, etc. Pseudomonas aeruginosa is one of the main pathogens involved in nosocomial infections. It can cause endocarditis, empyema, meningitis, septicaemia and even death. However, the Pseudomonas classification system is currently inadequate and not well established. Results In this study, the whole genomes of 103 Pseudomonas strains belonging to 62 species available in GenBank were collected and the specificity of the 16S–23S ribosomal RNA internal transcribed spacer (ITS) sequence was analysed. Secondary structures of ITS transcripts determining where the diversity bases were located were predicted. The alignment results using BLAST indicated that the ITS sequence is specific for most species in the genus. The remaining species were identified by additional frequency analyses based on BLAST results. A double-blind experiment where 200 ITS sequences were randomly selected indicated that this method could identify Pseudomonas species with 100% sensitivity and specificity. In addition, we applied a universal primer to amplify the Pseudomonas ITS of DNA extracts from fish samples with next-generation sequencing. The ITS analysis results were utilized to species-specifically identify the proportion of Pseudomonas species in the samples. Conclusions The present study developed a species-specific method identification and classification of Pseudomonas based on ITS sequences combined NGS. The method showed its potential application in other genera.

Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry in veterinary medicine: Recent advances (2019–present)

“…At present, sequencing the 16S rRNA gene sequence is the gold standard utilized for pathogen detection and microecological research. The 16S technique has been reported to obtain as high as 100% sensitivity and specificity for determining Pseudomonas species [206]. In addition to the high sensitivity and specificity demonstrated, the ability to amplify sequences through the polymerase chain reaction helps assure a low copy number of genetic material may be detected.…”

Development of a real-time loop-mediated isothermal amplification method for monitoring Pseudomonas lurida in raw milk throughout the year of pasture

“…The Pseudomonas genus is a large group divided into five categories ( Bossis et al, 2000 ) with target genes, including the apr gene ( Dufour et al, 2008 ), gyrB gene ( Bablishvili et al, 2017 ), and intergenic spacer sequence of 16S-23S rRNA ( Hu et al, 2022 ). The gyrB gene ( Lu et al, 2016 ) is a protein-coding gene that is related to DNA replication, modification, restriction, and repair.…”

“…The apr gene ( D’Incecco et al, 2019 ) is a thermostable-alkaline-protease-coding gene, which exists in Pseudomonas and other spoilage bacteria that can hydrolyze proteins. Notably, a previous study engineered aprX gene primers based on Pseudomonas and detected the thermostable alkaline proteases of P. fluorescens ( Hu et al, 2022 ). P. lurida is usually identified using a combination of traditional isolation and culture methods (3–4 d) and 16S rRNA sequencing (1 d, excluding transportation time; Behrendt et al, 2007 ).…”

“…This method has several advantages, such as strong specificity, high sensitivity, simple operation, rapid reaction, and low cost; therefore, it is widely used in various fields ( Iwamoto et al, 2003 ). In addition, real-time fluorescence LAMP (RealAmp) technology has been established by adding SYBR Green I dye to the system before the reaction, which allows the data to be analyzed based on the amplification curve and peak time ( Hu et al, 2022 ). However, no RealAmp reaction system has been developed for P. lurida and its thermostable alkaline proteases detection to monitor their dynamic changes in raw milk throughout the year of pasture.…”

“…The present study aimed to develop a RealAmp reaction system for the early and rapid detection of P. lurida and its thermostable alkaline proteases in raw milk. Primers were designed based on the apr X ( Hu et al, 2022 ) and gyrB genes of P. lurida . and the RealAmp reaction system and conditions were optimized.…”