Introduction: Accurate identification of pathogenic bacteria is crucial for efficient therapy prescription. Culturing, biochemical tests, and molecular detection are among the common methods for the detection of bacteria in clinical labs. Nowadays, molecular diagnosis methods are becoming more prevalent. These methods are both sensitive and fast, making them extremely valuable in identifying different pathogens. Detecting bacteria at the molecular level can be achieved by amplifying the targeted sequences in the genome. Currently, isothermal amplification is gaining popularity, these techniques are better than PCR because they are faster and can be done in under an hour. This method operates at a constant temperature and does not require a thermocycler. It also uses enzymes that do not need temperature cycles. The primary objective of this research was to introduce a novel technique for the rapid and accurate detection of pathogenic bacteria at a constant temperature. In that regard, A novel isothermal amplification technique has been developed to detect Klebsiella aerogenes.
Methods: Two specific primers were designed to target a specific region in the whole genome of K. aerogenes and their specificity was checked using the Primer-BLAST tool in NCBI. Also, the bacterial samples were cultured and twelve different bacterial species including Citrobacter freundii, Serratia marcescens, Burkholderia cepacia, Yersinia enterocolitica, Pseudomonas aeruginosa, Proteus mirabilis, Morganella morganii, Enterococcus faecalis, Acinetobacter baumannii, Shigella boydii, Klebsiella pneumoniae, Salmonella typhi were utilized for specificity analysis. One colony from each species of cultured bacteria was selected to extract their genome. Nucleic acid contents were extracted using boiling and measured for equal standard concentration. The amplification reaction was set up to amplify the targeted region and detect K. aerogenes. To improve accuracy both betaine and Recombinase protein were used together with ATP. This method can be carried out at a consistent temperature in a heater block without any initial heating.
Results: The absorption of the extracted genomes was measured and no contamination was detected in the samples. Specific primers were designed based on the reaction temperature. Based on the results of the Primer-Blast Tool, the designed primer sequences were specific to the target bacterium, ensuring accurate identification. The forward and reverse primers were 31 and 32 bp, respectively and the targeted amplicon was 239 bp. The results of the amplification method showed that the entire process could be completed almost instantly in 20 minutes. The developed method successfully differentiated all tested bacteria with 100% specificity based on differentiation and color contrast between positive and negative samples.
Conclusion: In conclusion, the speed and accuracy of the method make it an appropriate choice for rapid detection tests, especially point-of-care testing and diagnostic kits. This approach significantly accelerates the treatment process and can even prevent pandemics in some cases by facilitating swift diagnoses.