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A dCas9/sgRNA complex-mediated competitive assay for accurate and sensitive Pseudomonas aeruginosa analysis
Buyi Wang1, Ziyao Yu1, Zhihao Zhang2
1The First Affiliated Hospital of Harbin Medical University, Harbin City, Heilongjiang Province, 150001, China.
Abstract:
Pseudomonas aeruginosa (P. aeruginosa), a Gram-negative pathogenic bacterium, is one of the most common bacteria that causes severe infectious diseases. However, accurate and efficient detection of P. aeruginosa in clinical samples is a huge challenge. Therefore, in this study, we developed a Cas9 derivative (dCas9)/sgRNA-mediated competitive assay for the sensitive and precise characterization of genomic materials from P. aeruginosa. Our approach involved the identification of target genomic sequences using the dCas9/sgRNA complex, which occupied the "sensing probe" (SP) binding site, resulting in an increased availability of free SP. SP subsequently facilitated DNA polymerase/endonuclease-mediated signal cycles and signal production, enabling highly sensitive detection of P. aeruginosa. The proposed competitive assay demonstrated a robust linear response to P. aeruginosa within a concentration range from 10 CFU mL-1 to 106 CFU mL-1, leveraging numerous signal amplification processes and competitive target recognition while exhibiting robust anti-interference capacity. Compared with former strategies, the proposed competitive assay enabled the accurate detection of P. aeruginosa by directly identifying and binding genomic sequences, which could be easily extended to the detection of other bacteria by simply changing the sgRNA. In addition, the proposed approach exhibits significant clinical potential for early disease diagnosis owing to its excellent sensitivity and accuracy.
Insights
We developed a novel competitive assay for sensitive and precise detection of Pseudomonas aeruginosa (P. aeruginosa) using dCas9/sgRNA technology. This method offers potential for early clinical diagnosis of infectious diseases.
Area of Science:
- Molecular Biology
- Bacteriology
- Diagnostic Assays
Background:
- Pseudomonas aeruginosa (P. aeruginosa) is a common Gram-negative bacterium causing severe infections.
- Accurate and efficient detection of P. aeruginosa in clinical samples remains a significant challenge.
- Existing detection methods may lack sensitivity or specificity for early diagnosis.
Purpose of the Study:
- To develop a sensitive and precise assay for detecting P. aeruginosa genomic material.
- To utilize a Cas9 derivative (dCas9)/sgRNA-mediated competitive approach for bacterial identification.
- To explore the potential for early disease diagnosis through enhanced bacterial detection.
Main Methods:
- Developed a dCas9/sgRNA-mediated competitive assay targeting specific P. aeruginosa genomic sequences.
- Utilized the dCas9/sgRNA complex to occupy sensing probe (SP) binding sites, increasing free SP availability.
- Employed DNA polymerase/endonuclease-mediated signal cycles for amplified signal production and detection.
Main Results:
- The assay demonstrated a robust linear response for P. aeruginosa detection from 10 to 10^6 CFU/mL.
- The method leveraged signal amplification and competitive target recognition, showing strong anti-interference capacity.
- The assay accurately detected P. aeruginosa by directly binding genomic sequences, adaptable for other bacteria via sgRNA modification.
Conclusions:
- The dCas9/sgRNA-mediated competitive assay provides sensitive and accurate detection of P. aeruginosa.
- This approach offers a promising tool for early clinical diagnosis of P. aeruginosa infections.
- The adaptable nature of the assay allows for potential application in detecting various bacterial pathogens.
