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A Rapid and Sensitive CRISPR-Cas12a for the Detection of Legionella pneumophila Cover

A Rapid and Sensitive CRISPR-Cas12a for the Detection of Legionella pneumophila

Polish Journal of Microbiology
Volume 74 (2025): Issue 4 (December 2025)
By: JIANGHAO LI,  XINYU WANG,  XINLING WANG,  HAI QU,  LIFEI GAO,  ZHONGLING ZHAO,  PEI LUO and  YEHUAN ZHENG  
Open Access
|Dec 2025

Figures & Tables

Fig. 1.

Lp genome map showing primers and crRNAs.
Lp genome map showing primers and crRNAs.

Fig. 2.

Optimization of RPA-CRISPR/Cas12a detection system. A) Screening for the best RPA primer. Lane 1 - Amplification fragments of primer 1-F&R; Lane2 - Amplification fragments of primer 2-F&R; Lane3 - Amplification fragments of primer 3-F&R. B) Screening for the best amplification time. Lanes 1-6 represent amplification time at 5, 10, 15, 20, 25, and 30 minutes. C) Real time fluorescence signal of two crRNA designed for RPA-CRISPR/Cas12a system. Blue - crRNA 1; red - crRNA 2; gray - without crRNA. D) The fluorescence curves of different concentrations of the fluorescent reporting probe. E) The image of RPA-CRISPR/Cas12a LFB system by different concentrations of the reporting probe. (The fluorescence reporting probe concentrations represented by samples 1-6 are 0.1 μmol/l, 0.2 μmol/l, 0.4 μmol/l, 0.8 μmol/l, 1.6 μmol/l, and 3.2 μmol/l, respectively. C - control line, T - test line.)
Optimization of RPA-CRISPR/Cas12a detection system. A) Screening for the best RPA primer. Lane 1 - Amplification fragments of primer 1-F&R; Lane2 - Amplification fragments of primer 2-F&R; Lane3 - Amplification fragments of primer 3-F&R. B) Screening for the best amplification time. Lanes 1-6 represent amplification time at 5, 10, 15, 20, 25, and 30 minutes. C) Real time fluorescence signal of two crRNA designed for RPA-CRISPR/Cas12a system. Blue - crRNA 1; red - crRNA 2; gray - without crRNA. D) The fluorescence curves of different concentrations of the fluorescent reporting probe. E) The image of RPA-CRISPR/Cas12a LFB system by different concentrations of the reporting probe. (The fluorescence reporting probe concentrations represented by samples 1-6 are 0.1 μmol/l, 0.2 μmol/l, 0.4 μmol/l, 0.8 μmol/l, 1.6 μmol/l, and 3.2 μmol/l, respectively. C - control line, T - test line.)

Fig. 3.

The sensitivity and specificity of the RPA-CRISPR/Cas12a fluorescent and LFB detection system. A) The fluorescence curves of different concentrations of target DNA and the fluorescence values of different concentrations of target DNA at 10 minutes. B) Sensitivity test of RPA-CRISPR/Cas12a LFB system by different concentrations of target DNA. NC - non-template control; PC - Legionella pneumophila target; C - control line, T - test line. C) Specificity of the RPA-CRISPR/Cas12a fluorescent assay for the detection of L. pneumophila. D) Specificity of the RPA-CRISPR/Cas12a LFB for the detection of L. pneumophila. NC - non-template control; PC - Legionella pneumophila; N1 - Legionella longbeachae; N2 - Legionella anisa; N3 - Legionella gormanii; N4 - Legionella feeleii; N5 - Legionella dumoffii; N6 - Fusobacterium nucleatum; N7 - Acinetobacter baumannii; N8 - Acinetobacter baumannii; N9 - Burkholderia cepacia; N10 - Haemophilus influenzae; N11 - Streptococcus pneumoniae; N12 - Moraxella catarrhalis; N13 - Klebsiella pneumoniae; N14 - Staphylococcus aureus; N15 - Escherichia coli; N16 - Pseudomonas aeruginosa. C - control line, T - test line.
The sensitivity and specificity of the RPA-CRISPR/Cas12a fluorescent and LFB detection system. A) The fluorescence curves of different concentrations of target DNA and the fluorescence values of different concentrations of target DNA at 10 minutes. B) Sensitivity test of RPA-CRISPR/Cas12a LFB system by different concentrations of target DNA. NC - non-template control; PC - Legionella pneumophila target; C - control line, T - test line. C) Specificity of the RPA-CRISPR/Cas12a fluorescent assay for the detection of L. pneumophila. D) Specificity of the RPA-CRISPR/Cas12a LFB for the detection of L. pneumophila. NC - non-template control; PC - Legionella pneumophila; N1 - Legionella longbeachae; N2 - Legionella anisa; N3 - Legionella gormanii; N4 - Legionella feeleii; N5 - Legionella dumoffii; N6 - Fusobacterium nucleatum; N7 - Acinetobacter baumannii; N8 - Acinetobacter baumannii; N9 - Burkholderia cepacia; N10 - Haemophilus influenzae; N11 - Streptococcus pneumoniae; N12 - Moraxella catarrhalis; N13 - Klebsiella pneumoniae; N14 - Staphylococcus aureus; N15 - Escherichia coli; N16 - Pseudomonas aeruginosa. C - control line, T - test line.

Bacterial strains_

Identification numberBacteria name
ATCC® 33152™Legionella pneumophila
ATCC® 33484™Legionella longbeachae
ATCC® 35292™Legionella anisa
ATCC® 33297™Legionella gormanii
ATCC® 35072™Legionella feeleii
ATCC® 33279™Legionella dumoffii
ATCC® 25586™Fusobacterium nucleatum
ATCC® 19606™Acinetobacter baumannii
ATCC® 17666™Stenotrophomonas maltophilia
ATCC® 25608™Burkholderia cepacia
ATCC® 19418™Haemophilus influenzae
ATCC® 49619™Streptococcus pneumoniae
ATCC® 25238™Moraxella catarrhalis
ATCC® 10031™Klebsiella pneumoniae
ATCC® 25923™Staphylococcus aureus
ATCC® 25922™Escherichia coli
ATCC® 27853™Pseudomonas aeruginosa

The results of two detection methods in environmental water samples_

Methods qPCRChi-square valuep-value
PositiveNegativeTotal
RPA-CRISPR/Cas12a LFBPositive230230.0250.875
Negative1136137
Total24136160

The sequence of RPA primers and crRNA_

NameSequences 5’-3’
Primer 1F: AACCGATGCCACATCATTAGCTACAGACAAGGR: GTTAAAGCCAATTGAGCGCCACTCATAGCGTC
Primer 2F: AATGTCAACAGCAATGGCTGCAACCGATGCCAR: AGAACGTCTTTCATTTGCTGTTCGGTTAAAGCC
Primer 3F: ACAAGGATAAGTTGTCTTATAGCATTGGTGCCGR: TTAAAGCCAATTGAGCGCCACTCATAGCGTCTTG
Lp-crRNA1UAAUUUCUACUAAGUGUAGAUAAAAUCAAGGCAUAGAUGUUA
Lp-crRNA2UAAUUUCUACUAAGUGUAGAUGCCAUUGCUUCCGGAUUAACA
DOI: https://doi.org/10.33073/pjm-2025-041 | Journal eISSN: 2544-4646 | Journal ISSN: 1733-1331
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Language: English
Page range: 484 - 493
Submitted on: Aug 1, 2025
|
Accepted on: Oct 29, 2025
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Published on: Dec 26, 2025
Published by: Polish Society of Microbiologists
In partnership with: Paradigm Publishing Services
Publication frequency: 4 issues per year
Keywords:
Legionella pneumophila,
RPA,
CRISPR-Cas12a,
rapid detection
Related subjects:
Life sciences,
Microbiology and virology

© 2025 JIANGHAO LI, XINYU WANG, XINLING WANG, HAI QU, LIFEI GAO, ZHONGLING ZHAO, PEI LUO, YEHUAN ZHENG, published by Polish Society of Microbiologists
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License.

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