Table I
Bacterial strains.
| Identification number | Bacteria 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 |
Fig. 1.
Lp genome map showing primers and crRNAs.
Table II
The sequence of RPA primers and crRNA.
| Name | Sequences 5’-3’ |
|---|---|
| Primer 1 | F: AACCGATGCCACATCATTAGCTACAGACAAGG R: GTTAAAGCCAATTGAGCGCCACTCATAGCGTC |
| Primer 2 | F: AATGTCAACAGCAATGGCTGCAACCGATGCCA R: AGAACGTCTTTCATTTGCTGTTCGGTTAAAGCC |
| Primer 3 | F: ACAAGGATAAGTTGTCTTATAGCATTGGTGCCG R: TTAAAGCCAATTGAGCGCCACTCATAGCGTCTTG |
| Lp-crRNA1 | UAAUUUCUACUAAGUGUAGAUAAAAUCAAGGCAUAGAUGUUA |
| Lp-crRNA2 | UAAUUUCUACUAAGUGUAGAUGCCAUUGCUUCCGGAUUAACA |
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.)
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.