Reagents

Primers for RPA were synthesized by TsingKe Biological Technology (Beijing, China). Recombinant RNase inhibitor, crRNA, and RNase-free water were obtained from Takara Bio (Beijing, China). The ssDNA reporter was synthesized by Sangon Biotech (Shanghai, China). The TIANamp Bacteria DNA kit was purchased from TIANGEN (Beijing, China). The TwistAmp Basic Kit for RPA reaction was purchased from TwistDx (Cambridge, UK). Lba Cas12a (Cpf1) and NEBuffer r2.1 were purchased from New England Biolabs (Beijing, China). Milenia HybriDetect for lateral flow detection was purchased from Milenia Biotec (Gießen, Germany).

Bacterial strain and clinical samples

N. gonorrhoeae reference strain (ATCC 43,069) was donated by Professor Xiaobing Zhang. N. gonorrhoeae clinical isolates (n = 8) were identified by Gram staining and mass spectrometry. 10 non-N. gonorrhoeae clinical isolates, including Neisseria sicca (N. sicca, n = 2), N. meningitidis (n = 2), Ureaplasma urealyticum (U. urealyticum, n = 1), Pseudomonas aeruginosa (P. aeruginosa, n = 1), Enterobacter cloacae (E. cloacae, n = 1), Klebsiella pneumoniae (K. pneumoniae, n = 1), Enterococcus faecium (E. faecium, n = 1), and Acinetobacter radioresistens (A. radioresistens, n = 1), were selected as negative controls. In addition, 24 clinical samples were collected from patients suspected of having gonorrhea, including cervical or vaginal discharge from women and urethral secretions from men. Sufficient clinical samples were manually separated into two equal halves, one for traditional culture (gold standard) and one for DNA extraction for molecular diagnosis using the RPA-Cas12a system. All the clinical isolates and samples described above were obtained from the First Affiliated Hospital of Chongqing Medical University.

RPA primers, crRNA and ssDNA reporter design

All the available porA pseudogene sequences of N. gonorrhoeae (Accession Numbers AJ010732.1, AJ010733.1, AJ223449.1, AJ223448.1, AJ223447.1, and AJ223446.1) were downloaded from the NCBI database (www.ncbi.nlm.nih.gov) and aligned by Mega-X (www.megasoftware.net) to identify the conserved regions. To distinguish N. gonorrhoeae from N. meningitidis, we aligned the conserved sequences of the porA pseudogene of N. gonorrhoeae with the porA gene of 228 N. meningitidis strains (the NCBI accession numbers of porA gene of N. meningitidis were listed in Additional file 1: Table S1). RPA primers (Additional file 1: Table S2) were designed in accordance with the TwistDx instruction manual (www.twistdx.co.uk) to cover the conserved porA pseudogene region that differs significantly from the porA gene sequences of N. meningitidis. IDT OligoAnalyzer (www.idtdna.com) (Kersting et al. 2014) was used to examine the primer dimer and hairpin structures. The primers’ specificities were verified by NCBI’s Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/ primer-blast). We designed crRNA (Additional file 1: Table S3) for Cas12a to recognize a 21-bp target sequence near the PAM site (TTTN, N = A/C/G) in the amplicon between RPA primer pairs, and checked the crRNA sequence using the BLAST algorithm. Fluorescent ssDNA reporter was labeled by fluorophore and quencher (Additional file 1: Table S4). Lateral flow ssDNA reporter was labeled by fluorophore and biotin (Additional file 1: Table S4).

Nucleic acid preparation

Bacterial genomic DNA was extracted with the TIANamp Bacteria DNA kit per the manufacturer's instructions. The DNA concentration ranged from 20 ng/µL to 100 ng/µL, and was stored at – 20 ℃ before use after being determined using a NanoDrop 2000 (Thermo, United States). For sensitivity testing, a solution containing genomic DNA from a N. gonorrhoeae reference strain was diluted to various concentrations with RNase free water.

The RPA reaction

To achieve robust assay performance and a specific RPA reaction, a comparative evaluation of the specificity of multiple candidate RPA primer pairs in identifying N. gonorrhoeae was carried out. Briefly, six candidate primer pairs were used to amplify N. gonorrhoeae and non-N. gonorrhoeae strain sequences, and the optimal primer pairs were evaluated by nucleic acid electrophoresis or the CRISPR/Cas12a-fluorescent assay. Using a commercial RPA kit, the RPA reaction was carried out at 39 ℃ for 15–20 min (Table 1) according to the instructions.

Table 1 The RPA reaction systemRPA-Cas12a-fluorescent assay

Fluorophore and quencher-labeled ssDNA reporter was introduced into the Cas12a-fluorescent system. Following the RPA reaction, the RPA product was added as substrate into the Cas12a-fluorescent system. The RPA-Cas12a-fluorescent reaction was carried out at 37 °C in a final volume of 20 µL (as specified in Table 2). The fluorescence signal was examined by TECAN infinite 200Pro (TECAN, Switzerland) with an excitation wavelength of 492 nm, and an emission wavelength of 522 nm. The optimization of cleavage time within the range of 5 to 55 min was undertaken to attain desirable performance of the CRISPR/cas12a system.

Table 2 The RPA-Cas12a assay systemRPA-Cas12a-LFD assay

Except for the ssDNA reporter, the RPA-Cas12a-lateral flow detection (LFD) system contained the same components as the RPA-Cas12a-fluorescent assay (Table 2). In addition, after CRISPR/Cas12a cleavage at 37 °C, the CRISPR/Cas12a reaction product was diluted proportionately in Hybri Detect Assay Buffer to meet the solution volume requirements for LFD. The strips were then inserted and incubated at room temperature for 2 min before being withdrawn and photographed. ImageJ (https://imagej.nih.gov/ij/) quantification and GraphPad (www.graphpad.com) visualization were used to examine the band intensities of the test line of the strips. Several parameters of the RPA-Cas12a-LFD system were optimized, such as the ssDNA concentration (3.125 nmol/L, 6.25 nmol/L, 12.5 nmol/L, 25 nmol/L, 62.5 nmol/L, 125 nmol/L, 250 nmol/L), the volume ratio of Cas12a cleavage product to Hybri Detect Assay Buffer (1:1, 1:3). Commercial strips were used for lateral flow detection. The Milenia HybriDetect kit contains the Hybri Detect Assay Buffer and lateral flow strips.

Specificity and sensitivity of the RPA-Cas12a system

Using DNA templates from the N. gonorrhoeae reference strain (ATCC43069), eight N. gonorrhoeae clinical isolates and ten non-N. gonorrhoeae clinical isolates (including N. sicca, N. meningitidis, U. urealyticum, P. aeruginosa, E. cloacae, K. pneumoniae, E. faecium, A. radioresistens), the specificity of the RPA-Cas12a system was evaluated. Following a 20-min RPA reaction, the RPA products were added into both the Cas12a-fluorescent system and the Cas12a-LFD system.

The sensitivity of the RPA-Cas12a system was evaluated using serial dilutions of genomic DNA of the N. gonorrhoeae reference strain (500 pg/µL, 50 pg/µL, 5 pg/µL, 500 fg/µL, 50 fg/µL, 5 fg/µL). The RPA products were added into both the Cas12a-fluorescent system and the Cas12a-LFD system following a 20-min RPA reaction.

Clinical validation of the RPA-Cas12a system

The feasibility of the RPA-Cas12a system was evaluated using 24 clinical samples from individuals suspected of having gonorrhea, with traditional culture as a reference method. Sufficient clinical samples were manually separated into two equal halves, one for traditional culture, and the other for DNA extraction for molecular diagnosis using RPA-Cas12a-fluorescent and RPA-Cas12a-LFD assays. In other words, all clinical samples were evaluated using the aforementioned three methods, and the test results were compared with those of the culture method. Culturing was performed on Thayer-Martin (TM) medium in 5% CO2 at 37 °C for 24–72 h (Su et al. 2011).