Strain acquisition
C. parapsilosis (C. parapsilosis means C. parapsilosis sensu stricto unless otherwise specified) ATCC 22019/90018/200954/7330 was purchased from Shanghai Covey Chemical Technology Co., Ltd. (Shanghai, China) and 15 strains of C. parapsilosis were isolated from clinical samples collected from 2020 to 2021, and the authenticity of the strain was verified by qPCR. The specificity of the RPA-LFS assay was verified based on the FSK2 gene (GenBank: EU221326.1) of 35 common pathogens stored in our laboratory, which included Candida tropicalis ATCC 20962, C. albicans ATCC 10231, Candida auris, Candida dubliniensis, Candida krusei, Candida glabrata, Aspergillus fumigatus, Cryptococcus neoformans ATCC 14116, Enterococcus faecium, E. coli O157, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus capitis, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus saprophyticus, Staphylococcus warneri, Stenotrophomonas maltophilia, Streptococcus pneumonia, Viridans streptococci, Klebsiella pneumoniae, and Acinetobacter baumannii ATCC 1960, Candida metapsilosis, Candida orthopsilosis, Cryptococcus gattii, Acinetobacter calcoaceticus, Acinetobacter lwoffi, Acinetobacter haemolytius, Acinetobacter junii, Acinetobacter johnsonii, Enterobacter cloacae, Mycobacterium tuberculosis H37Ra, Listeria monocytogenes, Neisseria meningitidis. In total, 281 samples (Blood specimen 124, sputum specimen 107, pus specimen 28, dermal specimen 22) were collected from patients with suspected Candida infection.
Genomic DNA extraction
All bacterial strains were boiled at 100°C for 10 min to release DNA for use as templates. If not otherwise specified, 1 μL of 105 colony-forming units (CFU)/mL of heat-treated culture was used as a template. For C. parapsilosis and other fungi, genomic DNA was extracted and purified from cultures or clinical samples using the GeneJET Genomic DNA Purification Kit (Thermo Fisher Scientific, Waltham, MA, USA) in accordance with the manufacturer's instructions and quantified using a Invitrogen™ Qubit™ 4 Fluorometer (Thermo Fisher Scientific).
Object and probe design and screening
Two primer pairs based on the FSK2 gene were designed for RPA using Primer Premier 5 software (Premier Biosoft, Palo Alto, CA, USA) with the following parameters: product size, 80–150 bp; primer size, 30–35 bp; complementary pairing, ≤ 3 consecutive bases at the 3ʹ end; maximum hairpin fraction, 5; maximum primer-dimer fraction, 5; and maximum poly-X, 5. The primers were designed based on sequences retrieved from the National Center for Biotechnology Information (NCBI) database (https://www.ncbi.nlm.nih.gov/). The species specificity of the primers and probes were confirmed using the NCBI primer designing tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast). The performance of the forward primer, which was extended backward by 15–23 bp, was evaluated to theoretically avoid the formation of dimers and hairpin structures using Primer Premier 5 software. The size of the probe was 46–53 bp with a GC content of 30%–80% and melting temperature of 57–80°C. The 5' end of the probe was labeled with FITC, the 3' end was closed with a C3 spacer, the bases in the middle of the probe (at least 30 bases before the THF site and at least 15 afterward) were replaced with THF, and the 5' end of the reverse primer was labeled with biotin.
RPA reaction
RPA reactions were performed using the TwistAmp® Liquid DNA Amplification Kit (TwistDx Inc., Maidenhead, UK) in accordance with the manufacturer's instructions. Each 50-µL reaction system contained 25 µL of 2× reaction buffer, 5 µL of 10× E-mix, 2.5 µL of 20/ core mix, 2.4 µL of 10 µM forward primer, 2.4 µL of 10 µM reverse primer, and 9.2 µL of distilled water, in addition to 2.5 μL of 280 mM magnesium acetate and 1 μL of template to the top of the reaction tube. After a short centrifugation step, the reaction mixture was incubated at 37°C for 30 min. The RPA amplification products were purified using the PCR Cleaning Kit (Shanghai MEIJI Biotechnology Co. Ltd.) and then separated by electrophoresis on a 2% agarose gel.
RPA-LFS assay
RPA reactions were performed using the Twist Amp® DNA amplification nfo kit (TwistDx Ltd.) in accordance with the manufacturer's instructions. Each 50-μL reaction mixture contained 2.1 μL of each primer (10 μM), 0.6 μL of the probe (10 μM), 2.0 μL of the template, and other standard reaction components. Primers and probes were synthesized by Anhui General Biotechnology Co., Ltd (Chuzhou, China). Magnesium acetate (280 mM, 2.5 μL) was added to initiate the reaction prior to incubation of the reaction mixture at 37°C for 20 min. Then, 5 μL of the amplification product were diluted 20-fold and spotted on the LFS (USTAR Biotechnology Co., Ltd., Hangzhou, China). The LFS consisted of a sample pad, a gold-labeled antibody pad (soaked with mouse-derived AuNP-labeled anti-FITC antibody), a test line (coated with streptavidin), a control line (coated with anti-mouse antibody), and an absorption pad, arranged by the solvent migration pathway. The RPA amplification product was added to the sample pad of the LFS and the LFS was submerged into 100 μL of solvent for approximately 2 min until the test and control lines became visible.
Sensitivity and specificity of the RPA-LFS assay
A 10-fold gradient dilution was tested from 100 to 105 CFU/µL (reaction volume of 50 µL containing 1 µL of C. parapsilosis inactivation solution) and 105 CFU/µL of an inactivating solution of another common pathogen (C. albicans) were prepared for the RPA-LFS reaction. The lower limit of detection (LOD) of the method was determined with a probit regression analysis of 10 independent experiments. To verify the specificity of the assay system, 35 common clinical fungi and bacterial were selected for RPA-LFS testing.
Quantitative PCR (qPCR) analysis
The primers and probes for qPCR analysis are listed in Table 1. Specific primers and probes were targeted to FSK2 of Candida subtilis for qPCR detection. Each qPCR reaction mixture consisted of 12.5 μL of MonAmpTM Taqman qPCR mixture (Tiangen Biotechnology Co., Ltd., Beijing, China), 0.5 μM forward and reverse primers, 0.2 μM probe, 1 μL of genomic DNA, and distilled water to a final volume of 25 μL. The cycling program consisted of an initial denaturation step at 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and 55°C for 60 s. The qPCR reactions were conducted using a LightCycler® 480 System (Roche Diagnostics GmbH, Mannheim, Germany).