Recombinase Polymerase Amplification Assay with Lateral Flow Strips for Rapid Diagnosis of Candidiasis due to Candida parapsilosis

Abstract

Background

Candida parapsilosis is a common cause of candidiasis among hospitalized patients, often surpassing Candida albicans. Due to the recent increase in C. parapsilosis infections, there is an urgent need for rapid, sensitive, and real-time on-site detection of nucleic acids for timely diagnosis of candidiasis.

Methods

We developed an assay for detection of C. parapsilosis by combining recombinase polymerase amplification (RPA) with a lateral flow strip (LFS). The RPA-LFS assay was used to amplify the beta-1,3-glucan synthase catalytic subunit 2 (FSK2) gene of C. parapsilosis with a primer-probe set optimized by introducing base mismatches (4 bases modified by the probe and one by the reverse primer) to achieve specific and sensitive detection of clinical samples.

Results

The RPA assays can rapidly amplify and visualize a target gene within 30 min, while the entire process can be completed within 40 min by pre-processing the sample.The sensitivity and specificity of the RPA-LFS assay were determined by analysis of 35 common clinical pathogens and 281 clinical samples against quantitative PCR.

Conclusions

The results confirmed that the proposed RPA-LFS assay is a reliable molecular diagnostic method for the detection of C. parapsilosis to meet the urgent need for rapid, specific, sensitive, and portable field testing.

Background

Candida parapsilosis is a common cause of candidiasis in low birth weight neonates and critically ill patients who are mainly infected during invasive procedures, such as central venous catheter placement, surgery, and parenteral nutrition [1, 2]. The incidence of invasive candidiasis has increased in recent decades and the detection rate of candidiasis caused by C. parapsilosis exceeds that of Candida albicans in some hospitals in Europe, Asia, and South America [3].

Traditional fungal culture methods are the gold standard for the diagnosis of fungal infectious diseases. With the continued development of molecular diagnostic techniques, such as polymerase chain reaction (PCR)-restriction fragment length polymorphism based on DNA amplification [4], random amplified polymorphic DNA [5], real-time PCR [6], PCR for intron length analysis of polymorphisms [7], and matrix-assisted laser desorption ionization time-of-flight mass spectrometry [8], sequencing analysis of pan-fungal markers has been applied for clinical detection of C. parapsilosis [9]. Meanwhile, isothermal amplification is more frequently utilized in field testing, as this method is not limited by instrumentation and laboratory settings. The main isothermal amplification techniques currently available include loop-mediated isothermal amplification(LAMP) [10], nuclear acid sequence-based amplification(NASBA) [11], rolling circle amplification (RCA) [12], single primer isothermal amplification (Yang et al., 2021)(SPIA), helicase-dependent isothermal DNA amplification(HDA) [13], and strand displacement amplification (SDA) [14]. Recombinase polymerase amplification (RPA) is a recently developed technique that merges the advantages of thermostatic amplification and compensating for the shortcomings as a rapid, specific, sensitive, and portable diagnostic assay.

RPA uses recombinase junctions that bind specific primers to open the double-stranded helix to bind to the target fragment, single-stranded DNA binding protein wraps single-stranded DNA with high affinity to protect against degradation and secondary structure formation, and the polymerase of Bacillus subtilis recognizes the 3' end of the primer for rapid amplification(Supplementary Figure S1A&B), which can produce a large number of amplification products in about 20 min at 30–45°C [15]. The amplification products of RPA can be detected by gel electrophoresis, a fluorescence detector, or with the use of a lateral flow strip (LFS). However, gel electrophoresis and fluorescence detection is limited to laboratory settings. In contrast, LFS is suitable for in situ detection in field conditions and the results can be directly observed without a dedicated display device or the need for sophisticated thermal cycling equipment and highly trained technicians. Visualization of a LFS is dependent on labeling of the 5' and 3' ends of the probe with fluorescein isothiocyanate (FITC) and C3 blocking sites, respectively, while the reverse primer is labeled with biotin. Binding of the probe to the amplification strand is performed by cleavage of the middle tetrahydrofuran (THF) site of the probe by endonuclease IV (nfo gene of Escherichia coli K-12) to release the 3' end of the probe for extension and the 5' and 3' ends of the final amplification product are labeled with FITC and biotin, respectively. Gold nanoparticle (AuNP)-labeled anti-FITC antibody binds to the amplification product and is subsequently captured and aggregated by streptavidin on the detection line to produce a red positive signal, while the secondary antibody on the quality control line captures the AuNP-labeled anti-FITC antibody without binding the amplification product and produces a red signal regardless of the presence or absence of the amplification product(Supplementary Figure S1C) [16, 17]. The RPA-LFS assay can achieve fast response times and good accuracy for the diagnosis of various infectious diseases.

In our study, RPA and qPCR primers were designed for the diagnosis of C. parapsilosis by FSK2 sequence. To test the effectiveness of the assay system, primer and probe specificity screening, sensitivity testing, specificity analysis, and testing of clinical samples was performed to ensure that our established method could be used for the diagnosis of Candida infections caused by C. parapsilosis.

Methods

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 MonAmp^TM 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).

Results

Primer screening and validation

FSK2 was selected from the C. parapsilosis genome as a target gene for RPA-LFS detection. NCBI Primer-BLAST yielded two potential primer pairs for amplification of FSK2 (Table 1). These primers were initially screened by amplification of target gene fragments and a no template control (NTC). The amplification products were separated by electrophoresis on an agarose gel to compare the amplification performance of the target gene with the formation of primer-dimers by the NTC. The best amplification performance was obtained with the primer pair F2/R2 and no primer dimer was formed (Figure 1A). The candidate probe was obtained by extending the 3' end of the forward primer F2 by 16 bp. All possible cross-dimers generated by this probe and the reverse primer were predicted for subsequent modification of the base sequence (Table 1) until no dimer could be formed (Figure 1B). Finally, the five forward primers were screened and tested upstream of the probe. The LFS results showed that the primer/probe sets F3/P/R2B, F4/P/R2B, F5/P/R2B, and F6/P/R2B effectively amplified the target gene fragment, while the negative controls F3/P/R2B, F4/P/R2B, and F5/P/R2B (NTC) produced false-positive signals. Hence, only F6/P/R2B met the requirements of the assay and had effectively amplified the FSK2 fragment without amplification as a NTC (Figure 1C). Therefore, the primer/probe set F6/R2/P was used in the subsequent experiments.

Sensitivity of the RPA-LFS assay

To determine the LOD of C. parapsilosis, a 10-fold gradient dilution was tested from 10⁰ to 10⁵ CFU/µL. A red band appeared on the test line at 10 CFU/µL. Moreover, the red band became darker with an increasing concentration of the C. parapsilosis solution (Figure 2A). To determine whether the system is resistant to interference from other fungal DNA, 10⁵ CFU/µL of C. albicans were added to the RPA reaction. The results showed that the C. albicans inactivation solution did not interfere with the detection of C. parapsilosis (Figure 2B). Hence, the LOD of the RPA-LFS system was 10 CFU/50 µL per reaction and the presence of other fungi did not interfere with the detection sensitivity. However, not all assays yielded positive results when using 10⁰ (75% positive results, 6/8 samples) and 10 (12.5%, 1/8 samples) CFUs. To determine the exact LOD of the RPA-LFS assay, probit regression analysis of data from eight independent assays was performed using SPSS software (SPSS, Inc., Chicago, IL, USA). At 95% probability, the lowest LOD was 11.7 CFU per reaction (Figure 2C).

Interspecies specificity of the RPA-LFS assay

To confirm the inclusivity and specificity of the primer-probe set F6/P/R2B, four reference strains, 17 clinical isolates, and other pathogenic bacteria were amplified（Table 2）. The four reference strains and 17 clinical isolates produced positive results (Figure 3), while the results for all other pathogenic cultures were negative (Figure 4A&B), indicating that the primer-probe set was specific for C. parapsilosis without cross-reactivity with other pathogens.

Analysis of clinical samples

For verification, 281 clinical samples were collected for the RPA-LFS and qPCR assays, respectively. The results are shown in Table 3. The detection rate of 89 C. parapsilosis isolates was 31.7%, consistent with the detection results of qPCR and traditional culture methods. The experimental results showed that the accuracy of the RPA-LFS assay was the same of that of qPCR and consistent with traditional culture methods.

Discussion

The incidence of hospital-acquired infections caused by Candida spp. continues to increase, most dramatically with C. parapsilosis, which is responsible for 10–25% of Candida infections of neonates and intensive care patients. C. parapsilosis infections are associated with the use of medical devices, especially central venous catheters, parenteral nutrition administration, and exposure to healthcare workers [18, 19].

In addition to traditional fungal culture methods to identify C. parapsilosis, molecular diagnosis based on nucleic acid detection is gaining importance, in particular, isothermal amplification technology and real-time qPCR technology. Isothermal amplification techniques can be divided into two categories based on the purpose of amplification: specific amplification and non-specific amplification. Besides RPA, LAMP, NASBA, SPIA, SDA, HDA, and RCA are also included. Among these amplification methods, only RPA is the only one that can automate isothermal amplification, while the others require DNA annealing and other necessary initial operations to achieve isothermal amplification, so RPA has a significant advantage in practical applications [20]. Real-time qPCR is specific and sensitive for large-scale detection of nucleic acids, especially for detection of novel coronaviruses, which are currently prevalent worldwide [21, 22]. Due to the large-scale screening of nucleic acids conducted in various locations, the demand for laboratory testing by trained professionals continues to increase. In addition, most screening methods require expensive instruments for nucleic acid extraction and amplification. Moreover, aerosol contamination is a potential problem associated with testing of nucleic acids in a laboratory setting.

RPA is an effective complement to qPCR for testing of nucleic acids and can be used for home testing of patients or on-site testing in harsh conditions. Notably, this method allows for exponential amplification of nucleic acids at 37°C without the need for expensive thermal sequencing instruments and professional laboratory personnel. Enzyme-linked immunosorbent assays are suitable for in vitro quantitative detection of proteins as markers of pathogens in cell cultures, serum, plasma, and other biological fluids. The double antibody sandwich method to detect antigenic proteins employs a microtiter plate with wells coated with a high-affinity biotinylated antibody for detection of the sample or standard. The presence of the target protein in the sample is detected by a colorimetric reaction with a substrate solution that is terminated by the addition of a termination solution [23]. However, this method does not allow effective detection at the early stage of disease, thus delaying prognosis and threatening the health of the patient. To compensate for this shortcoming, RPA is a highly sensitive, rapid, specific, and portable method for detection of C. parapsilosis.

Molecular detection techniques require the selection of diagnostic amplification targets to effectively detect specific species. Many studies have evaluated various methods for detection of C. parapsilosis. In the present study, a primer-probe set was designed to target FKS2 of C. parapsilosis. The results showed that there was no significant effect on the LOD for base mismatching and that the RPA-LFS assay accurately detected C. parapsilosis. The LOD of the RPA-LFS assay was 10 CFU, which was more sensitive than qPCR at 10⁰–10 CFU per reaction. In addition, the RPA-LFS assay for detection of C. parapsilosis is simple and rapid, as the assay can be performed in 30 min or less. In addition, this method requires an isothermal temperature of only 37°C, whereas PCR, qPCR, and loop-mediated isothermal amplification require temperature control equipment and relatively long reaction times. Evaluation of clinical samples showed that the RPA-LFS assay was specific. Testing of samples from different patients showed that the results of the RPA-LFS and qPCR assays were comparable, demonstrating that the RPA-LFS assay is a rapid and useful alternative.

Conclusion

The RPA-LFS assay developed in this study is a rapid highly specific and sensitive molecular technique for diagnosis of C. parapsilosis infection. Importantly, the test results are available within 40 min without the use of expensive instruments or trained laboratory personnel. Since testing can be conducted on-site, the proposed assay is useful for rapid detection of Candida spp. The established RPA-LFS assay is simple, rapid, and accurate, does not require laboratory facilities, and can be combined with a minimal and rapid DNA extraction method for home detection of C. parapsilosis infections for timely diagnosis to facilitate early treatment.

Declarations

Ethics approval and consent to participate

The study protocol was approved by the Medical Ethics Committee of the Second People's Hospital of Lianyungang City (Lianyungang, Jiangsu, China) (permit number 2020013) and informed consent was obtained from patients prior to collection of clinical samples. All methods were carried out in accordance with relevant guidelines and regulations.

Consent for publication

Not applicable.

Availability of Data and Material (ADM)

The data presented in this study are included in the article. Further inquiries should be directed to the corresponding authors.

Competing interests

The authors declare that they have no competing interests.

Funding

This study was supported by grants from the Jiangsu Postgraduate Research and Practice Innovation Program in 2022(grant no. KYCX22_3855), the China Agriculture Research System of MOF and MARA, the Guangxi Innovation-driven Development Project (grant no. AA19182012-2), National Key R&D Program of China, the Key Projects of International Scientific and Technological Innovation Cooperation (grant no. 2021YFE0111100), the Zhenjiang Science and Technology Support Project (grant no. GJ2021015), the Lianyungang Science and Technology Bureau, Municipal Science and Technology Plan (Social Development) Project (grant no. SF2140), the Lianyungang City Health Science and Technology Project (grant no. 202122), and the Jiangsu University Clinical Medicine Science and Technology Development Fund Project (grant no. JLY2021088).

Authors' contributions

LW, XZW, and KW designed the experiments and wrote the manuscript.YYL and YW collected the clinical samples. YW, SHF, and LW performed the main experiments. LH analyzed the data. All authors reviewed and approved the final version of the manuscript.

Acknowledgment

We thank International Science Editing (http://www.internationalscienceediting.com) for editing this manuscript.

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Tables

Supplementary Figures

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