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Rapid and supersensitive allele detection of Plasmodium falciparum chloroquine resistance via a Pyrococcus furiosus argonaute-triggered dual-signal biosensing platform

Parasites & Vectors volume 17, Article number: 488 (2024) Cite this article

Abstract

Background

Malaria remains a serious public health problem worldwide, particularly in Africa. Resistance to antimalarial drugs is an essential issue for malaria control and elimination. Currently, polymerase chain reaction (PCR) combined with Sanger sequencing is regarded as the gold standard for mutation detection. However, this method fails to meet the requirements of point-of-care testing (POCT) because of its time-consuming, expensive instruments and professional dependence. To support this strategy, we developed a novel diagnostic platform that combines recombinase polymerase amplification (RPA) with the Pyrococcus furiosus argonaute (PfAgo) protein and was designed to detect gene mutations related to antimalarial drug resistance. The Pfcrt haplotypes CVMNK and CVIET of chloroquine resistance (CQR) were used as examples and were assessed in this study.

Methods

By meticulously designing strategies, RPA primers, guide DNAs, and probes were screened, the reaction was optimized, and the resulting parameters were employed to ascertain the genotype of Pfcrt. The recombinant plasmids pUC57/Pfcrt-CVIET and pUC57/Pfcrt-CVMNK were constructed and diluted for sensitivity detection. The pUC57/Pfcrt-CVIET plasmid mixture was added to the pUC57/Pfcrt-CVMNK plasmid mixture in different additions to configure several specific proportions of mixed plasmid mixtures. The RPA-PfAgo platform was used, and the mixed plasmid was detected simultaneously via nest-PCR (nPCR) and Sanger sequencing. The platform was then evaluated on 85 clinical samples and compared with Sanger sequencing.

Results

The entire process achieves the key mutation Pfcrt-CVMNK/CVIET genotype identification of CQR within 90 min. The platform achieved 1.8 × 104 copies/μL sensitivity and could detect as little as 3% CVIET in mixed plasmids, which is a higher sensitivity than that of Sanger sequencing (5%). Notably, the platform shows 100% concordance with the gold standard method when 85 clinical samples are tested. The sensitivity and specificity were 100% for the 85 clinical samples.

Conclusions

This study established an RPA-PfAgo platform for genotyping the key mutation Pfcrt-CVMNK/CVIET of CQR. This method can rapidly produce reliable results and avoid the disadvantages of nPCR with sequencing. This approach has the characteristics of a short operation time, low device dependence, and a good match to the POCT strategy, suggesting that the platform can be easily applied locally or on site.

Graphical abstract

Background

Malaria is recognized as a major public problem worldwide, and it is estimated that more than 600,000 individuals will die annually [1]. Most of them are centralized in resource-poor countries and areas, particularly in sub-Saharan Africa and Southeast Asia [2]. For malaria control, treatment with effective, safe and affordable antimalarial drugs is largely needed. The emergence and rapid spread of drug resistance have severely compromised this strategy and have been linked to increases in malaria-associated morbidity and mortality. Chloroquine (CQ), the least expensive basic antimalarial drug, has lost its efficacy in most parts of Africa [3]. Pfcrt haplotypes associated with CQ resistance (CQR) in natural parasite isolates harbor threonine (T), as opposed to lysine (K), at amino acid 76 P. falciparum CQR strains carrying the CVIET haplotype (residues 72–76), and chloroquine-sensitive strains with the CVMNK haplotype are commonly detected in Africa. In contrast, CQR strains with the SVMNT haplotype are rarely detected in Africa [4]. Accurate confirmation of these single-nucleotide polymorphism (SNP) loci and alleles will aid in medication guidance [5, 6].

Nest-PCR (nPCR) and Sanger sequencing are considered the recommended methods for identifying Plasmodium parasite mutations related to antimalarial drug resistance [7]. Compared with authentication by sequencing DNA fragments, PCR-restriction fragment length polymorphism (PCR–RFLP), allele-specific diagnostic PCR (AS-PCR) [2, 8], and real-time PCR are practical and effective, but they require sophisticated thermal cyclers with trained personnel, which makes their use difficult in underequipped laboratories and low-resource field settings [7, 9, 10]. Thus, they fail to meet the requirements of the point-of-care testing strategy.

To overcome these limitations, new alternative genotyping methods for key mutations, especially isothermal amplification technologies combined with programmable endonuclease-based methods, have been developed [11,12,13,14]. Furthermore, prokaryotic argonaute is the key protein in the host defense system that functions by mediating nucleic acid molecules [15,16,17]. As a DNA-guided endonuclease from Pyrococcus furiosus, the Pyrococcus furiosus argonaute (PfAgo) prefers to cut target cognate DNA under the guidance of short 5′-phosphorylated single-strand DNA without the need for a protospacer-adjacent motif (PAM) or PFS (protospacer flanking site) in the target sequence, which largely extends its application in the selection of available target DNA sequences [18, 19]. Recently, it has been applied to the molecular detection of the key SARS-CoV-2 mutation L452R [14], which was identified in delta and omicorn BA.5 variants [20, 21]. However, PfAgo-based technology has yet to be applied to identify the genotypes of antimalarial drug resistance genes.

In this study, a PfAgo-triggered dual-signal biosensor combined with recombinase polymerase amplification was developed and evaluated for rapid and sensitive genotyping of the key mutation Pfcrt-CVMNK/CVIET haplotype of CQR. The platform can also be applied to identify Plasmodium species and the genotypes of other antimalarial drug resistance genes.

Methods

Overall workflow of the RPA-PfAgo platform

For rapid and sensitive genotyping of the Pfcrt mutant haplotype in the field, a dual-signal biosensor detection platform triggered by RPA combined with PfAgo was developed (Fig. 1). After blood samples were collected from suspected malaria patients, genomic DNA was rapidly extracted via the Chelex-100 method [22]. The target sequence is amplified via RPA technology. The product was subsequently identified by the PfAgo cleavage system. With the guidance of gDNA mutation (gDNA-M) and gDNA-common (gDNA-C), PfAgo can specifically cleave one strand of the amplified product fragment that is complementary to the gDNA-M sequence (the first cleavage). This cleavage produces another ssDNA fragment with 5′ phosphorylation, which serves as the new gDNA to guide PfAgo to perform the second cleavage. The substrate of the second cleavage is the fluorescent probe, the ssDNA reporter; its markers are the ROX fluorophore and the BHQ2 quencher. The fluorescent reporter was designed to confirm the production of the first cleavage. The specific cleavage of the fluorescent reporter by PfAgo releases ROX from BHQ2, producing a fluorescent signal. For the amplified CVMNK fragment, because there is no strand complementary to gDNA-M, the first cleavage cannot occur, and there is no fluorescence signal. Moreover, we designed the corresponding gDNA-wild-type (gDNA-W) strain and another reporter with the FAM fluorophore and the BHQ1 quencher for the wild-type strain. Once the CVMNK amplification product is present, it will produce another kind of fluorescent signal. By using these methods, we detected the CVMNK and CVIET fragments simultaneously in the same reaction mixture containing orthogonal PfAgo proteins, and the orthogonal system yielded two independent channel signals without interfering with each other.

Fig. 1
figure 1

A dual-signal biosensor detection platform triggered by RPA-PfAgo for Pfcrt haplotype detection

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PfAgo protein expression and purification

The purification of the PfAgo protein (NCBI-Protein ID: WP_011011654.1) was performed following a protocol reported previously [17]. In brief, codon optimization of the PfAgo gene was performed via JCat software (http://www.jcat.de/). The PfAgo gene was subsequently obtained via gene synthesis and subsequently cloned and inserted into the pET28a ( +) plasmid for recombinant protein expression. Finally, the expression of the His-tag fusion protein was induced in the Escherichia coli BL21 (DE3) strain with 1 mM IPTG at 37 °C, after which the protein was purified through Ni-affinity chromatography on an AKTA Prime Plus system (GE Healthcare Life Sciences, Boston, MA). Storage buffer (20 mM of Tris–HCl, pH 8.0; 300 mM of NaCl; 0.5 mM of MnCl2; 15% (v/v) glycerol) was used to maintain the eluted purified protein, and aliquots were stored at −80 °C for further use.

Primer design and screening

The sequence of the Pfcrt gene (PF3D7_0709000) of the P. falciparum 3D7 strain was obtained from PlasmoDB (http://plasmodb.org/plasmo/, Release 56, 15 Feb 2022). A series of RPA primers (F1-F3, forward; R1-R3, reverse) were designed according to the RPA design manual (www.twistdx.co.uk) and Primer-BLAST, combining Primer Premier 5.0 software (Premier Biosoft, San Francisco, CA, USA) and BLAST (Basic Local Alignment Search Tool, National Center for Biotechnology Information) global alignment. Three forward primers and three reverse primers were cross-assembled into nine combinations (Table S1) and used for RPA amplification, which was conducted via a TwistAmp® Basic Kit (TwistDx, Cambridge, UK). The 50 μL final reaction mixture contained 2.4 μL of each forward primer and reverse primer (10 μM), 2.0 μL of MgAc (280 mM), 29.5 μL of rehydration buffer, 2.0 μL of genomic DNA, lyophilized enzyme pellets, and free water. The reaction mixture was turned up and down 10 times to ensure full mixing. The reaction time and temperature were 20 min and 37 °C, respectively. Isothermal amplification was carried out on a Life Touch Thermal Cycler (Bioer Technology Co., Ltd., China), after which all amplification products were purified via a TIANquick Midi Purification Kit (TIANGEN Biotech Co., Ltd., Beijing, China) and analyzed on a 2% agarose gel.

Design and screening of guide DNA and ssDNA reporters

Guide DNA (gDNA) is the key factor in the PfAgo cleavage system because it guides the specific recognition of a particular sequence (in the case of this study, a sequence containing the mutant CVIET fragment) in the target by PfAgo, which in turn activates the nuclease. According to the sequence of the amplification region of the optimal primers, two combinations, 5′-phosphorylated gDNA for PfAgo (16 nt in length) and ssDNA reporters with FAM-BHQ1 or ROX-BHQ2 modifications at the two ends, were manually designed for a total of three groups (Table S1). The ssDNA reporter had a 16 nt region to potentially align to the gDNA cleavage product for the second cleavage. All of the oligonucleotides used are listed in Table S1 and were synthesized by GENEWIZ (Suzhou) Co., Ltd., China. The amplification products of the selected primers were used as target fragments to screen three groups of gDNAs via PfAgo cleavage experiments. The optimal gDNA combination should be able to clearly distinguish between Pfcrt-CVMNK and Pfcrt-CVIET, and the target fragment should be clearly cleaved.

Plasmid construction

Two kinds of plasmids containing 515 bp fragments of the Pfcrt gene were designed and constructed by GENEWIZ (Suzhou) Co., Ltd., China. The two recombinant plasmids used were pUC57/Pfcrt-CVMNK (wild-type) and pUC57/Pfcrt-CVIET (mutant-type). The genes were subsequently validated through Sanger sequencing. DNA concentrations were quantified via a NanoDrop™ ND-2000 spectrophotometer (Thermo, Wilmington, USA). The quantity of plasmid DNA was calculated via the following formula: plasmid DNA copy number (copies/μL) = (OD260 × 10−9 × 6.02 × 1023)/(n × 660), where n represents the plasmid length. Serial tenfold dilutions were performed with the above plasmids, covering a wide range of concentrations (from 1.8 × 104 to 1.8 × 1010 copies/μL).

RPA-PfAgo platform cleavage assay

By referencing the results of other studies [19, 23,24,25,26], the reaction system of the RPA-PfAgo platform was initially established. The 25 μL PfAgo reaction mixture contained 4 μL of purified RPA products, 6 μL (200 U/L) of PfAgo, 2 μL (20 μM) of gDNA-common (gDNA-C1), 1 μL (20 μM) of gDNA-WT (gDNA-W), 1 μL (20 μM) of gDNA-MT (gDNA-M), 1 μL (10 μM) of each signal-producing ssDNA reporter (Probe-W1 and Probe-M1), 3 μL (40 mM) of MnCl2, 4 μL of nuclease-free H2O and 2 μL of endonuclease reaction buffer (15 mM of Tris/HCl pH 8.0, 250 mM of NaCl). The fully mixed reaction mixture was incubated for 30 min at 95 °C in a SLAN-96S Real-Time PCR Detection System (Shanghai Hongshi Medical Technology Co., Ltd., Shanghai, China), after which the FAM and ROX dual fluorescence signals were recorded at 30 s intervals. After the reaction, the reaction tubes were observed by a BLT GelView 6000Plus (Guangzhou Biolight Biotechnology Co., Ltd., Guangzhou, China) with a blue emission filter (470 nm/520 nm) and a green emission filter (530 nm/600 nm) or directly irradiated by a simple ultraviolet (UV) lamp in dark, confined space and observed with the naked eye.

Optimization of the PfAgo cleavage reaction

To improve the overall performance of the RPA-PfAgo reaction, the concentrations of gDNA, probes, MnCl2, and PfAgo were optimized (Supplementary Materials). In this study, the optimal conditions were determined by observing the fluorescence curve and fluorescent tube signal. After a series of optimization experiments, as described in the Supporting Information, an integrated RPA-PfAgo cleavage platform was established.

Urea-denaturing PAGE

The PfAgo cleavage products were verified via a urea-PAGE gel. The urea-PAGE mixture consisted of three layers: the lowest layer was 15% urea-PAGE (2.5 mL of 15% urea-PAGE gel solution, 12.5 μL of 10% APS, 2.5 μL of TEMED); the middle layer was 12% urea-PAGE (12% urea-PAGE gel solution, 4.0 mL; 10% APS, 20.0 μL; TEMED, 4.0 μL); and the top layer was a concentrated gel (urea, 1.47 g; PAA, 350 μL; 5% TBE, 700 μL; APS, 35 μL; and TEMED, 3.5 μL of ddH2O added to 3.5 mL). Urea-PAGE was carried out at 200 V for 30 min before use, and the sample was loaded at 200 V for 15 min and 150 V for 30 min. After electrophoresis, the urea-PAGE gel was decolorized in PAGE staining solution (Zhongke Ruitai (Beijing) Biotechnology Co., Ltd., Beijing, China) for 30 min and imaged.

Nested PCR and sequencing

The classical nested PCR primers targeting Pfcrt were synthesized as previously described [27, 28] by GENEWIZ (Suzhou) Co., Ltd., China. TaKaRa Taq™ HS Perfect Mix (TaKaRa, Carlsbad, CA) was used as the master mixture, and the mixture was supplemented with each primer at a concentration of 0.2 μM. The 25 μL primary PCR mixture contained 12.5 μL of master mix plus 1 μL of DNA template. 1 L μL of PCR products obtained from the primary round were used as DNA templates for the second round of amplification. The 50 μL reaction mixture included 25 × master mix plus 1 μL of primary PCR product. The reaction conditions for rounds one and two are presented in Table S2. All the PCR products were analyzed via 1.0% agarose gel electrophoresis, and DNA sequencing was performed via an ABI 3730XL automated sequencer (PE Biosystems, CT, USA). Chromas software v2.6.6 was used, and the obtained sequences were aligned via the BioEdit Sequence Alignment Editor (version 7.0.5) for variations.

Sensitivity validation

The pUC57/Pfcrt-CVIET and pUC57/Pfcrt-CVMNK plasmids were gradient diluted (concentrations ranging from 1.8 × 104 to 1.8 × 1010 copies/μL) and used to validate the sensitivity of the RPA-PfAgo platform. In addition, we mixed the pUC57/Pfcrt-CVIET and pUC57/Pfcrt-CVMNK plasmids and performed gradient dilutions (concentrations ranging from 1.8 × 104 to 1.8 × 1010 copies/μL). The sensitivity of the RPA-PfAgo platform for simultaneous detection of the wild-type and mutant strains in a single reaction system was validated. The RPA-PfAgo platform was validated under optimal conditions. The limit of detection (LOD) was defined as the concentration of the plasmid with the lowest fluorescence signal and the longest reaction time. The sensitivity of the RPA-PfAgo platform was measured in terms of detection thresholds and was also used as an evaluation criterion. In addition, we diluted the concentrations of the pUC57/Pfcrt-CVMNK and pUC57/Pfcrt-CVIET plasmids to 1.8 × 105 copies/μL, and 1 μL, 3 μL, 5 μL, 7 μL, and 10 μL of the pUC57/Pfcrt-CVIET plasmid were mixed with the pUC57/Pfcrt-CVMNK plasmid for a total volume of 100 μL. Using these mixed plasmids as templates, the performance of the RPA-PfAgo platform for detecting low-concentration mutant types was evaluated.

Clinical evaluation

P. falciparum samples were prepared as described in previous studies [28]. Genomic DNA was extracted via the methods described in the supporting information. Nested PCR followed by Sanger sequencing was used to confirm the PCR genotyping results, and the reaction conditions are presented in Table S2. We subsequently randomly selected 85 samples, including wild-type, mutant, and mixed-type Pfcrt, and detected them via the RPA-PfAgo platform. The results were interpreted according to the color of the fluorescent signal and verified by comparison with the sequencing results.

Data analysis

The experimental data were analyzed with SPSS 22.0 (SPSS, Inc., Chicago, IL, USA). A P value < 0.05 indicated a significant difference. The sensitivity was calculated as the number of true positives/ (number of true positives + number of false negatives), and the specificity was calculated as the number of true negatives/ (number of true negatives + number of false positives). The false negative rate was calculated as 1—sensitivity—and the false positive rate was calculated as 1—specificity. The 95% confidence intervals (CI) for sensitivity and specificity were calculated via SPSS 22.0.

Results

Confirming the optimal use of the RPA primer and gDNA

The primers (Table S1) used were designed for Pfcrt and were cross-assembled into nine combinations. On the gel after RPA, the primer set F2R1 produced single, clear, bright expected bands without nonspecific bands, which is the optimal option (Fig. 2A). Three sets of 5′-phosphorylated gDNAs and ssDNA reporters were subsequently designed and reacted with the RPA amplification products of the F2R1 primer. The results are displayed in Fig. 2C–E. The fluorescence signals of the first group of gDNA were the strongest, and the wild type and the mutant type could be identified. To verify the accuracy of the cleavage, urea PAGE was carried out on the cleavage products of the first set of gDNA. With the guidance of the first set of gDNA, PfAgo can accurately cleave the target product, producing two fragments of different lengths (Fig. 2B).

Fig. 2
figure 2

Screening of primers and guide DNA. A Screening of RPA primers. B Results of urea PAGE of the first group of guide DNAs (gDNAs) after cleavage of the target fragment. Lane 1 is the urea PAGE result of the first gDNA-guided Pfcrt-CVMNK cleavage product, lane 2 is the urea PAGE result for gDNA-W only, lane 3 is the urea PAGE result of the PfcrtCVMNK-CVMNK amplification products, lane 4 is the urea PAGE result of the first gDNA-guided Pfcrt-CVIET cleavage product, lane 5 is the urea PAGE result for gDNA-M only, and lane 6 is the urea PAGE result of the Pfcrt-CVIET amplification products. (C, D, and E) The results of the first, second, and third groups of gDNA and reporters, respectively. “a,” “d,” and “g” indicate that the target fragment cleaved by the RPA-PfAgo platform is the amplification product of the wild-type and mutant strains; “b,” “e,” and “h” indicate that the target fragment cleaved by the RPA-PfAgo platform is the amplification product of the wild-type strain; and “c,” “f,” and “i” indicate that the target fragment cleaved by the RPA-PfAgo platform is the amplification product of the mutant strain

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Optimization of the PfAgo cleavage reaction

To improve the overall performance of the RPA-PfAgo reaction, the concentrations of gDNA, probes, MnCl2, and PfAgo were optimized. Since the fluorescence intensities in the tubes were similar among all the concentrations, the condition with the highest fluorescent signal for the two channel signals at 30 min was selected as the optimal one. gDNA concentrations ranging from 0.4 to 2.4 μM were tested, and fluorescence curves (Fig. 3A) and fluorescent tube signals (Fig. 3B) were observed. The optimal gDNA concentration was 2.4 μM. The reporter concentrations of the reporter-wild-type (Reporter-W) and reporter-mutant (Reporter-M) strains were tested in six groups in different proportions. The fluorescence curve (Fig. 3C) and fluorescence tube signal (Fig. 3D) indicated that 1.2 μM/1.0 μM was the optimal concentration. MnCl2 is an activator of the PfAgo cleavage reaction. In this study, six concentrations of MnCl2 were used. The fluorescence curve Fig. 3E and fluorescence tube Fig. 3F signals indicated that 4.8 nM was the optimal concentration. Similarly, PfAgo concentrations ranging from 16 to 56 U/μL were tested. Two channel signals achieved a high fluorescence signal simultaneously at 32 U/μL, which was determined to be the optimal concentration Fig. 3G, H.

Fig. 3
figure 3

Optimizing PfAgo cleavage conditions. A and B Optimization of the concentration of guide DNA (gDNA). C and D Optimizing the concentration proportions of reporter-FAM and reporter-ROX. The numbers “1–6” indicate proportions 1 to 6, respectively. E and F Optimizing the concentration of MnCl2. G and H Optimizing the addition of PfAgo. A, C, E, and G show the fluorescence signal values of the reaction; B, D, F, and H show the results of the fluorescent tubes at the end of the reaction

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Sensitivity of the RPA-PfAgo platform

To explore the sensitivity of the RPA-PfAgo platform, the pUC57/Pfcrt-CVIET and pUC57/Pfcrt-CVMNK plasmids were tested, and the concentrations ranged from 1.8 × 104 to 1.8 × 1010 copies/μL. According to the fluorescence signals, concentrations as low as 1.8 × 105 copies/μL of pUC57/Pfcrt-CVMNK (Fig. 4A, B) and 1.8 × 104 copies/μL of pUC57/Pfcrt-CVIET (Fig. 4C, D) were detected, which is consistent with the sensitivity of the mixed plasmids (Fig. 4E, F). Furthermore, we diluted the concentrations of the pUC57/Pfcrt-CVMNK and pUC57/Pfcrt-CVIET plasmids to 1.8 × 105 copies/μL, and 1 μL, 3 μL, 5 μL, 7 μL, and 10 μL of the pUC57/Pfcrt-CVIET plasmid were mixed with the pUC57/Pfcrt-CVMNK plasmid for a total volume of 100 μL. The RPA-PfAgo platform was used, and nPCR was combined with Sanger sequencing to detect the mixed plasmid simultaneously. The RPA-PfAgo platform detected as little as 3% of the pUC57/Pfcrt-CVIET mixed plasmids (Fig. 4G, H). However, sequencing identified up to 5% of the pUC57/Pfcrt-CVIET mixed plasmids Fig. 4I.

Fig. 4
figure 4

Sensitivity test. A and B Sensitivity of the Pfcrt-CVMNK plasmid. C and D Sensitivity of the Pfcrt-CVIET plasmid. (E and F) Sensitivity results of the mixed plasmids. The numbers “1–7” indicate 1.8 × 104 to 1.8 × 1010 copies/μL, respectively. G and H Fluorescence curve and fluorescence tube signal for the two-plasmid mixture based on RPA-PfAgo. I Sanger sequencing results for the two-plasmid mixture. Red or green bimodal peaks were not observed at the locations indicated by the arrows for 3% of the CVIET mixed plasmids. Extraneous noise occurred in the 5% and 7% CVIET mixed plasmids. A, C, and E show the fluorescence signal values of the reaction; G shows the fluorescence signal values at the end of the reaction; B, D, F, and H show the results of the fluorescent tubes at the end of the reaction

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Clinical evaluation

The efficacy of the platform was further confirmed via the use of clinical samples to test its reliability. The ultimate genotyping results for each allele were visually interpreted by changes in the curves on the RPA-PfAgo platform. In total, 85 DBSs were used for clinical evaluation (Table S3). These samples were genotyped via Sanger sequencing; moreover, the RPA-PfAgo platform was used. The results are presented in Fig. 5. When the template was the wild type, reporter-W released a fluorescent signal; when the template was the mutant type, reporter-M released a fluorescent signal; and when the template was the mixed type, two fluorescent signals were generated (partially explaining the samples). The results of the methodological assessment and analysis of the assay results of the RPA-PfAgo platform with the Sanger sequencing results are displayed in Table 1. For these clinical samples, the sensitivity and specificity of CVMNK, CVIET, and the CVM/I N/E K/T were 100%. All the results indicated that the platform was consistent with the nPCR results.

Fig. 5
figure 5

Results for clinical samples (using partial clinical samples as an example). A and B The results of the Pfcrt RPA-PfAgo platform are shown (partially) for three groups of clinical samples: CVMNK, CVIET, and CVM/I N/E K/T. A shows the fluorescence signal values at the end of the reaction. B shows the results of the fluorescent tubes at the end of the reaction. C Typical representation of sequencing results from CVMNK, CVIET, and CVM/I N/E K/T, three different types of clinical samples. The arrows point to the mutated sites

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Table 1 Methodological comparison of RPA-PfAgo and nested PCR with Sanger sequencing
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Discussion

For the detection of Plasmodium parasite mutations, nPCR combined with sequencing is regarded as the “gold standard” technique [29,30,31]; this technique is relatively accurate and is used extensively [32, 33]. However, these materials are prone to technological malfunctions [34]. The quality of the PCR products used for sequencing determines the accuracy and reliability of Sanger sequencing results [35], especially concerning the amplification and purification of PCR products before sequencing [34]. However, in clinical samples, when the concentration of the mutation template is low, the quality of the PCR products is not effectively controlled.

Despite the many sequencing standards that have been put forward in recent years to lessen the limitations of Sanger sequencing, several challenges inherent in actual sequencing reads, such as extraneous noise, have not been adequately addressed [35]. An important technical challenge in the interpretation of DNA chromatograms from sequencing data is the existence of extraneous noise in the data. This interference can cause mistakes in base calling and increase the difficulty in distinguishing between real signals and background noise [35]. This was also found in our research (5% and 7% CVIET mixed plasmids, as shown in Fig. 4I). On the other hand, fewer ideal peaks can call for more fine-tuning or troubleshooting to improve the quality of the data [36]. However, fine-tuning may result in the loss of certain low signals. As shown in Fig. 4I, for 3% CVIET mixed plasmids, we did not observe red or green bimodal peaks at the locations indicated by the arrows. Additionally, another technical challenge arises from the double peaks present within DNA chromatograms [35]. These dual peaks might stem from genuine heterozygosity, technical anomalies, or the occurrence of double infections. Determining the characteristics of these double peaks might be difficult, and their existence may cause inaccuracies when performing sequence analysis (5% and 7% CVIET mixed plasmids in Fig. 4I).

Because the RPA-PfAgo platform does not have a unique advantage in the sensitive detection of individual plasmids, it might be unsatisfactory at detecting low-density infections. However, it is satisfactory in clinical applications. The sensitivity and specificity were 100% when 85 clinical samples were used. The methodological advantage of our method is the ability to detect a low proportion of the mutant in mixed infections. The RPA-PfAgo platform achieved 3% sensitivity for genotyping the CVMNK/CVIET mutation of the Pfcrt gene, with a higher sensitivity than nPCR combined with Sanger sequencing, which is advantageous over other detection methods (Table 2). An important feature of this platform is its convenience. The amplification step used RPA, which was conducted isothermally at 37 °C; the temperature of PfAgo cleavage was 95 °C [24]. Precise thermal cycling equipment is not needed. The platform only needs minimum laboratory support, such as pipettors, a portable spin, a heat block, a blue-light source or UV light. The entire platform procedure was completed within 1.5 h. Because the PfAgo enzyme is thermostable and the RPA reagent is lyophilized, the reagents used in this platform do not require cold chain transport or storage. The RPA-PfAgo platform is a rapid and sensitive genotyping platform for Pfcrt that can be easily applied locally or on site [14].

Table 2 Molecular methods used for identifying mutations in antimalarial drug resistance genes
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The RPA-PfAgo platform not only has wide application value in resource-poor areas and clinical research but also offers a feasible platform for haplotype detection [14, 23]. The other advantage of the RPA-PfAgo platform is that it can detect CVMNK and the mutant CVIET simultaneously via a dual-signal biosensor system. Monitoring population changes and controlling malaria epidemics should benefit greatly from the identification of mixed infections via an integrated test.

However, the RPA amplification products must be purified by a purification kit (centrifugal column type) to remove the proteins, ions, and other impurities involved in the RPA reaction, which requires slightly tedious steps. Otherwise, PfAgo cleavage will not occur, which reduces the portability of the POCT method. We have tested existing rapid purification methods; however, a rapid purification method adapted to our platform has yet to be found. For example, organic solvent residues in the RPA product are purified via the phenol‒chloroform extraction method, which denatures the PfAgo enzyme and prevents cleavage from occurring. The thermal denaturation method can only denature and inactivate the recombinase and polymerase by heat but fails to achieve complete separation of the protein, ions, and other impurities from the DNA amplification product by high-speed centrifugation, which affects the PfAgo reaction system and prevents the digestion reaction from occurring [37]. We will focus on solving this problem and making the POCT method more portable in future research.

Conclusions

This study established an RPA-PfAgo platform for genotyping the key mutation Pfcrt-CVMNK/CVIET of CQR. This approach has the characteristics of a short operation time, low device dependence, and good match to the POCT strategy, suggesting that the platform can be easily applied locally or on site. These findings strongly support the epidemiological investigations of P. falciparum, as they can not only be used to indicate resistance to CQ and other 4-aminoquinoline antimalarial drugs but also be used to monitor population changes in P. falciparum.

Availability of data and materials

All the data generated or analyzed during this study are included in this published article and its supplementary information files.

Abbreviations

PfAgo:

Pyrococcus furiosus Argonaute

Pfcrt:

Plasmodium falciparum chloroquine resistance transporter

POCT:

Point-of-care testing

RPA:

Recombinase polymerase amplification

SNP:

Single-nucleotide polymorphism

CQ:

Chloroquine

CQR:

Chloroquine resistance

nPCR:

Nest-PCR

PCR–RFLP:

PCR-restriction fragment length polymorphism

AS‒PCR:

Allele-specific diagnostic PCR

PAM:

Protospacer-adjacent motif

PFS:

Protospacer flanking site

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Acknowledgements

The authors are very grateful to Chaozhou People’s Hospital for providing the experimental site for this study.

Funding

This study was supported by the Guangxi Provincial Natural Science Foundation (grant number 2019JJD140052 and grant number 2020JJA140656), the Guangdong Provincial Key Laboratory of Functional Substances in Medicinal Edible Resources and Healthcare Products (grant no. 2021B1212040015), Scientific Projects of Key Disciplines in Guangdong Province (grant no. 2021ZDJS042), the Huizhou Science and Technology Research and Development Plan Social Development Field Research and Development Project (grant no. 210426104574869) and 2023 Innovation Project of Youjiang Medical University For Nationalities Graduate Education (grant no. YXCXJH2023025).

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Author notes

  1. Liying Chen, Wencheng Chen and Huagui Wei have contributed equally to this work.

Authors and Affiliations

  1. Department of Reproductive Medicine, Affiliated Hospital of Youjiang Medical University for Nationalities, Baise, Guangxi, China

    Liying Chen, Huagui Wei, Wenai Lin, Cheng Zhang, Junli Wang, Zongyun Lin, Yuxia Wei & Min Lin

  2. School of Basic Medicine Science, Hubei University of Medicine, Shiyan, Hubei, China

    Daiqian Zhu & Jian Li

  3. Laboratory Medical Center, Affiliated Hospital of Youjiang Medical University for Nationalities, Baise, Guangxi, China

    Wencheng Chen, Hongfei Hu & Chunfang Wang

  4. Modern Industrial College of Biomedicine and Great Health, Youjiang Medical University for Nationalities, Baise, Guangxi, China

    Liying Chen, Huagui Wei, Wenai Lin, Cheng Zhang, Chunfang Wang, Junli Wang, Zongyun Lin, Yuxia Wei & Min Lin

  5. Guangxi Medical and Health Key Discipline Construction Project of the Affiliated Hospital of Youjiang Medical University for Nationalities, Baise, Guangxi, China

    Junli Wang, Yuxia Wei & Min Lin

  6. Key Laboratory of Research On Clinical Molecular Diagnosis for High Incidence Diseases in Western Guangxi of Guangxi Higher Education Institutions, Baise, Guangxi, China

    Wencheng Chen

  7. Laboratory Medical Center, Huizhou Municipal Central Hospital, Huizhou, Guangdong, China

    Jiangtao Chen & Xueyan Liang

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  1. Liying Chen
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Contributions

L.C., W.C., and H.W. contributed equally to this article; M.L. and L.C. conceptualized the study; W.C., W.L., and C.Z. designed the study; H.W., H.H., and C.W. conducted the laboratory work; J.C., X.L., and D.Z. collected the samples; J.W. interpreted the data; Z.L. and Y.W. drafted the manuscript; J.L. critically revised the work for important intellectual content. All the authors have read and approved the final manuscript.

Corresponding authors

Correspondence to Jian Li or Min Lin.

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Ethics approval and consent to participate

The studies involving humans were approved by the medical ethics committee of the Affiliated Hospital of Youjiang Medical University for Nationalities. The studies were conducted in accordance with local legislation and institutional requirements. Written informed consent for participation in this study was provided by the participants’ legal guardians.

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Chen, L., Chen, W., Wei, H. et al. Rapid and supersensitive allele detection of Plasmodium falciparum chloroquine resistance via a Pyrococcus furiosus argonaute-triggered dual-signal biosensing platform. Parasites Vectors 17, 488 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13071-024-06575-0

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Keywords

Parasites & Vectors

ISSN: 1756-3305

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