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. 2026 May 25;26:650. doi: 10.1186/s12866-026-05216-z

Highly sensitive and rapid determination of Coxsackievirus A16 using restriction endonuclease‐mediated reverse transcription multiple cross displacement amplification

Qi Liang 1,2,3, Yumei Cao 1,4, Xujian Zhang 1,5, Rui Ye 1,2,3, Mao Liu 1,2,3, Shu Zhang 2,3,✉, Yu Wang 1,2,✉
PMCID: PMC13383146  PMID: 42185971

Abstract

Background

As a major etiological agent of hand, foot, and mouth disease in humans, Coxsackievirus A16 (CVA16) exerts a detrimental impact on the health of infants and toddlers. In the absence of effective antiviral drugs and vaccines targeting CVA16, a diagnostic strategy with sensitivity, specificity and rapid response is indispensable for the prevention and management of CVA16 infection.

Results

In this study, a novel molecular diagnostic approach, coupling real-time fluorescence technique with reverse transcription multiple cross-displacement amplification (RT-MCDA), was developed for the sensitive, rapid, and specific identification of CVA16 (termed CVA16-E-RT-MCDA). To construct the system, a unique set of CVA16-E-RT-MCDA primers was successfully designed targeting the VP1 gene of CVA16, and the optimal conditions for CVA16-E-RT-MCDA were determined to be 63 ℃ for 35 min. The limit of detection for the CVA16-E-RT-MCDA assay was 14 copies/µL (approximately 70 copies per reaction) for the RNA standard template of CVA16. The CVA16-E-RT-MCDA assay exhibited high specificity and anti-interference. The findings demonstrated that the applicability of the CVA16-E-RT-MCDA method was promptly and successfully confirmed using clinical samples from patients with suspected CVA16 infections.

Conclusions

The CVA16-E-RT-MCDA assay established in the present study exhibits sensitivity, specificity and rapidity for CVA16 detection, rendering it a promising diagnostic tool for clinical applications.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12866-026-05216-z.

Keywords: Coxsackievirus A16, Multiple cross displacement amplification, Restriction endonuclease, Limit of detection

Background

Coxsackievirus A16 (CVA16), which belongs to the enterovirus genus of the Picornaviridae family, is a non-enveloped positive single-stranded RNA molecule of approximately 7,400 nucleotides (nt) packaged in a protein shell termed a capsid [1–4]. Hand, foot, and mouth disease (HFMD) is linked to multiple key pathogens, among which CVA16 is particularly widespread among infants and young children [5]. In 1957, Toronto saw the initial appearance of common symptoms like fever and a vesicular rash on hands and feet due to Coxsackievirus, mainly CVA16 [6]. Although infection with CVA16 typically presents with mild, self-limiting manifestations, it may occasionally progress to severe clinical complications, such as encephalitis, myocarditis, pneumonitis, and even death [7–9]. Additionally, CVA16 often co-circulates with other etiological agents of HFMD, such as Enterovirus A71 (EVA71), Coxsackievirus A10 (CVA10), and Coxsackievirus A6 (CVA6) [2, 10]. This phenomenon gives rise to co-infection and viral genetic recombination, which collectively pose greater challenges to the comprehensive prevention and control of HFMD. The absence of effective antiviral drugs and vaccines targeting this virus exacerbates concerns regarding its potential outbreak [11]. Precise and rapid detection of CVA16 is a significant step toward addressing the challenges posed by this virus.

Viral isolation culture and serological examination are traditional methods for CVA16 detection [12]. Nevertheless, characterized by elaborate procedural requirements and extended detection duration, viral isolation poses practical constraints in rapid diagnostic scenarios [13]. However, the misinterpretation of serological results may be caused by cross-reactivity with other enteroviruses [14]. Recently, molecular methods have become the state-of-the-art detection technologies. As a rapid and dependable diagnostic modality, the quantitative reverse transcription polymerase chain reaction (qRT-PCR) is frequently acclaimed as the current “gold standard” of CVA16 confirmation because of the exceptional sensitivity and specificity [15–17]. However, the widespread adoption of qRT-PCR has been impeded because of the reliance on sophisticated equipment and the necessity for operators with specialized expertise, particularly in primary healthcare facilities and resource-constrained regions [18]. To address these limitations, researchers have developed various isothermal amplification techniques, including recombinase polymerase amplification (RPA) [19], single-primer isothermal amplification (SPIA) [20], rolling circle amplification (RCA) [21], and loop-mediated isothermal amplification (LAMP) [22]. RPA enables rapid amplification (20 min at 37–42 °C) with high sensitivity (1–10 copies/reaction) and simple primer design (30–35 nt) [23], but is prone to non-specific amplification due to primer-dimer formation at low temperatures, and its patent-protected core enzymes limit reagent availability [24]. SPIA uses a single chimeric RNA–DNA primer to produce ssDNA products at 47 °C within 30–90 min, facilitating downstream analysis [25]. However, chimeric primer synthesis is costly and complex, and the ssDNA products cannot be effectively detected with double-stranded DNA dyes (e.g., SYBR Green), requiring gel electrophoresis or specific probes [26]. RCA achieves single-molecule sensitivity and an ultra-low error rate via phi29 polymerase proofreading activity at 30–65 °C [27]. Nevertheless, it requires circular templates; linear templates must be ligated, and uncircularized templates cannot be amplifie; circularization conditions need optimization for different templates, increasing complexity, and multimerization or incorrect ligation may cause non-specific amplification [28]. The extremely high sensitivity of LAMP, which can achieve detection limits as low as single-digit to double-digit copies per reaction under optimized conditions, makes it highly susceptible to aerosol contamination during tube opening operations [29]. In addition, non-specific primer interactions may produce false amplicons, resulting in false-positive outcomes when sequence-non-specific intercalating dyes or pH-sensitive indicators are employed for detection [30].

Recently, multiple cross-displacement amplification (MCDA) technology [31], which plays a critical role in isothermal amplification, has been remarkably successful in the detection of numerous pathogens [32–35]. MCDA is an innovative amplification method that relies on isothermal chain displacement polymerization reactions, utilizing a set of 10 specific primers to cover 10 distinct parts of the target sequence [31]. Operation of the MCDA assay is straightforward, requiring merely an isothermal heater or thermostatic water bath and no extra specialized instrumentation are needed for the assay [36]. Lateral flow biosensors and chromogenic assays represent the primary approaches for monitoring MCDA amplification products [37]. However, there are certain limitations, like the contamination risk associated with biosensor use and the subjectivity of the outcomes. Environmental factors can also impact the stability of nanoparticles [38]. The CVA16-E-RT-MCDA assay devised here provided a resolution to these problems.

To address these limitations, a modified MCDA primer incorporating a restriction endonuclease cleavage site was designed, thereby establishing a novel CVA16-E-RT-MCDA detection system featuring real-time fluorescent signal output in a closed-tube format. As the entire reaction is performed in a closed tube without any post-amplification opening, the amplicons can be visualized simply under blue light, effectively eliminating aerosol-induced carryover contamination [39]. The clinical accuracy of the CVA16-E-RT-MCDA assay for CVA16 detection was assessed, demonstrating that it is a promising diagnostic tool for accurate viral identification.

Materials and methods

Reagents and facilities

DNA/RNA extraction kits were obtained from Xi’an Tianlong Technology Co., Ltd. (Xi’an, China). The 10 × isothermal amplification buffer II, MgSO4, Bst 3.0 DNA polymerase, deoxynucleotide (dNTP) solution mix, and restriction endonuclease (Nb.BsrDI) were obtained from New England BioLabs (USA). The T7 High Yield RNA Transcription Kit was acquired from Vazyme Biotech Co., Ltd. (Nanjing, China). The restriction enzyme SpeI, SMART®MMLV Reverse Transcriptase, and TaKaRa MiniBEST DNA Fragment Purification Kit Ver. 4.0 was produced by TaKaRa Biomedical Technology Co., Ltd. (Beijing, China). The RNA Clean & Concentrator™−5 was obtained from Zymo Research (USA). Applied Biosystem Inc. (USA) provided the Applied Biosystems 7500 Fast Real-Time PCR System. The qRT-PCR detection kits for CVA16 were produced by Jiangsu Uninovo Biological Technology Co., Ltd. (Jiangsu, China). The concentration and purity of nucleic acid were evaluated using a WD-2112A Ultra-micro spectrophotometer (Beijing Liuyi Biotechnology Co., Ltd.) by assessing the A260/A280 ratio. The E-Box CX5 stand-alone gel documentation system was procured from Vilber Co., Ltd. (France).

Primer design

With the exception of adding a restriction endonuclease recognition sequence to the 5' end of the D1 primer, the MCDA primer sequences used in this study are identical to those from our previous report [40]. A panel of ten primers was developed on the basis of the nucleotide sequence of the VP1 gene (GenBank Accession: GQ279371.1) of CVA16 using Primer Explorer 5 (https://primerexplorer.jp). The primer set was composed of one pair of cross-primers (CP1, CP2), one pair of displacement primers (F1, F2), and three pairs of amplification primers (C1, C2, D1#, D2, R1, R2). In parallel, the D1 primer was modified with the addition of a short sequence (5’-TGCAATG-3’) and a FAM fluorophore at the 5’ end, with Black Hole Quencher 1 (BHQ1) being embedded in the middle segment of the primer (termed D1#). Multiple sequence alignment was conducted with MEGA 12 software (available at https://www.megasoftware.net/), and the results verified that the target VP1 genomic region exhibits high sequence conservation among diverse CVA16 isolates (Fig. S1). All primers were synthesized by Tsingke Biotechnology Co., Ltd. (Beijing, China) and were of HPLC purification grade. An integrated DNA technology design tool was employed to assess the potential of these primers to form self-dimerization and hairpin structures. The sequences, positional data, and modification profiles of all primers are summarized in Fig. 1 and Table S1.

Fig. 1.

Fig. 1

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Primers for the VP1 gene used in the CVA16-E-RT-MCDA assay in this study. Position and sequences of the primers used in this research targeting the VP1 gene of CVA16. Right and left arrows indicate the sense and complementary sequences used, respectively

Preparation of RNA standards

To obtain the RNA standard template required for the CVA16-E-RT-MCDA assay, a 344 bp genomic fragment from the VP1 gene of CVA16 was cloned into the pGEM-T easy vector which contained the T7 promoter. The inserted fragment within the plasmid was verified by means of Sanger sequencing and restriction enzyme digestion, which was accomplished by Beijing Tianyi Huiyuan Biotechnology Co., Ltd. (Beijing, China). The plasmid construct with insertion sequence of VP1 gene was linearized by using the SpeI restriction enzyme at 37 ℃ and incubated for 6 h to secure comprehensive digestion. The linearized plasmid constructs were thereafter subjected to verification via agarose gel electrophoresis and purified with TaKaRa MiniBEST DNA Fragment Purification Kit Ver.4.0, according to the instructions of the manufacturer. Based on the recommendations of the manufacturer, linearized plasmids were employed as template for IVT reaction, which was implemented using the T7 High Yield Transcription Kit at 37 ℃ for 5 h. After 5 h incubation, newly transcribed RNA was subjected to a 15-min digestion with DNase I at 37 ℃ and was purified using RNA Clean & Concentrator™−5. The RNA standard template was quantified by measuring the absorbance at 260/280 nm using a WD-2112A Ultra-micro spectrophotometer to determine the concentration of the purified RNA. The copy number of RNA standards was computed using the following formula: RNA copy number (copies per microliter) = [(6.02 × 1023) × (RNA concentration, in nanograms per microliter) × 10−9]/[(fragment length, in nt) × 340].

Preparation of samples

Enterovirus strains were obtained from individuals with HFMD in Guizhou Province. All pathogens are presented in Table S2. Moreover, 96 clinical samples (48 anal and 48 throat swab samples) were acquired from patients suspected of HFMD at the First People’s Hospital of Guiyang. To assess the clinical relevance of the CVA16-E-RT-MCDA assay, all clinical samples were identified concurrently with both the qRT-PCR and the CVA16-E-RT-MCDA assay. This research was ratified by the ethics committee of the First People’s Hospital of Guiyang (Ethical approval No. G2024‐S022). Due to the retrospective nature of the study and the use of de-identified residual clinical specimens, the ethics committee granted a waiver of written informed consent. All samples were anonymized prior to analysis.

Nucleic acid extraction

Sterile flocked swabs were used to collect clinical anal swab specimens. Prior to collection, patient information was verified, and the patient was assisted into a lateral decubitus or knee-chest position to expose the anal area. After donning gloves, the swab tip was inserted approximately 2–3 cm into the anus and gently rotated 3–5 times to collect pathogens from the rectal mucosa and crypts. Following withdrawal, the swab was immediately placed vertically into a tube containing transport medium, broken at the break point, and the cap was tightened. Specimens were submitted for testing at room temperature or under refrigeration (2–8 ℃). Each tube was supplemented with 1 mL of sterile normal saline and vigorously vortexed to achieve complete sample suspension. Genomic RNA and DNA were extracted using commercial kits. RNA was eluted in 50 μL of RNase-free water and stored at −80 ℃, while DNA was stored at −20 ℃ until further use. The purity and concentration of the extracted samples were evaluated using a WD-2112A Ultra-micro spectrophotometer at A260/280.

Amplification reaction of qRT-PCR assay

The qRT-PCR reaction was performed using a dual nucleic acid detection kit for Enterovirus 71 and Coxsackievirus A16 (real-time fluorescent PCR method). The FAM channel was selected. The reaction system consisted of 14.2 μL of real-time PCR reaction mix, 0.8 μL of enzyme mix, and 5 μL of template. The reaction conditions were set as follows: reverse transcription at 42 ℃ for 10 min; initial denaturation at 94 ℃ for 10 s; pre-amplification for 5 cycles of 94 ℃ for 5 s, 50 ℃ for 20 s, and 72 ℃for 20 s; followed by PCR amplification for 40 cycles of 94 ℃ for 5 s, 56 ℃ for 50 s, and 72 ℃ for 15 s. The results were analyzed using a 7500 real-time fluorescence PCR instrument.

Standard CVA16-E-RT-MCDA reaction

Standard amplification mixtures for CVA16-E-RT-MCDA were performed in a total volume of 25 μL, which contained 0.4 µM each of displacement primers (F1 and F2), 0.8 µM each of amplification primers (C1, C2, R1, R2, D1#, and D2), 1.6 µM each of cross primers (CP1 and CP2), 2.5 μL 10 × isothermal amplification buffer II, 1.5 µL 6 mM MgSO4, 3.5 µL 1.4 mM dNTP, 1 μL of Bst 3.0 DNA polymerase (8U), 1 µL (10U) of Nb.BsrDI, 1 µL (10U) of SMART®MMLV reverse transcriptase, and 5 µL template (RNA template and clinical sample), then added RNase-free water to achieve a final volume of 25 µL. Additionally, the viral RNA template of CVA10 served as a negative control (NC), and 5 µL of RNase-free water was employed as a blank control (BC). Amplification was performed using real-time fluorescence instrumentation, featuring an initial hold stage at 50 ℃ for 2 min, followed by 40 cycles, setting the mode to 63 ℃ for 50 s. Simultaneously, the CVA16-E-RT-MCDA reaction results were real-time monitored by a fluorescence PCR platform and visually inspected under blue light. Agarose gel electrophoresis was employed to authenticate the products.

Optimal temperature of the CVA16-E‐RT‐MCDA assay

The optimal temperature for the CVA16-E-RT-MCDA reaction was verified by performing the CVA16-E‐RT‐MCDA amplification at temperatures from 61 to 68 ℃ at 1 ℃ intervals. The amplification reactions were performed and monitored in real time using a quantitative PCR instrument. Each procedure was executed at corresponding temperature and maintained for 35 min, employing 1.4 × 106 copies/µL of RNA standard template of CVA16 as a positive control, whereas the extracted RNA template of CVA10 served as an NC, and RNase-free water served as a blank control (BC). The temperature generating the highest amplification efficiency was considered optimal. Three independent replicates were performed at each temperature.

Sensitivity and specificity verification of the CVA16-E-RT-MCDA assay

The analytical sensitivity of CVA16-E-RT-MCDA was estimated employing a serially diluted RNA standard template for CVA16. Tenfold serial dilutions (1.4 × 106, 1.4 × 105, 1.4 × 104, 1.4 × 103, 1.4 × 102, 1.4 × 101, and 1.4 × 100 copies/µL) were utilized to measure the limit of detection (LoD) of the CVA16-E-RT-MCDA assay. Accordingly, 5 μL of the diluted RNA standard template of CVA16 was provided as the template, which was added to the CVA16-E-RT-MCDA reaction systems, and RNase-free water served as the BC. Triplicate replicates were set up for every reaction to guarantee data reliability. The LoD was defined as the lowest concentration that could be consistently detected across all replicates (n = 15). For the specificity evaluation of the CVA16-E-RT-MCDA assay, five CVA16 strains and 19 non-CVA16 pathogens were tested. All results were revealed using the real-time PCR platform and visually observed under blue light.

Reproducibility of the CVA16-E-RT-MCDA assay

Three plasmid concentrations (1.4 × 10⁶, 1.4 × 104, and 1.4 × 102 copies/µL) were used to evaluate reproducibility. For intra-batch assessment, each concentration was tested in triplicate within a single run. For inter-batch assessment, the same concentrations were tested across three separate experiments performed on different days. The coefficient of variation (CV) was calculated based on the obtained fluorescence values using the following formula: CV (%) = (standard deviation/mean) × 100.

Evaluation of clinical performance of the CVA16-E-RT-MCDA assay

The CVA16-E-RT-MCDA assay was subjected to clinical feasibility evaluation to validate its diagnostic potential, utilizing 48 clinical anal swab samples and 48 throat swab samples from the First People’s Hospital in Guiyang. Both the CVA16-E-RT-MCDA assay and qRT-PCR methodology were employed for the evaluation of all samples. The performance of the CVA16-E-RT-MCDA assay was compared with the qRT‐PCR assay. Based on the specifications of the manufacturer, the qRT‐PCR reactions were executed using the 7500 real-time PCR platform. Mplementation of the CVA16-E-RT-MCDA reaction adhered to the previously established procedure. The CVA16-E-RT-MCDA and CVA16-qRT-PCR assay were assessed using online statistical software MedCalc (http://www.medcalc.org/calc/diagnostic_test.php) (version 23.0.5) [41].

Statistical analysis

Statistical analyses were conducted using GraphPad Prism software (version 10.5.0). Standard errors represent the uncertainties in the mean values. Significance levels were defined as follows: *p < 0.05, **p < 0.01, and ns indicates no significance. Cohen’s kappa coefficient (κ) was calculated to evaluate the agreement between the CVA16-E-RT-MCDA assay and the reference qRT-PCR method.

Results

Schematic mechanism of the CVA16‐E-RT‐MCDA assay

The CVA16-E-RT-MCDA test was created by integrating the RT-MCDA reaction with restriction enzyme cleavage, and a real-time fluorescence quantification PCR setup was used for interpreting the results. The RT-MCDA assay was executed using the selected primer set, which targeted VP1 gene of CVA16 (Fig. 1). As illustrated in Fig. 2A, the RT-MCDA reaction with the primer D1#, which was modified with FAM at 5’ terminus and a dark quencher (BHQ1) inserted in the intermediate region, generated abundant amplicons carrying specific restriction endonuclease recognition motifs. These amplicons were further subjected to recognition and cleavage by the enzyme Nb.BsrDI. Then, fluorescent signals were generated owing to the separation of FAM and BHQ1. Therefore, the CVA16-E-RT-MCDA assay was capable of detecting CVA16 in a single tube using real-time fluorescence PCR. The complete workflow, spanning from template extraction to result explanation, was finalized within 52 min (Fig. 2B).

Fig. 2.

Fig. 2

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Schematic illustration of CVA16-E-RT-MCDA assay. A real-time fluorescent signal is generated in the CVA16-E-RT-MCDA system by the separation of FAM from BHQ1, which occurs when Nb.BsrDI cleaves its recognition site upon double-strand formation. This system uses a FAM-labeled primer (VP1-D1.#) targeting the VP1 gene, featuring Nb.BsrDI recognition sites and an internal BHQ1 quencher, and amplification is triggered by primer binding under isothermal conditions. A Schematic diagram of MCDA reactions using modified primers. Primer D1 carries a 5’-terminal endonuclease recognition site, a 5’-end FAM fluorophore (F), and an internal BHQ1 quencher (Q). Amplification generates double-stranded products containing the recognition motif. B The procedure of CVA16-E-RT-MCDA assay. Three steps, incorporating rapid viral RNA extraction (15 min), CVA16-E-RT-MCDA reaction (35 min), and results interpretation (2 min), are requested for the CVA16-E-RT-MCDA assay, and the total workflow can be completed within 52 min. The picture was drawn by using Generic BDiagramming Platform (GDP, https://BioGDP. com) [49]

Confirmation of the primer set for the CVA16-E-RT‐MCDA assay

To confirm the effectiveness of primers for CVA16 detection, the RNA standard template of CVA16 served as the positive control for CVA16-E-RT-MCDA reactions, which were conducted at a constant 63 ℃ for 35 min. The real-time fluorescence PCR platform showed distinct fluorescence values from fluorescent signals released by positive reactions (Fig. 3A), a light yellow colour under blue light (Fig. 3B), and ladder-like bands on agarose gel (Fig. 3C), whereas no fluorescent signal was detected for NC or BC by the real-time fluorescence PCR instrument or under blue light, and no bright ladder-like bands were observed on the gel. These outcomes demonstrated that the selected primer set is both valid and reliable for the CVA16-E-RT-MCDA assay.

Fig. 3.

Fig. 3

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Identification of CVA16-E-RT-MCDA method. A A real-time fluorescence PCR platform was applied to analyze fluctuations in the fluorescent signals of CVA16-E-RT-MCDA amplification products. Curve 1, positive control (PC) of RNA standard template of CVA16; curve 2, negative control (NC) of virus CVA10; curve 3, blank control (BC). B The products of CVA16-E-RT-MCDA were visually observed under blue light. Tube 1, positive control (PC) of RNA standard template of CVA16; tube 2, negative control (NC) of virus CVA10; tube 3, blank control (BC). “ + ”, positive; “-”, negative. C Agarose gel electrophoresis used to detect CVA16-E-RT-MCDA products. Lane M, DL 2,000 bp DNA marker; lane 1, positive control (PC) of RNA standard template of CVA16; lane 2, negative control (NC) of virus CVA10; lane 3, blank control (BC)

Optimal temperature of CVA16-E‐RT‐MCDA assay

The CVA16-E-RT-MCDA assay’s optimal temperature was further assessed by performing the reaction at temperatures ranging from 61 to 68 ℃, with 1 ℃ intervals. Experimental results were recorded by utilizing a real-time fluorescence PCR platform, from which eight fluorescence analysis plots corresponding to different temperature were retrieved (Fig. 4A-H). Based on analysis of the fluorescence values, the optimal temperature of 63 ℃ was confirmed because the reaction exhibited the highest amplification efficiency at 63 ℃ (Fig. S2). Consequently, subsequent experiments were conducted at 63 ℃.

Fig. 4.

Fig. 4

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Optimal amplification temperature for CVA16-E-RT-MCDA assay. A real-time fluorescent PCR platform was employed to monitor the emission of fluorescent signals during CVA16-E-RT-MCDA amplification reactions. The corresponding curves were shown in the panels. The negative control (NC) was virus CVA10, and the blank control (BC) was RNase-free water. Abscissa represents cycles, ordinate represents fluorescence value. Positive amplification was identified on the basis of an obvious fluorescence measurement. Eight amplification curves (A-H) were revealed from 61 to 68 ℃ (1 ℃ intervals), with 1.4 × 10.6 copies/µL of RNA standard template of CVA16

Sensitivity and specificity verification of the CVA16-E-RT-MCDA assay

For the purpose of determining the sensitivity of the CVA16-E-RT-MCDA assay, a serial dilution of the RNA standard template of CVA16 (1.4 × 106, 1.4 × 105, 1.4 × 104, 1.4 × 103, 1.4 × 102, 1.4 × 101, and 1.4 × 100 copies/µL) was employed in the CVA16-E-RT-MCDA assay. As illustrated in Fig. 5 and Fig. S3, the CVA16-E-RT-MCDA assay consistently generated detectable fluorescence signals, indicating that the concentrations of RNA standard template of CVA16, ranging from 1.4 × 106 to 1.4 × 101 copies/µL, were positive, whereas concentrations under 14 copies/µL and the BC were negative. Therefore, the LoD of the CVA16-E‐RT‐MCDA assay was 14 copies/µL (approximately 70 copies per reaction). As detailed in Table S2, a panel of five isolated CVA16 strains and 19 non‐CVA16 pathogens was employed to assess the specificity of the CVA16-E‐RT‐MCDA assay. As expected, when analyzed using a real-time fluorescence PCR platform, only the CVA16 strains emerged with positive results, whereas all non-CVA16 strains emerged with negative results (Fig. 6A). When exposed to blue light, only the amplification products derived from the five CVA16 strains exhibited a light yellow color, with the 19 non-CVA16 pathogenic strains yielding no corresponding color signal (Fig. 6B). Ultimately, the CVA16-E-RT-MCDA assay proved to be 100% specific within the tested panel of 19 non-CVA16 pathogenic strains.

Fig. 5.

Fig. 5

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The sensitivity of CVA16-E-RT-MCDA assay. A series of dilutions (1.4 × 10⁶, 1.4 × 105, 1.4 × 104, 1.4 × 103, 1.4 × 102, 1.4 × 101 and 1.4 × 10.0 copies/µL) of RNA standard template of CVA16 and a blank control (BC) were operated according to standard CVA16-E-RT-MCDA reactions. The fluorescence signals from CVA16-E-RT-MCDA were registered by the real-time fluorescence PCR (A, C) and visually identified under blue light (B). D Ct values were mapped relative to the nucleic acid copy numbers corresponding to positive reaction outcomes. The ordinate implies the number of PCR cycles required to reach the threshold and the abscissa implies the copy number of CVA16 (log10). The error bars symbolize the SDs (n = 3)

Fig. 6.

Fig. 6

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Analytic specificity of the CVA16-E-RT-MCDA assay. The specificity was assessed using templates from five CVA16 strains and 19 non-CVA16 pathogens. All products were detected by the real-time fluorescence PCR equipment (A) and observed visually under blue light (B). Curves/tubes 1 − 5, five CVA16 strains; curves/tubes 6 − 24, 19 non-CVA16 pathogens

Reproducibility of the CVA16-E-RT-MCDA assay

The intra-assay coefficients of variation (CV%) for ranged from 5.61% to 7.87%, and the inter-assay CV% ranged from 11.05% to 14.70%. These findings indicated the excellent stability of the established assay (Table S3).

Feasibility of CVA16-E‐RT‐MCDA assay in clinical samples

A set of 48 anal and 48 throat swab samples which were previously tested using the qRT-PCR assay, was used to assess the clinical utility of the CVA16-E-RT-MCDA system for CVA16 detection. The CVA16-E-RT-MCDA assay results indicated that 13 anal swab samples and 15 throat swab samples tested positive, while the remaining 68 were negative (Figs. S4-5). The experimental results were concordant with the corresponding measurements obtained using the qRT-PCR technique (Fig. 7). Our assay demonstrated 100% sensitivity and specificity (95% CI: 87.66%−100% and 94.72%−100.00%, respectively) within this preliminary sample set of 96 clinical specimens collected from a single center when compared to the qRT-PCR method (Table 1). These observations indicate that the CVA16-E-RT-MCDA assay serves as a valuable diagnostic tool for CVA16 detection in clinical environments, especially for screening patients with HFMD during the early stages of the disease.

Fig. 7.

Fig. 7

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Comparison of the performance of the qRT-PCR assay and CVA16-E-RT-MCDA assay in detection of 48 clinical anal swab samples and 48 clinical throat swab samples. A1-A48 represented 48 anal swab samples. T1-T48 represented 48 throat swab samples. +, positive; -, negative

Table 1.

Comparison of qRT-PCR and CVA16-E-RT-MCDA methods to identify CVA16 in clinical samples

CVA16-E-RT‐MCDA assay CVA16‐qRT‐PCR Total Sensitivity (%) Specificity (%) Kappa
Positive Negative value 95% CI value 95% CI
Positive 28 (13 anal swab samples and 15 throat swab samples) 0 28 100 87.66 ~ 100 100 94.72 ~ 100 1
Negative 0 68 68
Total 28 68 96
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Discussion

CVA16 is one of the principal etiological agents of HFMD [42]. For nearly twenty years, CVA16 has been the predominant causative agent of HFMD worldwide, particularly in Southeast Asian countries [43]. Since 2013, CVA16 has always had two sub-genogroups (B1a and B1b) prevalent in China. The use of the inactivated EVA71 vaccine has led to a swift decline in HFMD cases linked to EVA71, while the rate of HFMD cases associated with CVA16 has stayed the same. Currently, no specific drugs or vaccines are available for HFMD caused by CVA16 infection [44]. Thus, there is an urgent requirement for precise, quick, and sensitive diagnostic tools to support immediate clinical action and disease control.

In this work, a novel real-time fluorescent RT-MCDA detection platform was established, which combined RT-MCDA with real-time fluorescence technique to implement valuable diagnosis for CVA16 infection. Through modification of the selected MCDA primer D1 using the short sequence 5’-TGCAATG-3’, restriction endonucleases were employed to execute cleavage of the products, generating fluorescence signals that could be captured by the real-time fluorescence detector. By comparing the analytical features of previously reported CVA16 diagnostic methods, it confirmed that the CVA16-E-RT-MCDA assay was suited for CVA16 detection (Table S4). Serological assays [45] cannot detect early acute infections due to the antibody window period, whereas our assay directly targets viral RNA for early detection. Real-time PCR [46], despite being a gold standard, requires expensive instruments and trained personnel, while our assay needs only a water bath and a blue light transilluminator. The previously reported RT-MCDA-LFB [40] and LAMP-LFD [47] methods require tube opening for detection and poses a risk of aerosol contamination. CRISPR assay [48] relies on Cas proteins or complex probes and require multi-step handling. In contrast, the assay is performed in a closed tube, allows direct visual readout under blue light, enables real-time monitoring, and incorporates restriction endonuclease-based signal conversion for enhanced specificity. To identify the optimal reaction conditions for the CVA16-E-RT-MCDA assay, various reaction temperatures were assessed. As illustrated in Fig. 4, a reaction temperature of 63 ℃, with the highest amplification efficiency for CVA16, was deemed to be the most optimal. As demonstrated in this study, the primers we used enabled precise and proper detection of the RNA of CVA16 strains with optimum conditions, and no cross-reaction was output with non-CVA16 pathogens or BC, indicating the excellent specificity of the CVA16-E-RT-MCDA method. Additionally, serial dilutions of the RNA standard template of CVA16 were utilized to measure the LoD of the assay. Notably, the sensitivity of the developed assay was determined to be 14 copies/µL (approximately 70 copies per reaction). The optimized CVA16-E-RT-MCDA process can identify CVA16 within 52 min, comprising rapid nucleic acid extraction (15 min), CVA16-E-RT-MCDA based reaction (35 min), and fluorescence signal interpretation (2 min), which allowed for prompt treatment and infection control. These findings highlight the potential of this technology as a rapid, sensitive, and specific method for CVA16 detection. Additionally, 48 clinical anal swab and 48 throat swab samples from suspected HFMD patients were utilized to estimate the suitability of the CVA16-E-RT-MCDA assay for nucleic acid detection, in comparison to the qRT-PCR technique. These results indicate a high degree of consistency between the outcomes of the CVA16-E-RT-MCDA assay and those of qRT-PCR, which validated the reliability of this method.

However, this study has several inherent limitations. The clinical validation cohort was limited to a single center (n = 96) with no geographic diversity. Given that sample characteristics may vary across regions and populations, caution is warranted when extrapolating the current findings. Larger multicenter studies are therefore needed to further validate the assay performance. Moreover, the absence of independent external or multi-center validation affect the generalizability of the results. Furthermore, while this study preliminarily demonstrates the method's performance in a single-center setting, its robustness under different operators and equipment conditions remains unexplored. Therefore, future large-scale, multi-center prospective validation encompassing diverse populations and geographic regions is necessary to fully evaluate the method’s applicability in real-world clinical scenarios.

In brief, this investigation established a CVA16-E-RT-MCDA diagnostic tool for CVA16, characterized by timeliness, simplicity, and reliability. This innovative assay not only emerges as a robust candidate tool for the rapid, sensitive, and specific diagnosis of CVA16 infections but also provides a viable approach to enhance recognition capacities.

Conclusion

In this study, featured by the integration of fluorescence detection and RT-MCDA methodology, the CVA16-E-RT-MCDA assay presented in this study can accurately recognize CVA16 with the limit of detection of 14 copies/µL (approximately 70 copies per reaction), whereas the lack of cross-reactivity further verified the assay’s outstanding specificity for CVA16 detection. The full workflow can be completed within 52 min. In this pilot cohort (n = 96), the CVA16-E-RT-MCDA system achieved 100% sensitivity and specificity within the limits of the current sample size. These preliminary results suggest that the proposed method has the potential to achieve high diagnostic accuracy, warranting large-scale prospective validation.

Supplementary Information

Supplementary Material 1. (12.7MB, docx)

Acknowledgements

We thank thank Home for Researchers editorial team (www.home-for-researchers.com) for language editing service.

Authors’ contributions

Q.L., S.Z. and Y.W. conceived and designed the experiments. Q.L., Y.C., X.Z., R.Y. and M.L. conducted the trials. Q.L., Y.C., X.Z., R.Y., M.L. and S.Z. analyzed the data. Y.W. contributed the reagents and analysis tools. Q.L. and Y.W. drafted the manuscript. Y.W. revised the manuscript. The article’s submitted version was reviewed and approved by all authors.

Funding

This study was supported by grants from the National Natural Science Foundation of China (82460393 and 81860594), Zhu ke he tong [2021]-43–25 and GCC[2023]030 from the Science and Technology Department of Guiyang City of Guizhou Province.

Data availability

Data availability The datasets generated during the current study have been deposited in the National Center for Biotechnology Information (NCBI) [https://www.ncbi.nlm.nih.gov/] under accession number GQ279371.1 for CVA16, AF081300.1 for CVA10, MN233767.1 for CVA6, AY421762.1 for CVA4, AF465515.1 for CVA21, AY421760.1 for CVA2, AF081340.1 for ECHO30, AF081324.1 for ECHO7, AF081316.1 for ECHO3, and other datasets generated during this study are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

The study was approved by the Human Ethics Committee of the First People’s Hospital of Guiyang (Approval No. G2024-S022) and acts following the Declaration of Helsinki. Before collecting samples/isolates and conducting the study, monitoring stations removed all identifying details from suspected HFMD patients. The First People’s Hospital of Guiyang’s Human Ethics Committee exempted patients from providing informed consent.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Shu Zhang, Email: zhangshu@gmc.edu.cn.

Yu Wang, Email: wangzhongyuwy@163.com.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1. (12.7MB, docx)

Data Availability Statement

Data availability The datasets generated during the current study have been deposited in the National Center for Biotechnology Information (NCBI) [https://www.ncbi.nlm.nih.gov/] under accession number GQ279371.1 for CVA16, AF081300.1 for CVA10, MN233767.1 for CVA6, AY421762.1 for CVA4, AF465515.1 for CVA21, AY421760.1 for CVA2, AF081340.1 for ECHO30, AF081324.1 for ECHO7, AF081316.1 for ECHO3, and other datasets generated during this study are available from the corresponding author upon reasonable request.

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