Universal deoxyribonucleic acid isothermal amplification kits and visual MG reagents (malachite green) were purchased from Tian-Jin Huidexin Technology Development Co., Ltd. (Tianjin, China). Viral qEx-DNA/RNA extraction kits and universal bacterial genomic DNA extraction kits were obtained from Xi′an Tianlong Science &Technology Co., Ltd. (Xi′an, China). These biomaterials, including backing card, sample pad, conjugate pad, nitrocellulose membrane (namely, NC membrane), and absorbent pad were purchased from Jie-Yi Biotechnology. Co., Ltd. (Shanghai, China). Biotinylated bovine serum albumin (biotin-BSA) and rabbit anti-FITC antibody (anti-FITC) were obtained from Abcam. Co., Ltd. (Shanghai, China). Dye (crimson red) streptavidin-coated gold nanoparticles (SA-AuNPs) were provided by Bangs Laboratories, INC. (Indiana, USA). Real-time turbidimeter (LA-500) was provided by Eiken chemical Co., Ltd. (Japan). The ChemiDoc MP imaging system obtained from Bio-Rad (USA).

The AuNPs-LFB biosensor was designed according to the principle of FAM’s specific binding to the anti-FITC antibodies (Li S. et al., 2020; Yang et al., 2022). The AuNPs-LFB biosensor is mainly composed of a sampling region, conjugate region, binding region (NC membrane) and absorbent region. A total of two lines separated by 5 mm, including the test line (TL) and the control line (CL), were constructed on the NC membrane of the AuNPs-LFB biosensor. Specifically, the rabbit anti-FITC antibody with a concentration of 0.15 mg/ml in 0.01 M PBS (pH 7.4) was deposited on an NC membrane close to the sample pad to form the TL line; other side, the biotinylated bovine serum albumin (biotin-BSA) with a concentration of 2.5 mg/ml in 0.01 M PBS (pH 7.4) was deposited to form the CL line. The SA-AuNP nanoparticles (129 nm, 10 mg ml⁻¹, 100 mM borate, pH 8.5 with 0.1% BSA, 0.05% Tween 20, and 10 mM EDTA) were collected in a conjugate pad located in the middle of the sample pad and the NC membrane. Subsequently, these components, including the sample pad, conjugate pad, NC membrane, and adsorbent pad, were fixed on the backing card using plastic adhesive. According to our design, AuNPs-LFB biosensors are assembled by TianJin HuiDeXin Biotech. Co., Ltd. (Tianjin, China) and stored at room temperature (dry environment) away from light. The detection workflow (including the design principle) of the AuNPs-LFB biosensor for validating MCDA amplicons was shown in Figure 1.

In this EBV-MCDA-LFB reaction system, five sets of specific MCDA primers targeted repetitive regions of the EBNA-LP gene (GenBank accession number, MK973061.1), including F1, F2, C1, C2, R1, R2, D1*, and D2, CP1*, and CP2 primers, were designed using the Primer Premier software (5.0). The sequence alignment of designed EBV-MCDA primers was performed using the BLAST software (basic local alignment search tool software). Subsequently, five sets of MCDA primers were synthesized to screen the optimal set of specific primers used in this study. According to the reaction principle of AuNPs-LFB biosensor, the FAM and biotin groups were engineered at the 5 ‘end of CP1* primer and D1* primer, respectively. Details of EBV-MCDA primers were shown in Table 1. The EBV-MCDA primers (HPLC purification grade) were synthesized by Tianyi-Huiyuan Biotech Co., Ltd. (Beijing, China). Moreover, the EBNA-plasmids used in this study were prepared and quantified by synthesizing EBNA-LP gene (GenBank accession number, MK973061.1), and subsequently cloning them into a pUC57 vector. The entire preparation process (i.e., cloning and extraction) was carried out by Tianyi-Huiyuan Biotech Co., Ltd. (Beijing, China). After quantification, the EBNA-plasmids with initial concentrations of 3 × 10¹⁰ copies were diluted to prepare a series of diluents (10⁶, 10⁵, 10⁴, 10³, 10², 10¹, 10⁰, and 10⁻¹ copies).

A total of 22 pathogens, including 11 viruses and 11 bacteria, were used to evaluate the specificity of the EBV-MCDA-LFB assay. These nucleic acid templates extracted from various organisms were prepared using the viral qEx-DNA/RNA extraction kits and universal bacterial genomic DNA extraction kits, respectively. All extraction steps are performed according to the manufacturer’s instructions. The prepared nucleic acid template is stored at −20°C and avoids repeated freeze–thaw. Details of the organisms, including names, sources, numbers, etc. were presented in Table 2. In addition, DNA templates were prepared from 107 whole blood samples to evaluate the EBV-MCDA-LFB assay. Briefly, after 1 ml of the whole blood sample is diluted with 0.9% NaCl solution, slowly add it to a glass test tube containing 500 μl of lymphocyte separation medium (Tianjin Haoyang Biological Manufacture Co., Ltd., Tianjin, China); and the tubes were centrifuged at 2,000 rpm for 10 min. Then, the leukocyte-rich solution was pipetted into a 1.5 ml centrifuge tube and centrifuged at 12,000 rpm for 5 min; then the supernatant was discarded, 50 μl of DNA extraction solution (Daan Gene Co., Ltd., Guangzhou, China) was added and mixed, and heated at 100°C for 8 min. Finally, the tubes were centrifuged at 12,000 rpm for 2 min, and the supernatant was stored at −20°C.

The 25-μl amplification mixture of the EBV-MCDA assay contain the following: 12.5 μl of 2 × BF (reaction buffer), 1 μl of Bst thermostatic enzyme (2.0), 0.4 μM each of displacement primer (EBNA-F1 and EBNA-F2), 0.8 μM each of amplification primer (EBNA-C1, EBNA-C2, EBNA-R1, EBNA-R2, EBNA-D1*, and EBNA-D2), 1.6 μM each of cross primer (EBNA-CP1* and EBNA-CP2), 1 μl of MG reagent, 1 μl of nucleic acid templates extracted from pure culture and 2 μl of nucleic acid templates extracted from whole blood samples, and double-distilled water were added to 25 μl. The mixed system of EBV-MCDA assay was continuously amplified at 63°C for 60 min. Then, the amplification products of the EBV-MCDA assay were verified using an AuNPs-LFB biosensor, MG visualization indicator, 1.5% agarose gel electrophoresis, and real-time turbidimeter (LA-500). In addition, a one-microliter DNA template extracted from human cytomegalovirus was used as a negative control. One microliter of environment sample was used as a laboratory internal control. One microliter of double-distilled water was used as a blank control.

In the current study, the amplification conditions of the EBV-MCDA-LFB assay mainly included amplification temperature and time. Generally, amplification temperature, as a critical factor that can affect amplification efficiency, needs to be optimized in EBV-MCDA-LFB tests. Optimization tests of the EBV-MCDA assay were performed to explore the optimal incubation temperature. One microliter of EBNA-plasmid was used as the amplification template. The mixed tubes of the EBV-MCDA assay were incubated using different reaction temperatures (62–70°C with 1°C intervals) according to the EBV-MCDA-LFB reaction system. Then, the amplicons of the EBV-MCDA assay were validated using a real-time turbidimeter. In addition, an optimization tests of reaction time for the EBV-MCDA assay were implemented using 1 μl of each diluent of EBNA-plasmids as a template (10⁶, 10⁵, 10⁴, 10³, 10², 10¹, and 10⁰ copies). According to the EBV-MCDA reaction system, this time-optimized test was performed by setting different reaction times (10–60 min, 10 min intervals). Finally, the amplicons of the EBV-MCDA assay were verified using an AuNP-LFB biosensor.

To evaluate the performance of the MCDA-LFB assay for the detection of low-concentration EBV-DNA templates, the sensitivity test was performed according to the optimal reaction system. One microliter of series diluents of EBNA-plasmids (10⁶, 10⁵, 10⁴, 10³, 10², 10¹, 10⁰, and 10⁻¹ copies) was used as MCDA amplification templates. Next, the MCDA amplification products were validated using AuNPs-LFB biosensors, MG visualization reagents, 1.5% agar-gel electrophoresis, and a real-time turbidimeter. Finally, the lowest concentration that can be detected by the MCDA-LFB test after more than three repetitions are defined as its limit of detection (LoD).

Here, 25 nucleic acid templates extracted from 22 pathogens and three EBV-positive whole blood samples were examined to evaluate the detection specificity of the MCDA-LFB assay (Table 2). According to the optimal MCDA reaction condition, 1 μl nucleic acid template was used as the detection object for MCDA-LFB test. Then, the MCDA amplicons of specificity tests were detected using AuNPs-LFB biosensors.

In the current study, 107 whole blood samples collected from patients with suspected EBV infection were used to evaluate the detection applicability of MCDA-LFB assay. The whole blood samples were prepared for DNA template using a commercial extraction kit, and all extraction procedures were followed by the kit’s instructions (as mentioned earlier). In order to better evaluate the detection performance of the MCDA-LFB assay, a conventional real-time PCR assay was used as a reference method. The MCDA-LFB assay was implemented based on the MCDA reaction system and optimal reaction conditions, and then the amplicons were validated using the AuNPs-LFB biosensors. Meanwhile, the real-time PCR assay was performed according to the commercial kits. Five microliters of DNA prepared from whole blood samples were used as amplification templates for MCDA-LFB and real-time PCR assays. Finally, the results of the real-time PCR assay were used as criteria to evaluate the applicability of the MCDA-LFB assay to detect whole blood samples with EBV infection.

In the MCDA-AuNPs-LFB reaction system, one set of MCDA primers can recognize 10 different complementary regions on the target (P1s, P2s, C1s, C2s, P1s, P2s, D1s, D2s, R1s, R2s; Figure 2, step 1). The prepared double-stranded DNAs of EBV were amplified specifically in the presence of Bst DNA polymerase and MCDA primers. That is, the CP1* primer labeled with FAM can combine with complementary P1s site on the target and extends to synthesize new FAM-labeled double-stranded amplicons (Figure 2, step 2). Subsequently, the D1* primer labeled with biotin binds to the complementary D1s site on the newly synthesized MCDA amplicons contained FAM group and extends further (Figure 2, step 3). As a result, the exponentially amplified FAM-amplicons-biotin products were produced in the MCDA reaction mixture (Figure 2, steps 4, 5, and 6). In other words, the whole detection process, including preparation of the EBV template (Figure 1A, step 1), reaction reagents premixing and MCDA amplification (Figure 1A, steps 2 and 3), and AuNPs-LFB-mediated validation of EBV-MCDA amplicons (Figure 1B, steps 1, 2, and 3), can be completed in a short time (within 60 min). In the AuNPs-LFB detection system, the FAM-amplicons-biotin complex combined with SA-AuNPs (streptavidin-coated gold nanoparticles) in the conjugated pad can be captured by anti-FITC on the NC membrane to form a TL line (Figure 1B, R1). Meanwhile, the superfluous SA-AuNPs, driven by the running buffer, can be captured by biotin-BSA on the NC membrane to form CL lines to verify the effectiveness of AuNPs-LFB biosensors (Figure 1B, R1/R2).

To verify the feasibility of the MCDA-LFB assay designed in the current study, a confirmation test was performed based on the EBV-MCDA reaction system. One microliter EBNA-plasmids with a concentration of 10⁶ copies was used as an amplification template. Next, the amplification results were validated using the four validation methods (i.e., AuNPs-LFB biosensor, MG amplification indicator, 1.5% agarose gel electrophoresis, and real-time turbidimeter). As expected, when AuNPs-LFB biosensor was used to verify the EBV-MCDA results, both TL and CL regions were red in the positive results (Figure 3A); In contrast, only the CL region was red, and the TL region was colorless for negative reactions (Figure 3A). After adding the MG amplification reagent to the reaction system, it can be observed by the naked eye that the positive reaction mixture is blue, and the negative reaction mixture is light blue or colorless (Figure 3B). The MCDA amplification products were visualized by staining with ethidium bromide in a 1.5% agarose gel under ultraviolet light, and the positive results showed bright bands with characteristic gradients (Figure 3C). In addition, MCDA amplification products can also be monitored by real-time turbidimeters; usually, the turbidity threshold is set to 0.1; greater than 0.1 is considered positive amplification (Figure 3D).

The optimal amplification temperature was explored by setting a series of temperature gradients (62–70°C, 1°C intervals) to improve the detection efficiency (Figure 4). In the current study, when the reaction temperature was 67°C, the amplification efficiency of the EBV-MCDA assay was the highest by observing the real-time dynamic drawings of real-time turbidity (Figure 4F). In addition, amplification time is an essential factor that needs to be optimized to achieve rapid detection of EBV. The lowest concentration of a series of diluted EBNA-plasmids (10⁶, 10⁵, 10⁴, 10³, 10², 10¹, and 10⁰ copies) that the EBV-MCDA-LFB assay can detect was 10 copies when tested against the reaction time of 30, 40, 50, and 60 min (Figure 5). Thereby, the optimal amplification time of the EBV-MCDA-LFB assay was found as 30 min (Figure 5C). According to the above optimization tests, the optimal amplification condition of EBV-MCDA-LFB was 67°C for 30 min in the current study.

To explore the ability of the EBV-MCDA-LFB assay to detect EBV templates with low concentrations, this sensitivity test was performed using a series of diluents of 1 microliter EBNA-plasmids as templates. The LoD of the EBV-MCDA-LFB assay was 10 copies/reaction after repeated trials (Figure 6).

A total of 25 nucleic acids extracted from different pathogens and EBV-positive samples were used as amplification templates to evaluate the specificity of the EBV-MCDA-LFB assay (Table 2). As shown in Figure 7, the EBV-MCDA-LFB assay can detect all representative EBV organisms, including inactivated EBV standard culture (BioBDS), EBV (CHCIP), EBNA-plasmids, EBV-positive clinical samples, and can exclude other non-EBV-pathogens. That is, the detection specificity of the EBV-MCDA-LFB assay is 100% in the current study.

Here, a total of 107 whole blood samples were used to evaluate the clinical applicability of the EBV-MCDA-LFB assay by comparing it with the results of the real-time PCR assay. Thirty whole blood samples (30/107) were tested as EBV-positive, and 77 were EBV-negative utilizing the real-time PCR assay (Table 3). Similarly, 30 clinical specimens tested as EBV-positive by real-time PCR assay were also detected as EBV-positive by the EBV-MCDA-LFB assay, and 77 were EBV-negative (Table 3). The positive rate of EBV-MCDA-LFB and real-time PCR was consistent in the current study. Of note, the MCDA-LFB has excellent detection capability for whole blood samples with different viral loads, including samples with lower viral loads quantified by real-time PCR assay (e.g., 532 copies, 589 copies, 778 copies, 967 copies, etc.). In general, these data confirm that the MCDA-LFB can be used as a competitive potential detection technique for EBV infection in clinical settings.