Target gene and clinical sample preparation

The full-length S sequences from five dominant HBV genotypes in China (B, C, D, recombinant B/C, and recombinant C/D; GenBank accession Nos. AB014366.1, AB014360.1, AB090268.1, KC774178.1, and AY800249.1) [28], and the HCV 5′-UTR target genes from five dominant subtypes in China (1b, 2a, 3a, 3b, and 6a; GenBank accession Nos. EU781827.1, HQ639944.1, D17763.1, JQ065709.1, and AY859526.1) [29] were synthetically produced, cloned and inserted into pUC57 plasmids, the concentration of each plasmid was adjusted to 108 copies/mL. All the plasmids were applied to positive controls.

Participants at the Second Affiliated Hospital, Guizhou University of Traditional Chinese Medicine provided 73 serum samples for HBV and/or HCV examination between February 2023 and October 2023. The nucleic acids from each of the above samples were isolated using a TIANamp Virus DNA/RNA Fast Kit (TIANGEN BIOTECH Co., Ltd.; Beijing, China). Briefly, 200 µL of each serum sample was treated with 20 µL of protease K, 250 µL of the lysis agent RLC, and 250 µL of isopropanol and incubated at 56 °C for 5 min. Then, the nucleic acids were enriched with a centrifugal RNase-free absorption column. After washing with PWT buffer, the nucleic acids were eluted in 50 µL of RNase-free ddH2O. The extracted nucleic acids were stored at -80 °C before use.

LAMP primers design and synthesis

The S and 5′-UTR sequences were selected as the target genes to detect HBV and HCV, respectively. The S genes from HBV genotypes B, C, D, B/C recombinant, and C/D recombinant and the 5′-UTR genes from HCV subtypes 1b, 2a, 3a, 3b, and 6a were aligned by DNASTAR software (Madison, WI, USA), respectively. The conserved sequences were applied to HBV- and HCV-LAMP primer design using Primer Explorer version 5 (http://primerexplorer.jp/e/) and Primer Premier version 5.0 according to the LAMP mechanism. The specificity of primer pair was confirmed through BLAST analysis tool. Table 1 lists the primer sequences and modifications, and Supplementary Figure S1 presents the primer locations. All primers were synthesized and purified via high-performance liquid chromatography grade by TsingKe Biotech Co., Ltd. (Beijing, China).

Table 1 The mLAMP-AuNPs-LFB degenerate primers used in this study

Preparation of AuNPs-based LFB The AuNPs-based LFB consists of 4 elements: a sample pad, a conjugation pad, a nitrocellulose (NC) membrane, and an absorption pad. Crimson red dye-conjugated streptavidin-coated AuNPs (SA-AuNPs; 40 ± 5 nm, 10 mg/mL; Bangs, Laboratories, Inc., Indiana, USA) were deposited on the conjugation pad. Anti-FAM antibody (0.2 mg/mL, Abcam Co., Ltd.), anti-Dig antibody (0.25 mg/mL, Abcam Co., Ltd.) and biotinylated bovine serum albumin (biotin-BSA, 4 mg/mL; Abcam Co., Ltd.) were immobilized at the NC membrane (detection region) of test line 1 (TL1), test line 2 (TL2) and the control line (CL), respectively, which were separated by 5 mm. All the elements were packaged in a plastic cartridge. Our AuNPs-LFBs used here were manufactured through Tian-Jin HuiDeXin Biotech Co., Ltd. (Tianjin, China) according to our devise manual.

LAMP reaction

The single LAMP reactions for HBV or HCV were performed in a one-step 25 µL reaction volumes. Each reaction contained 12.5 µL of 2×reaction buffer (40 mM Tris-HCl (pH 8.8), 16 mM MgSO4, 2 M betaine, 20 mM (NH4)2SO4, 40 mM KCl, and 0.2% Tween-20); 1 µL of Bst 2.0 DNA polymerase (8 U); 0.1 µM F3 and B3 (each), 0.2 µM LF or LF* (for AuNPs-LFB only) and LB (each), 0.4 µM FIP or FIP* (for AuNPs-LFB only) and BIP (each); 1 µL of standard plasmid template (5 µL of clinical sample template); 1 µL of AMV reverse transcriptase (10 U) (only for RNA templates); 2 µL of colorimetric indicator (MG) (for colorimetry only); and double-distilled water (ddH2O) up to 25 µL. The reactions were performed at 65 °C for 60 min.

The one-step multiplex LAMP reaction was also performed in a 25 µL reaction volumes containing 12.5 µL of 2×reaction buffer; 1 µL of Bst 2.0 DNA polymerase (8 U); 0.1 µM S-F3 and S-B3 (each), 0.2 µM S-LF or LF* (for AuNPs-LFB only) and S-LB (each), 0.4 µM S-FIP or S-FIP* (for AuNPs-LFB only) and S-BIP (each), 0.25 µM 5′UTR-F3 and 5′UTR-B3 9 (each), 0.5 µM 5′UTR-LF or LF* (for AuNPs-LFB only) and 5′UTR-LB (each), 1.0 µM 5′UTR-FIP or 5′UTR-FIP* (for AuNPs-LFB only) and 5′UTR-BIP (each); 1 µL of each standard plasmid template (5 µL of the clinical sample template); 1 µL of AMV reverse transcriptase (10 U) (only for RNA templates); 2 µL of colorimetric indicator (MG) (for colorimetry only); and ddH2O up to 25 µL. The reactions were performed at 65 °C for 60 min.

Monitoring methods, including AuNPs-based LFB, real-time turbidity measurements (LA-500, Lumiprobe; Japan), 2% agarose gel electrophoresis, and visual reagent (MG) were utilized for analyzed the LAMP amplification products. For AuNPs-LFB-based evaluation, an HBV-positive result was indicated when both the CL and TL1 of the AuNPs-LFB turned red. An HCV-positive result was indicated when both the CL and TL2 of the AuNPs-LFB turned red. For a negative outcome, only the CL turned red. For real-time turbidity measurements, turbidity > 0.1 indicated a positive outcome. For agarose gel electrophoresis detection, ladder-like bands on the agarose gel manifested a positive result, while no bands indicated a negative outcome. For visual reagent MG analysis, reaction mixtures that turned bright green indicated a successful reaction, while a colorless reaction indicated a negative result.

Optimization of reaction temperature and time for the mLAMP-AuNPs-LFB assay

The optimal amplification temperature for our assay at LAMP reaction stage was test by incubating the HBV-LAMP and HCV-LAMP at 60 to 67 °C (in1°C intervals), and their amplification results were monitored with real-time turbidity. Then, the reaction time for multiplex-LAMP was optimized from 25 to 40 min (at 5 min intervals) under the optimal amplification temperature, the amplification results were analyzed through AuNPs-LFBs. Each assay was conducted in triplicate.

Sensitivity of the mLAMP-AuNPs-LFB assay

Two synthetic plasmids, HBV-S and HCV-5′-UTR plasmids, were serially diluted by a factor of ten to provide copy numbers ranging from 2.0 × 104 copies to 1 copy. Each test was performed under optimal reaction conditions, and the results were monitored through visual reagent MG and biosensor AuNPs-LFB. Each test was repeated three times.

Specificity of the mLAMP-AuNPs-LFB assay.

The specificity of our assay was evaluated by assaying various templates, including HBV-S plasmids, HCV-5′UTR plasmids, HBV/HCV-positive clinical nucleic acid samples (confirmed by real-time PCR), and other microbe nucleic acids, including HIV, Human papillomavirus, Influenza A virus, Influenza B virus, Epstein-Barr virus, Human enterovirus EV71, Coxsackie virus CAV16, Cytomegalovirus, Herpes zoster virus, Escherichia coli, Staphylococcus aureus, Mycobacterium tuberculosis, Klebsiella pneumoniae, Cryptococcus neoformans, and Mycobacterium leprae (Table 2), distilled water (DW) was served as the blank control (BC). Each test was performed independently in triplicate on different days under the optimal reaction conditions.

Table 2 Microbial strains used in this studyFeasibility of the mLAMP-AuNPs-LFB assay for clinical specimens

Serum samples were collected from 73 patients at the Second Affiliated Hospital Guizhou University of Traditional Chinese Medicine, who were suspected to have viral hepatitis caused by HBV and/HCV. The Genomic nucleic acid templates were rapidly isolated through a TIANamp Virus DNA/RNA Fast Kit (TIANGEN BIOTECH Co., Ltd., Beijing, China) according to the manufacture′s guidelines. We compared our assay to commercially available real-time TaqMan PCR Kit for HBV and HCV (Xi’an Tianlong Technology Co. Ltd., Xi’an, China) on an Applied Biosystems™7500 Real-Time PCR System (Life Technologies; Singapore). The results with HBV > 5 IU (~ 30 copies) and HCV > 50 IU (~ 45 copies) were regarded as positive according to the manufacturer’s instructions. The Ethics Committee of The Second Affiliated Hospital of Guizhou University of Traditional Chinese Medicine approved the lawful collection of these genomic nucleic acid templates (Approval No. KYW2022034). The results of our mLAMP-AuNPs-LFB assay were compared with that of conventional quantitative real-time PCR (qPCR) method.