Rapid, visual, label-based biosensor platform for identification of hepatitis C virus in clinical applications

Abstract

Objectives: In the current study, for the first time, we reported a novel HCV molecular diagnostic approach termed reverse transcription loop-mediated isothermal amplification integrated with a gold nanoparticles-based lateral flow biosensor (RT-LAMP-AuNPs-LFB), which we developed for rapid, sensitive, specific, simple, and visual identification of HCV.

Methods: A set of LAMP primer was designed according to 5'untranslated region (5'UTR) gene from the major HCV genotypes 1b, 2a, 3b, 6a, and 3a, which are prevalent in China. The HCV-RT-LAMP-AuNPs-LFB assay conditions, including HCV-RT-LAMP reaction temperature and time were optimized. The sensitivity, specificity, and selectivity of our assay were evaluated in the current study. The feasibility of HCV-RT-LAMP-AuNPs-LFB was confirmed through clinical serum samples from patients with suspected HCV infections.

Results: An unique set of HCV-RT-LAMP primers were successfully designed targeting on the 5'UTR gene. The optimal detection process, including crude nucleic acid extraction (approximately 5 min), RT-LAMP reaction (67℃, 30 min), and visual interpretation of AuNPs-LFB results (~ 2 min), could be performed within 40 min without specific instruments. The limit of detection was determined to be 20 copies per test. The HCV-RT-LAMP-AuNPs-LFB assay exhibited high specificity and anti-interference.

Conclusions: These preliminary results confirmed that the HCV-RT-LAMP-AuNPs-LFB assay is a sensitive, specific, rapid, visual, and cost-saving assay for identification of HCV. This diagnostic approach has great potential value for point-of-care (POC) diagnostic of HCV, especially in resource-challenged regions.

Keywords: Biosensor; Hepatitis C virus; Limit of detection; Point-of-care platform; Reverse transcription loop-mediated isothermal amplification.

Fig. 1

Schematic diagram of HCV-RT-LAMP-AuNPs-LFB assay’s principle. (A) Schematic diagram of AuNPs-LFB principles for the interpretation of HCV-RT-LAMP outcomes. I, HCV-RT-LAMP amplification products (2.0 µl) and running buffer (100 µl) are added simultaneously on the sample pad. II, Due to capillary action, the running buffer containing HCV-RT-LAMP products move forward onto the conjugate pad and nitrocellulose (NC) membrane. The dye streptavidin-coated gold nanoparticles (streptavidin-AuNPs) are rehydrated and integrate with FAM/biotin labeled HCV-RT-LAMP products. III, The FAM/biotin-labeled HCV-RT-LAMP products are captured by anti-FAM at the test line (TL). Streptavidin-AuNPs are captured by biotin-BSA at the control line (CL). IV, Interpretation of the HCV-RT-LAMP-AuNPs-LFB assay. HCV-positive results are indicated by CL and TL bands on the AuNPs-LFB. Negative results are indicated when only the CL band appears on the AuNPs-LFB. (B) Workflow of the HCV-RT-LAMP-AuNPs-LFB assay. The workflow comprises the following closely linked steps: rapid genomic RNA extraction (step 1), RT-LAMP amplification (step 2), and AuNPs-LFB visual interpretation (step 3). The entire detection process is complete within 40 min

Fig. 2

Verification of HCV-RT-LAMP products. The HCV-RT-LAMP products were identified simultaneously using (A) 2% agarose gel electrophoresis, (B) color change (L-HNB), and (C) AuNPs-LFB. Templates of 1–8 were HCV-1b plasmid, HCV-2a plasmid, HCV 3b plasmid, HCV-6a plasmid, HCV-3a plasmid, hepatitis B virus (HBV), human immunodeficiency virus (HIV), and distilled water (DW), respectively. CL: control line; TL: test line

Fig. 3

Temperature optimization for HCV-RT-LAMP amplification. The RT-LAMP amplifications for detection of HCV were monitored using real-time turbidity (LA-500, Eiken Chemical Co., Ltd., Japan). The corresponding curves of amplicons are displayed in the graphs. Turbidity > 0.1 indicated a positive value. Eight kinetic graphs were obtained at different temperatures (63–70 °C, 1 °C intervals) with 2 × 10³ copies of the target gene (A-H). The graphs from E (67 °C) showed robust amplification

Fig. 4

Sensitivity analysis of the HCV-RT-LAMP-AuNPs-LFB assay with serial dilutions of nucleic acid template. Visual reagent (L-HNB) and AuNPs-LFB approaches were simultaneously used to readout the HCV-RT-LAMP outcomes. L-HNB (A)/AuNPs-LFB (B); 1–6 represent the HCV plasmid concentrations of 2.0 × 10³ copies, 2.0 × 10² copies, 2.0 × 10¹ copies, 2.0 × 10⁰ copies, and 1 copy per test and distilled water (DW), respectively. The limit of detection (LoD) of the HCV-RT-LAMP assay was 20 copies per test. CL: control line; TL: test line

Fig. 5

Amplification time optimization for the HCV-RT-LAMP-AuNPs-LFB assay. Four RT-LAMP reaction times, including 10 min (A), 20 min (B), 30 min (C), and 40 min (D), were evaluated at optimal reaction conditions. Tube/biosensor 1–6 represent HCV plasmid concentrations of 2.0 × 10³ copies, 2.0 × 10² copies, 2.0 × 10¹ copies, 2.0 × 10⁰ copies, and 1 copy per test and distilled water (DW), respectively. The results were analyzed simultaneously using visual reagent L-HNB and AuNPs-LFB. The signal of the LoD appeared with a 30 min reaction time through AuNPs-LFB. CL, control line; TL, test line

Fig. 6

HCV-RT-LAMP-AuNPs-LFB assay specificity with different strains. Assay specificity was evaluated using different nucleic acid templates. Each result was tested through AuNPs-LFB: 1–5, HCV 1b, 2a, 3b, 6a, and 3a 5’UTR plasmids; 6–11, HCV clinical samples; 12, Hepatitis B virus; 13, Human immunodeficiency virus; 14, Epstein-Barr virus; 15, Coxsackie virus CAV16; 16, Human papillomavirus; 17, Human enterovirus EV71; 18, Influenza A virus; 19, Influenza B virus; 20, Cryptococcus neoformans; 21, Enterococcus faecium; 22, Salmonella enteritidis; 23, Shigella bogdii; 24, Staphylococcus aureus; 25, Streptococcus pyogenes; 26, Streptococcus pneumoniae; 27, Escherichia coli; 28, Pseudomonas aeruginosa; 29, Klebsiella pneumoniae; 30, Mycobacterium tuberculosis; 31, Brucella; 32, Other microbial nucleic acid mixtures containing HCV-1b plasmids; 33, Other microbial nucleic acid mixtures containing HCV-2a plasmids; 34, Other microbial nucleic acid mixtures containing HCV-3b plasmids; 35, Other microbial nucleic acid mixtures containing HCV-6a plasmids; 36, Other microbial nucleic acid mixtures containing HCV-3a plasmids; 37, Other microbial nucleic acid mixtures; 38, distilled water (DW). CL, control line; TL, test line