Bridging the Diagnostic Gap: A Rapid, Cost-Effective, and Equitable Hepatitis C Virus RNA Detection Method for Resource-Limited Settings

Abstract

Hepatitis C virus (HCV) remains a major global health challenge, particularly in resource-limited settings where access to molecular diagnostics is restricted. The loop-mediated isothermal amplification (LAMP) assay offers a rapid, cost-effective alternative to polymerase chain reaction (PCR) for HCV RNA detection. Unlike PCR, LAMP uses isothermal amplification (60-65°C), eliminating the need for expensive thermal cyclers and yielding results in under 60 minutes. We evaluated two LAMP detection methods - hydroxynaphthol blue (HNB, colorimetric) and calcein (fluorescent) - against an in-house TaqMan qPCR assay. Both LAMP methods demonstrated high sensitivity (99.6%) and specificity (95.6% for HNB, 99.2% for calcein), with a broad dynamic range (10-10⁶ copies/mL). Clinical validation against real-time PCR showed strong agreement, with a positive predictive value of 98.6% and a negative predictive value of 94.4%. The HNB-LAMP assay, in particular, provides a simple visual readout (blue to sky blue), requiring no specialized equipment, while calcein-LAMP offers fluorescence-based detection under UV light. Both methods showed no significant correlation (p > 0.5), confirming their reliability. LAMP's advantages - minimal infrastructure, ambient temperature stability, and low cost (<$5 per test) - make it ideal for decentralized testing in low-resource settings. This approach could revolutionize HCV diagnosis by enabling same-day test-and-treat strategies, improving linkage to care, and supporting global HCV elimination efforts. Future steps include field validation in remote clinics, manufacturing scale-up, and integration into point-of-care platforms to maximize accessibility. By bridging the diagnostic gap, LAMP can potentially transform HCV management in underserved regions worldwide.

Keywords: bland-altman plot; calcein; hcv rna; hnb; icc; in-house qpcr; lamp methods.

Figure 1. Schematic representation of HCV detection using the LAMP method.

(1) Blood sample collection: A patient’s blood sample is collected for HCV testing. (2) HCV viral RNA extraction: The viral RNA is extracted from the blood sample to serve as the template for amplification. (3) LAMP amplification: The extracted RNA undergoes loop-mediated isothermal amplification (LAMP) using specific primers and a heating system to facilitate amplification. The top inset illustrates the LAMP primer binding sites and amplification mechanism. (4) Naked eye visualization with HNB dye: The results are visualized by the addition of hydroxynaphthol blue (HNB) dye and calcein, where a color change indicates a positive reaction (HCV+). This method provides a rapid, sensitive, and visual detection of HCV infection using LAMP technology.

Figure 2. Gel electrophoresis analysis of DNA amplification using LAMP and PCR primers.

(A) Gel electrophoresis results of loop-mediated isothermal amplification (LAMP) primer-based amplification. The DNA ladder (50 bp) is included for size reference. Distinct bands at 198 bp confirm successful amplification using LAMP primers (F3 and B3). (B): Polymerase chain reaction (PCR) amplification targeting HCV. The DNA ladder (100 bp) provides molecular weight markers. Clear bands at 377 bp indicate successful amplification using HCV-specific PCR primers.

Figure 3. The amplification plot represents fluorescence intensity (dRn) as a function of the number of polymerase chain reaction (PCR) cycles.

Each color in the plot corresponds to a different sample or condition, with curves representing the amplification process over time.

Figure 4. Gel electrophoresis analysis of DNA amplification under different conditions.

Evaluation of primer efficiency and amplification success. Lanes include no template control (NTC), individual primer sets (F3/B3, FLP/BLP), a DNA ladder for size reference, and amplification using all primers with 10² and 10³ DNA copies, showing distinct bands indicating successful amplification. The absence of bands in the NTC confirms no contamination. (B) Optimization of primer concentration. Lanes represent reactions with increasing primer volumes (0.5 µL, 1.0 µL, 1.5 µL, and 2.0 µL). The intensity of the amplified bands increases with primer volume, suggesting an optimal range for efficient amplification. A DNA ladder is included for size comparison, and the NTC shows no amplification, ensuring reaction specificity. These results validate primer performance, amplification efficiency, and optimal conditions for DNA detection.

Figure 5. Spectrophotometric analysis and visual detection of hepatitis C virus (HCV)-positive and healthy samples using hydroxynaphthol blue (HNB) dye.

(A) UV-vis absorption spectra of LAMP-amplified HCV samples with HNB dye, showing distinct absorbance peaks at 580 nm for the healthy control and 648 nm for HCV-positive samples. The color shift in absorbance spectra differentiates positive and negative samples. (B) Visual detection of HCV amplification in LAMP reaction tubes. A distinct color change from violet (negative) to sky blue (positive) confirms the presence of HCV RNA in the test samples. This simple, naked-eye detection method facilitates rapid and reliable identification of HCV-positive cases.

Figure 6. Spectrophotometric analysis and visual detection of hepatitis C virus (HCV)-positive and negative samples using calcein dye.

(A) UV-vis absorption spectra of LAMP-amplified HCV samples in the presence of calcein dye, showing distinct absorbance patterns. Positive samples (HCV+) exhibit higher absorbance intensities, while negative samples (HCV−) and the no-template control (NTC) display lower absorbance values. (B) Visual detection of LAMP-amplified HCV samples in reaction tubes. A color change to bright yellow indicates successful amplification in HCV-positive samples, whereas negative samples and NTC remain unchanged. This method enables rapid, naked-eye detection of HCV infection.

Figure 7. Scatter plot illustrates the correlation between the loop-mediated isothermal amplification (LAMP) assay and real-time polymerase chain reaction (PCR) for quantifying hepatitis C virus (HCV) samples.

Each data point represents an individual sample analyzed by both methods. The coefficient of determination 𝑅²=0.929 indicates a strong positive correlation, demonstrating the reliability of the LAMP assay as a quantitative tool for HCV detection.

Figure 8. The agreement between LAMP assay and real-time PCR. Agreement was assessed by both intraclass correlation and Bland-Altman methods on log-transformed viral load measurements.

(A) Scatter plot with ICC annotation. Each black dot represents one sample’s loop-mediated isothermal amplification (LAMP) assay result plotted against its corresponding real-time PCR value. The red dashed line is the line of identity (y = x). The one-way random-effects intraclass correlation coefficient is ICC (1, 1) = 0.938 (95% CI: 0.907-0.959), indicating excellent absolute agreement between the two methods. (B) Bland-Altman plot. The difference (PCR - LAMP) is plotted against the mean of the two methods for each sample. The central red line shows the mean bias (-0.01 log₁₀ units), and the upper (purple) and lower (green) dashed lines indicate the 95% limits of agreement (-0.24 to 0.22 log₁₀ units). Most points lie within these bounds, demonstrating minimal systematic bias and tight concordance across the full measurement range.