A comparative study of extraction free detection of HBV DNA using sodium dodecyl sulfate, N-lauroylsarcosine sodium salt, and sodium dodecyl benzene sulfonate

Abstract

This study aimed to develop an extraction-free method for quantitative and qualitative detection of HBV DNA compared to traditional nucleic acid extraction. Paired serum and dried blood spot (DBS) samples from 67 HBsAg-positive and 67 HBsAg-negative individuals were included. Two samples with known HBV DNA titers (~ 10⁹ copies/mL) were examined by extraction-free detection using three surfactants (0.2 to 1% of Sodium dodecyl sulfate:SDS, N-Lauroylsarcosine sodium salt:NL, Sodium dodecyl benzene sulfonate:SDBS), two stabilizing agents (0.1 or 0.01% 2-Mercaptoethanol:2ME and 3.5 or 7% Bovine Serum Albumin:BSA) and two Taq polymerases (Fast Advanced and Prime Direct Probe). HBV DNA in all 67 HBsAg-positive and 67 HBsAg-negative serum and DBS samples was directly quantified by Rt-PCR using 0.4% SDS or 0.4% NL with Fast Advance or Prime Direct Probe Taq. Extraction-free amplification was also performed. Detection limits were varied by different surfactants and Taq. SDS combined with Fast Advanced Taq showed lower detection limits, while SDS with Prime Direct Probe Taq outperformed NL or SDBS-based detection. Adding 2ME to SDS improved detection limit with Prime Direct Probe Taq but not significantly compared to SDS alone. BSA did not significantly enhance detection limits but provided insights into sample stability. The senitivity and specificity of 0.4% SDS and NL in combination with either Fast advanced or Prime Direct Probe Taq polymerase in serum samples were > 90% and 100% resepctively, while it was > 80% and 100% respectively in DBS samples. Extraction-free HBV DNA amplification provided 100% identity with original genomes. Our study suggests that SDS, NL or SDBS-based extraction-free HBV DNA detection strategies using Prime Direct Probe Taq have potential to simplify and accelerate HBV DNA detection with high sensitivity and specificity in both serum and DBS samples, with implications for resource-limited settings and clinical applications. Utilizing surfactants with 2ME is optional, and further research and validation are necessary to broaden its application in real-world diagnostics.

Keywords: HBV DNA; N-Lauroylsarcosine sodium salt; Sodium dodecyl benzene sulfonate; Sodium dodecyl sulfate; Surfactant; qPCR.

Fig. 1

Quantitative amplification of HBV DNA by real-time polymerase chain reaction(qPCR). Quantitative amplification by qPCR of HBV DNA was performed on paired serum and DBS samples at varying concentrations of surfactants: (a) Sodium dodecyl sulfate (b) N-Lauroylsarcosine sodium salt and (c) Sodium dodecyl benzenesulfonate in serum samples and (d) Sodium dodecyl sulfate (e) N-Lauroylsarcosine sodium salt and (f) Sodium dodecyl benzenesulfonate in DBS samples. The HBV viral load is expressed as copies per milliliter on the y-axis, while surfactant concentrations, ranging from 0.2% to 1%, are presented on the x-axis. The red line indicates the Prime Direct Probe Taq measurement, and the blue line represents the Fast Advance Taq measurement. The round shapes and triangles depict measurements for the first and second samples, respectively.

Fig. 2

Quantitative amplification of HBV DNA with the effect of 2-Mercaptoethanol (2-ME) on Sodium dodecyl sulfate (SDS) surfactant. Quantitative amplification by qPCR of HBV DNA was performed on paired serum and DBS samples with the addition of different 2-ME concentration (0.1 to 0.4%) on the SDS surfactant. The HBV viral load is expressed as copies per milliliter on the y-axis, while surfactant concentrations, ranging from 0.2 to 1%, are presented on the x-axis. The red line corresponds to the Prime Direct Probe Taq measurement, while the blue line represents the Fast Advance Taq measurement. Circular and triangular markers indicate results for the first and second samples, respectively.

Fig. 3

Quantitative amplification by real time polymerease chain reaction of HBV by serial dilution of different medium. Quantitative amplification by qPCR of HBV DNA was performed by serial dilution( No dilution to 10,000) of the different medium including water(long line), 3.5% Bovine serum albumin (ellipsis) and 7% Bovine serum albumin (long dash). The HBV viral load is expressed as copies per milliliter on the y-axis. The red color corresponds to the Prime Direct Probe Taq measurement, while the blue represents the Fast Advance Taq measurement. Circular and triangular markers indicate results for the first and second samples, respectively.

Fig. 4

Quantitative Detection of HBV DNA in 67 HBsAg-positive and 67 HBsAg-negative serum-DBS paired samples. Detection of HBV DNA by different medium and PCR-Taq among 67 HBsAg positive and 67 HBsAg-negative (a) Serum samples (b) DBS samples. The HBV viral load is expressed as copies per milliliter on the y-axis and different nucleic acid extraction and measurement are presented on the x-axis. The red circular markers indicate HBeAg positive while the blue circular markers indicate HBeAg negative.

Fig. 5

Receiver Operation Curve (ROC)analysis of extraction free detection of HBV DNA. Sensitivity and speicificity of extraction free detection of HBV DNA in serum and DBS samples showed by the ROC for different medium : (a) 0.4% SDS + Fast Advanced Taq , (b) 0.4% SDS + Prime Direct Probe Taq, (c) 0.4% NL + Prime Direct Probe Taq in serum samples and, (d) 0.4% SDS + Prime Direct Probe Taq and (e) 0.4% NL + Prime Direct Probe Taq in DBS samples. The HBV qPCR is expressed as copies per milliliter. The red circular markers indicate HBeAg positive while the blue circular markers indicate HBeAg negative.

Fig. 6

Amplification of HBV DNA by nested polymerase chain reaction (nested-PCR). Amplification of HBV surface partial genome using extraction-free (a) 0.4% SDS treated HBsAg positive serum samples and (b) 0.4% NL treated HBsAg positive serum samples shown by gel electrophoresis with 100 base pairs marker. The original gel photo is provided in Supplementary figure.