Development of a point-of-care test to detect hepatitis B virus DNA threshold relevant for treatment indication

The whole genome of HBV, strain ayw (NC_003977) [17] was used as a template for primer design (Primer Explorer 4.0, Eiken Chemical CO., LTD., Japan). Three sets of LAMP primers (S1, S2, and X) (Table 1) were selected based on the following conditions: (1) the primer length of 18–25 base pairs, (2) the melting temperature of 60–65°C, and (3) the estimated Gibbs free energy of below −4 kJ/mol. Sequences were checked for self-priming and specificity before synthesis (Integrated DNA Technologies, USA). Each primer set was evaluated for amplification efficiency using a standard 2% agarose electrophoresis.

The plasmid pUC19 containing a whole HBV genome AB246345 [18] was propagated in MAX Efficiency^® DH5α™ competent cells (Thermo Fisher Scientific, USA). The plasmid was extracted and purified using a High-Speed Plasmid Mini Kit (Geneaid Biotech Ltd., Taiwan) and the concentration was measured using a NanoDrop 1000 (Thermo Scientific, USA). The plasmid was adjusted to the concentration of 1 mg/mL and stored as aliquots at −20°C until use.

A HBV LAMP reaction consisted of 2.5 mL of 10× isothermal amplification buffer (New England Biolabs, USA), 150 mM MgSO_4, 0.14 mM dNTPs, 5 mM of F3 and B3 primers, 40 mM of forward inner primer (FIP) and backward inner primer (BIP) primers, 10 mM of loop forward primer (LF) and loop backward primer (LB) primers, eight units of Bst 2.0 Polymerase (New England Biolabs, USA), 2 mL of DNA sample, and deionized distilled water to a final volume of 25 mL. The reaction was incubated at 60°C for 60 min before inactivating the enzyme at 95°C for 2 min. Turbidity was detected using 8-well turbidimeter (Mobilis Automata co., Ltd., Thailand) [7] and the results were analyzed using an LAMP-Turbidity plotter version 1.0 (National electronics and computer technology center, NECTEC, Thailand).

Assay sensitivity was tested from serially diluted standard HBV DNA to the final concentrations of 107, 106, 105, 104, 103, 102, and 10 copies and performed under optimized condition as described above. The reactions were incubated in turbidimeter at 60°C for 60 min and correlation between turbidity detection and HBV genome concentration was analyzed. In the spiked serum experiment, the standard HBV DNA at 10⁸ to 10² copies was spiked into fetal bovine serum (1:100 v/v). Samples were diluted with equal amount of deionized distilled water and incubated at 95°C for 5 min and 100°C for 3 min before proceeding to turbidity-based HBV LAMP. Results were confirmed by four independent experiments.

The specificity of the HBV–LAMP assay was tested under optimized condition with six viral DNAs provided as positive controls as follows: HBV (Abbott, USA), human immunodeficiency virus (HIV) (Abbott, USA), Epstein–Barr virus (EBV, cytomegalovirus (CMV), hepatitis C virus (HCV), and herpes simplex virus (HSV) (Qiagen, Germany). The LAMP products were detected using agarose gel electrophoresis. All standard viral DNAs were kindly gifted from King Chulalongkorn Memorial Hospital and were added at 2 × 10⁵ copies to the reaction.

All plasma samples and their automated qPCR results (Abbott, USA) were courtesy of Virology unit, King Chulalongkorn Memorial Hospital with IRB approval (certificate of approval no. 972/2016) from Ethical Review Board, Faculty of Medicine, Chulalongkorn University. The samples were leftovers from routine diagnosis and stored at −70°C. The frozen plasma was rapidly thawed at 37°C and the DNA was extracted from the samples using the heat treatment method [19]. Briefly, the sample was diluted with equal amount of deionized distilled water, followed by incubation at 95°C for 5 min and 100°C for 3 min. DNA was collected from supernatant after centrifugation at 12,000 g for 5 min. Turbidity-based HBV LAMP assay was performed and analyzed as previously indicated. The LAMP product was also analyzed by gel electrophoresis to confirm the end-point results. Results from turbidimeter and gel electrophoresis were compared with those of qPCR diagnostic records.

Three sets of primers (S1, S2, and X) were generated according to the parameters described in Materials and methods section. Efficacy of primer sets was verified using the standard HBV DNA (Figure 1A). Results showed that S2 and X, but not S1, primer sets expressed positive amplification bands in ladder-like pattern, similar to previous report [19]. Next, we optimized the assay condition by varying 4 parameters as follows: temperature (55°C, 60°C, 63°C, and 65°C), betaine (0 M, 0.2 M, 0.4 M, and 0.8 M), dNTPs (1.2 mM, 1.4 mM, 1.6 mM), and MgSO₄ (4 mM, 6 mM, 8 mM). Each assay condition was analyzed using 10-fold serially diluted HBV plasmid (102–10⁵ copies). Results indicated that the optimal condition for S2 primer set was at 60°C with no betaine, 1.4 mM dNTPs, and 6 mM of MgSO₄ demonstrating the detection at 100 copies/reaction in the reaction using 2% agarose gel electrophoresis (Figure 1B and Table 2). Moreover, we verified the HBV DNA template as a standard positive control for further experiments by amplification with established universal primers [20]. PCR results showed that the HBV template was successfully detected by universal primers (Figure 1C); therefore, we reinstated that the plasmid containing HBV genome used in this assay development was a standard HBV DNA template.

The turbidity-based LAMP was set up using the optimal condition for S2 primer set as described and the turbidity was detected using hot plate-coupled, real-time turbidimeter [7]. This turbidimeter detected magnesium pyrophosphate by-product from the LAMP assay in real time. We compared sensitivity of both detection systems using serially diluted standard HBV DNA (Table 1) and results showed equal limit of detection (LoD) at 10² copies/reaction. Moreover, the time to turbidity threshold at the lowest and highest concentrations (10² and 10⁸ copies) was 1888.75 ± 84.94 s, and 1365.75 ± 94.68 s, respectively, suggesting that the positive results should be detected by this assay during 22.76 ± 1.58 and 31.48 ± 1.41 min. Also, a nonlinear correlation of HBV DNA and the threshold of time to positive turbidity were observed (Figure 2A) suggesting that this real-time HBV LAMP turbidity could be applicable in semiquantitative format. In addition to turbidimeter, the positive results can be detected from direct visualization and distinguishable from the no template control (NTC) (Figure 2B). In addition, results can also be reported in using direct visualization for turbidity at the reaction end point. This option is applicable to resource-limited area where only heat block and centrifuge were available.

Figure 1

Figure 2

Next, we tested the assay performance under the condition mimicking the actual serum samples in order to verify whether sera-derived proteins would interfere with the turbidity readout. Briefly, standard HBV DNA was serially diluted and spiked into fetal bovine serum (1:100 v/v), diluted 1:1 with deionized distilled water and heat treated before proceeding to turbidity-based HBV LAMP. The objective was to study possible turbidity interference from other contents in the serum when processing samples with heat treatment method. Our results were in accordance with the previous report comparing the heat treatment and standard DNA extraction methods [16]. LoD of this experiment was read at 10² copies/reaction with no significant difference from that of the sensitivity test previously described (P = 0.6571, Mann–Whitney test). In addition, the conventional HBV PCR amplified by universal primers (Figure 1C) was successfully detected the template at 10³ copies per reaction. We concluded that the heat treatment method coupled with turbidity-based LAMP analysis was adequate for analysis of clinical samples.

The HBV LAMP was tested with the standard DNA of HBV, HIV, HCV, CMV, EBV, and Herpes viruses standardized to 2 × 10⁵ copies/reaction as described in Materials and methods section. The assay was performed in turbidimeter for 60 min and the products were analyzed by gel electrophoresis. Positive results, or a ladder-like pattern, were found only in the HBV sample (Figure 3) suggesting that the HBV LAMP assay was exclusively specific to HBV.

Figure 3

A total of 270 clinical samples consisted of 162 significant (<2 × 10⁵ IU/mL) and 108 nonsignificant (<2 × 10⁵ IU/mL) viremia according to the automated qPCR results. Samples were thawed and DNA was extracted using heat treatment method. HBV LAMP was performed in real-time turbidimeter and results were obtained using turbidity measurement (A₆₅₀) and gel electrophoresis. The results were analyzed using 2 × 2 table for an accuracy of a diagnostic test [21] and compared with the diagnostic results from automated qPCR system. The turbidity-based LAMP method was sensitive, specific, and accurate at 90.12%, 83.33%, and 87.40%, respectively (Table 3), whereas results from the gel electrophoresis showed sensitivity, specificity, and accuracy of the test at 95.06%, 75.00%, and 87.03%, respectively. Interestingly, gel electrophoresis detection was more sensitive but less specific comparing to turbidity detection; in other words, the gel electrophoresis increased both detection rate and false-positive results. We concluded that the turbidity-based LAMP assay is applicable to discriminate the samples containing high viral titers (≥2 × 10⁵ IU/mL) from those of low viral titers (<2 × 10⁵ IU/mL) and negative results (undetectable) previously determined by qPCR (Abbott, USA). Moreover, the sensitivities, specificities, and accuracies of the tests were calculated using the other cutoff values (Table 4). Results showed that the test elicited the maximal accuracy at 10⁵ IU/mL.

In addition, significant viremic samples were further analyzed for correlation with time to turbidity thresholds derived from turbidity-based LAMP assay [12]. Unfortunately, linear regression pattern was not found; therefore, we concluded that the time to turbidity threshold could not be used to quantify HBV viral load in turbidity-based LAMP assay.

The major cost difference between qPCR and LAMP was the equipment used in each method. A quantitative thermal cycler machine price ranges between 30,000 and 50,000 USD, whereas a turbidimeter price used in this optimization was at 2,000 USD. In our design, we expected the local facilities to use a heat block (200–1200 USD), or a waterbath (50–100 USD). Since LAMP relied on an equipment capable of maintaining a single temperature for 30–60 min, the more economical choices of equipment selection can be considered. Moreover, the heat treatment method was not only feasible and time-saving, but also economical for omitting the DNA extraction process. Currently, DNA extraction kit price ranges between 2 and 5 USD/sample and requires about 2 h processing time. Cost and method comparison were summarized in Table 5.