Optimization of a rapid and sensitive nucleic acid lateral flow biosensor for hepatitis B virus detection

doi:10.21203/rs.3.rs-2692056/v1

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Optimization of a rapid and sensitive nucleic acid lateral flow biosensor for hepatitis B virus detection

https://doi.org/10.21203/rs.3.rs-2692056/v1

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Journal Publication

published 17 Aug, 2023

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Background and Objective

Rapid on-site testing with low sample volume and point-of-care diagnostics are very useful for preventive measures against hepatitis B virus. Therefore, the aim of this study is to develop a rapid, accurate, and cost-effective lateral flow biosensor for convenient HBV nucleic acid detection at the point-of-care.

Methods
Clinical serum samples with HBV-positive real-time PCR assays were used for the experiments. Spin column nucleic acid purification and serum heat treatment were used for sample preparation. A 250 bp fragment of the HBV polymerase gene was amplified with 3 pairs of specific biotin- and FITC-labeled LAMP primers. Different incubation temperatures (64-68° C) and times (30 min, 45 min, and 1 h) were explored for optimal LAMP assay, and results were assessed by fluorometric analysis, white turbidity, and lateral flow assay. Ready-to-use Milenia HybriDetect1 strips were used to visualize the LAMP amplicons. Subsequently, 10-fold serial dilutions of a secondary standard containing 10⁸ IU/ml viral load were used to evaluate lateral flow biosensor performance.

Results
The LAMP reaction was optimized at 67 ◦C, and appreciable turbidity was achieved after 30 minutes of incubation. Templates from 10⁸ IU/ml to 10¹ IU/ml exhibited distinct test bands when spin column approach was used as the purification method. However, the minimum viral load detectable by lateral flow was 10³ IU/ml when serum samples were treated with heat and the supernatant was directly used for LAMP. The result shows that the developed LAMP-LF assay was able to detect a viral load of 10¹ IU/ml only in 66.6% of cases.

Conclusion

The LAMP-LF assay is a potential solution for HBV testing in resource-limited settings. Overall, our study demonstrated that the integration of LAMP - LF assay can be a plausible approach for point-of-care application with high sensitivity and accuracy. However, direct amplification without DNA purification is excluded and may lead to poor performance of the approach.

Hepatitis B virus

point-of-care

Loop-mediated isothermal amplification

lateral-flow assay

Hepatitis B virus (HBV) is a global public health challenge. Approximately 250 million people worldwide are living with chronic HBV infection [1]. Nearly 15–40% of chronically infected patients develop serious health problems such as cirrhosis, liver failure, and hepatocellular carcinoma, and about one million people die annually from HBV-related complications [1, 2]. Despite this inherent challenge, approximately 90% of people living with hepatitis B worldwide are not diagnosed. Many of the affected populations have limited or no access to basic blood testing for hepatitis B. Approximately 68% of infections have been recorded primarily in developing countries in the medically underserved Western Pacific region and Africa [3, 4]. The lack of medical facilities in these areas contributes to the extremely low HBV detection rate. Therefore, not only high sensitivity but also point-of-care detection are prerequisites for HBV detection measures.

The diagnosis of HBV in practice is primarily based on the rapid detection of HBV surface antigen (HBsAg). This is the most important test, especially in screening studies. In some countries, anti-HBc tests are also used to detect chronic carriers with low viremia in whom HBsAg is undetectable [5–7]. To increase the accuracy, precision, and specificity of these tests, the applicability of metal nanoparticles has been investigated in recent years. Leading ones include gold nanoparticles, magnetic nanoparticles, quantum dot, or their combinations [8–11]. With the increase in serologically negative HBV infection groups and the rapid emergence of diagnostic escape mutants or the problem of non-detection of HBsAg due to low antigen levels, nucleic acid-based methods have proven to be a more effective approach as an adjunct or alternative to serodiagnostic tests [6].

In this direction, nucleic acid test (NAT) has been successfully used for screening in many developed countries in recent years [5]. The highly sensitive real-time polymerase chain reaction (PCR) is used as the gold standard in the currently used tests [12]. Because this method requires a high level of equipment and professional personnel, it can be performed correctly only by technically skilled and experienced personnel in well-equipped laboratories. Because single-sample testing is not cost-effective, they should be collected and stored until a sufficient number of samples have been collected to perform the test. On the other hand, HBsAg testing, anti-HBc, and NAT are required in parallel for comprehensive screening [7].

Rapid, low-sample-volume on-site testing and point-of-care (POC) diagnostics are very utilitarian for preventive measures [13]. Conventional nucleic acid tests cannot be readily applied in POC environments. Although PCR-based detection assays are the most widely used method for the detection of HBV deoxyribonucleic acid (DNA), isothermal techniques have recently gained rapid acceptance [6] Isothermal amplification techniques operate at a constant temperature, as opposed to thermal cycling, which is required at different temperatures. Numerous isothermal amplification techniques, including transcription-mediated amplification (TMA), loop-mediated isothermal amplification (LAMP), recombinase-polymerase amplification (RPA), nucleic acid sequence-based amplification (NASBA), and rolling circle replication (RCR), have been described in the literature. However, these technologies have only recently gained popularity as they are suitable for use in the field, at the point of care, or in the laboratory [6].

Bacillus stearothermophilus (Bst) DNA polymerase and a series of primers are used in a one-step amplification reaction at 58–68°C for 30–60 min to rapidly amplify DNA with high specificity and efficiency. This method also allows easy identification of substances by agarose gel electrophoresis, turbidity inspection, or fluorescence under ultraviolet (UV) light [6, 12, 14–16].

Although the studies performed show high sensitivity and accuracy for qualitative detection, a gel electrophoresis instrument and a UV transilluminator are still required for visualization. Several studies aim to eliminate these problems. The development of a portable fluorescence tube scanner, melting curve analysis in a real-time portable fluorometer, and a real-time fluorogenic protocol to measure time to positivity are well advanced in this regard [17–19]. However, the simplicity of lateral flow (LF) biosensing is potentially remarkable for hospitals, field studies, and POC applications. Therefore, this study aims to develop a rapid, accurate, and cost-effective lateral flow biosensor for convenient hepatitis B virus nucleic acid detection at the point-of-care.

Reagents, chemicals and machines

All primers were synthesized by Oligomer Biotechnology (Ankara, Turkey) (Table s1). LAMP Components such as Bst 2.0 WarmStart DNA Polymerase, Isothermal Amplification Buffer Pack, Deoxynucleotide (dNTP) Solution Mix, LAMP Fluorescent Dye were purchased from New England Biolabs (New England, USA). Ready-to-use Milenia HybriDetect1 LF Strips were provided by TwistDx Limited (United Kingdom). Eppendorf centrifuge, thermocycler, spectrophotometer, and portable Fluo-100 fluorimeter were from Eppendorf AG (Hamburg, Germany), BioRad (Marnes-la-Coquette, France), and Hangzhou Allsheng Instruments (Zhejiang, China), respectively. The kit for isolation of viral nucleic acid was purchased from Augenix Biotechnology (Istanbul, Turkey). Other solutions and buffers used in this study were prepared in our laboratory.

Clinical Sample Collection And Sample Preparation

The clinical serum samples used for the experiments were obtained from participants referred to the Marmara University Faculty of Medicine who underwent HBV-positive real-time PCR testing.

Two general approaches were used for sample preparation. The first was a spin column-based nucleic acid purification method, which was performed according to the manufacturer's instructions. The second method, which seems ideal for POC application, was the heat-treated method. For each sample, 25 µl of positive serum was diluted with 50 µl of nuclease-free water, vortexed, and heat-treated sequentially on a heating block for 5 minutes at 95◦C and 5 minutes at 100◦C. The lysed samples were collected and then centrifuged at 15000 rpm for 10 minutes to remove cell debris. Afterward, the supernatant containing viral DNA was collected for direct amplification of target DNA. Commercial real-time HBV PCR was used as a secondary standard to determine the detection range and performance of the developed LF biosensor.

Primer Design

A total of 46 sequences of the HBV polymerase coding region from known genotypes/subgenotypes of HBV (A-H) and common recombinant sequences were obtained from the NCBI database. Sequence alignment was performed using MEGA X software via the CLUSTALW algorithm. After sequence alignment, 3 pairs of LAMP primers were designed targeting HBV polymerase gene, the most conserved sequences of all genotypes/subgenotypes. The NEB LAMP primer design tool version 1.3.0 [20] was used for this purpose.

BLAST was used to analyze the specificity of the designed LAMP primers and target sequences for the HBV genome. The IDT Oligo Analyzer Tool was used for structural analysis of the primers for hairpin, self-dimer, and heterodimer construction. The sequence of the primers was modified with degenerate nucleotides to allow matching with templates containing diversity. The 5 ends of the forward and reverse primers were labeled with biotin and FITC, respectively.

Loop-mediated Isothermal Amplification (Lamp)

A 250 bp fragment of the HBV polymerase gene was amplified using specific biotin- and FITC-labeled primers LAMP (Table 1).

Table 1) Design of biotin- and FITC-labeled primers for loop-mediated isothermal amplification

The HBV-LAMP was performed in a reaction volume of 25 µL, including 12.5 µL 2 × reaction buffer (40 mM Tris-HCl (pH 8.8), 40 mM KCl, 16 mM MgSO4, 20 mM (NH4)2SO4, 2M betaine, and 0.2% Tween-20), 5 µL genomic template, 0.4 µM of outer primers F3 and B3, 1.6 µM of inner primers FIP and BIP, 0.8 µM of loop primers LF and LB, 1 µL (8U) Bst 2.0 DNA polymerase, and 0.5 µL colorimetric indicator (MG) and addition of double-distilled water to 25 µL. Negative serum and double distilled water were used as blank controls.

In the present study, different incubation temperatures (64–68° C) and times (30 min, 45 min, and 1 h) were investigated for an optimal LAMP assay, and the results were evaluated by fluorometric analysis, visual turbidity, and LF assay.

Visualization Of Products Of Loop-mediated Isothermal Amplification

Fluorometric analysis

Amplification was visualized by adding 0.5 µl of 50x LAMP dye to the reaction tube according to the instructions and measuring the fluorescence emission at blue 460 wavelengths using an Alshungh fluorometric instrument. Moreover, white turbidity of the reaction liquid in daylight or under UV light was checked by naked eyes.

Lateral flow test

Ready-to-use Milenia HybriDetect1, designed to detect amplicons labeled with biotin and FITC (or FAM), and primers containing these moieties were used to detect the LAMP amplicons. The test line was coated with biotin ligands, the control line with anti-rabbit antibodies, and the sample block contains gold-labelled anti-FITC antibodies. The products are capturing by the biotin ligands at the 5-end as the double-labelled amplicons (FITC and biotin) spread from the chromatographic membrane to the test line. On the other hand, the gold-labeled anti-FITC antibody binds to the FITC at the 3 end, resulting in red-pink staining. Non-captured particles are fixed with anti-rabbit antibody at the control line. In the absence of target amplicons, the color appears only at the control line. In total 100 µl fluid (20 µl of samples mixed with 80 µl analyte-specific solution which provided with kits) were load over application pad and incubate for 5–10 minutes.

Evaluation Of Performance Metrics

To determine the detection range and performance of the developed LF biosensor, secondary standards were determined using commercial real-time HBV PCR. 10-fold serial dilutions of a sample containing 10⁸ international units per milliliter (IU/mL) viral load were used as secondary standards. The experimental setup contained healthy negative serum, and HBV-positive samples at 10¹, 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, and 10⁸ IU/ml viral load, respectively, to evaluate the detection limit, and sensitivity.

Optimal reaction conditions

In the present study, different incubation temperatures (64–68° C) and times (30 min, 45 min, and 1 hour) were investigated for an optimal LAMP assay. The LAMP assay could be performed at a wide range of temperatures, and turbidity increased with increasing reaction time. However, the maximum Fluorescence emission intensity level was observed at 67 ◦C reaction temperature (Table 2). Therefore, the LAMP-LF test was performed at 67 ◦C to further determine the optimal reaction time. The appreciable turbidity was obtained after 30 minutes of incubation, indicating that 30 minutes is the optimal reaction time (Table 2).

Table 2

) Fluorescence emission intensity and concentration of isothermal amplification products for samples with 10⁶ IU/ml viral load. Data are the average of three replicate experiments for each condition.
Tm	30 min		45 min		1 hour
Tm	RFU	ng/µl	RFU	ng/µl	RFU	ng/µl
64^◦C	623854	3,2	656124	3.24	634727	3.41
65^◦C	763548	4.12	779608	4.58	740565	4.60
66^◦C	839859	4.94	750264	5.08	790459	5.15
67^◦C	856327	5,77	839364	5.71	842987	5.69
68^◦C	849859	5.67	848660	5.41	846901	5.60

Performance Metric Analysis

Sensitivity analysis and Detection limit

The templates from 10⁸ IU/ml to 10¹ IU/ml had distinct test bands when the spin column approach was used as a purification method (Fig. 1). However, the minimum viral load detectable by LF was 10³ IU/ml when serum samples were treated with heat and the supernatant was used directly for LAMP (Fig. 2). No test band was detected in the negative serum control.

To determine the minimum detection limit of the LF assay, 9 sample dilutions of 10¹ IU/ml viral load were detected by the LAMP-LF assay. The result indicates that the developed LAMP-LF assay could detect 10¹ IU/ml viral load in only 66.6% of cases (Fig. 3). None of the negative serum samples showed a clear band in the LAMP-LF assay.

Historically, the diagnosis of hepatitis B virus (HBV) has been based on serologic detection of HBsAg and, in some countries, anti-HBc. However, detection of HBV DNA is a prerequisite for licensure and initiation of medical treatment [5, 21]. Both technologies are critical, but their use is limited in resource-poor settings due to issues with cost, accessibility, manufacturing delays, and performance, which have contributed to global challenges in the fight against infectious diseases [22]. Serological tests are widely used but have some drawbacks, such as lower sensitivity and greater risk of test error due to manual performance. NATs have high sensitivity and specificity, but due to logistical and financial limitations, the test can only be performed in central laboratories [5, 22]. The use of point-of-care nucleic acid technologies is still comparatively new. It has been recommended that they be used in addition to traditional laboratory testing to improve test accessibility and clinical outcomes [22]. It may be possible to close diagnostic gaps for endemic diseases by increasing capacity for nucleic acid testing at the primary care level, where diagnostic capabilities are most limited. Investments in eradicating endemic infections such as HIV, HPV, and viral hepatitis may improve health only to a limited extent because access to reliable testing is inadequate [22].

Paper-based analyzers (PADs) have emerged as promising platforms for point-of-care testing (POCT) because they are user-friendly, fast, robust, affordable, and scalable in manufacturing. Efficient POCT using PADs are therefore finding widespread application, particularly in home and field settings such as clinical prognostics and diagnostics [23]. Developments at POC-NAT could potentially help achieve WHO 's 2030 goal of 90% diagnosis, 80% treatment, and 65% reduction in HBV-related deaths and eventual eradication of viral hepatitis. The current study addresses the development of a LF assay for HBV nucleic acid testing based on LAMP. In accordance with archival conditions and limited source search capacity, we aim to develop a simple and accurate alternative test. A LAMP method optimized with high sensitivity that can detect low viral loads up to 10¹ IU/ml. This detection limit is acceptable for clinical diagnosis. However, this result is consistent with several studies that indicated they were able to detect a viral DNA load as low as 10² − 10¹ IU/ml by the LAMP reaction for HBV DNA from HBV-positive plasma [12, 14, 15, 17, 24]. The optimized approach possesses a false-negative rate of 33.4%, which is explicitly high. A plausible key factor for the low sensitivity of the method at the very low viral load could probably be explained by passible viral heterogeneity.

However, the approach’s detection specificity and accuracy were seemingly high which could elucidate via primer design. Primers in this LAMP assay were designed that specifically targeted conserved sequences of polymerase regions that show about 96% sequence identity and homology across the HBV genotypes [15]. Another likely influential factor is LAMP approach itself which use multiple primers in reactions lead to more specific assay.

A simple and device-free approach is one of the prerequisites for POC diagnostics defined by the World Health Organization [25]. Therefore, several studies, including the current study, have attempted to simplify the testing procedure and perform it without elaborate equipment, consistent with the limited sources and field studies in which the conventional method has been discredited. In this regard, direct amplification of target DNA from the heat-treated samples is privileged and care has been taken to develop rapid, simple, and cost-effective procedures over DNA extraction approaches [12, 14, 15, 17].

Such approaches have been shown in several studies to be highly sensitive LAMP methods for detecting HBV DNA up to 10 copies/tube similar to qPCR performance and even more [12, 14, 15, 17]. In contrast to the arguments of studies claiming high sensitivity of direct amplification from heat-treated samples, the evidence in the current study shows that the nucleic acid extraction steps are critical and directly affect the sensitivity of the method. There was no significant resemblance between the two methods. The lower sensitivity of direct amplification from lysate compare to DNA purification approach naturally could describe via both method and sample volume which plays a critical role in detection sensitivity [15]. By far, the efficacy of the LAMP approach itself alone has long been accepted and there is a solid body of opinion regarding its applicability to POC diagnosis. In the current study, we optimize rapid (30 minutes) amplification of the HBV DNA fragment with a minimum detection limit of 10¹ IU/ml and 10³ IU/ml with and without DNA extraction, respectively, which can be visualized via LF strips. Apart from the fact that the sensitivity of direct LAMP from lysate is not in agreement with precede studies, direct LAMP from lysate may optimistically aid in the diagnosis of high viremia in high-risk groups such as pregnant women in resource-limited countries [26].

Isothermal incubation makes the LAMP assay easy to set up, and results can be read with the naked eye without the use of any tools. LAMP Results can be determined indirectly by changing the chemical composition of the reaction mixture. The pyrophosphate ion is released from deoxynucleotide triphosphate as a byproduct of DNA polymerization by DNA polymerase and precipitates with the magnesium ion in the reaction buffer, resulting in turbidity that is visible to the naked eye [27]. However, because white turbidity is not a direct signal of the primer-specific reaction, integration of LAMP with a field-ready amplicon-specific visualization method is essential.

The lateral flow assay is a popular POC diagnostic platform that fits well with the criteria of WHO's ASSURED and plays a critical role in combating infectious diseases in resource-limited settings [28]. Direct detection of nucleic acids in a rapid and portable format is essential for on-site detection. The integration of LAMP technologies with LF assay is a promising strategy to simplify traditional molecular testing and helps achieve on-site testing of viral nucleic acids with high sensitivity, where LF assay can enable direct visual interpretation of nucleic acid amplification results.

In this study, there were limitations related to genotypic variety validation, clinical approval, and cross-reactivity determination. The primers designed targeted conserved regions among the 8 different genotypes and common recombination, so speculation to detect common genotypes was promising. Nevertheless, validation with different genotypes and rigorous analysis is essential. Also, this study address to optimization of the approach, thus approval study with immense number of clinical samples and cross-reactivity with commonplace infectious diseases should be under consideration.

The HBV LAMP-LF assay optimized in the current study requires 30 minutes for amplification and has been validated to work well at 67°C, which is conducive for field application. The LAMP test had a detection limit of 10 IU/ml, which was sufficient for clinical samples. Therefore, the LAMP-LF test is a potential solution for HBV testing in resource-limited settings. Overall, our study demonstrated that the integration of LAMP - LF assay can be a plausible approach for point-of-care application with high sensitivity and accuracy. However, direct amplification without DNA purification is excluded and may lead to poor performance of the approach. Although the HBV LAMP detection assay, which uses heat treatment of plasma samples without a DNA extraction step, is not as sensitive as the purified DNA samples, the assay is a rapid, easy-to-use method for HBV detection, at least for detecting high viremia in at-risk groups.

Contributions of the authors
A.A.H. conceived the study, planned and performed the experiments, analyzed the data, and supervised the project. A.H.H. and S.Y.B. drafted the manuscript. All authors read and approved the final manuscript.

Funding

This study was supported by Istanbul Gelisim University, the office of Scientific Research Projects, Grant no: Grant No. KAP-050421-AHH.

Conflicts of interest: there are no conflicts of interest to declare.

Ethical consideration

All participants were provided with informed consent and the study protocol has been approved by the Ethics Committee of the Faculty of medicine, Marmara University in Istanbul, Turkey (07.05.2021 date & protocol no: 09.2021.577).

Data availability statement

Data sharing not applicable to this article as no datasets were generated or analysed during the current study

WHO. Global Hepatitis Report. https://www.who.int/publications/i/item/9789241565455. 2017.
Datta S, Chatterjee S, Veer V, Chakravarty R. Molecular Biology of the Hepatitis B Virus for Clinicians. J Clin Exp Hepatol [Internet]. 2012;2:353–65. Available from: https://www.sciencedirect.com/science/article/pii/S0973688312000801
Hellard ME, Chou R, Easterbrook P. WHO guidelines on testing for hepatitis B and C – meeting targets for testing. BMC Infect Dis [Internet]. 2017;17:703. Available from: https://doi.org/10.1186/s12879-017-2765-2
Clements CJ, Baoping Y, Crouch A, Hipgrave D, Mansoor O, Nelson CB, et al. Progress in the control of hepatitis B infection in the Western Pacific Region. Vaccine [Internet]. 2006;24:1975–82. Available from: http://europepmc.org/abstract/MED/16361001
Busch MP. Should HBV DNA NAT replace HBsAg and/or anti-HBc screening of blood donors? Transfusion Clinique et Biologique [Internet]. 2004;11:26–32. Available from: https://www.sciencedirect.com/science/article/pii/S1246782003001575
Datta S, Chatterjee S, Veer V. Recent advances in molecular diagnostics of hepatitis B virus. World J Gastroenterol [Internet]. 2014;20:14615–25. Available from: http://europepmc.org/abstract/MED/25356025
Kuhns MC, Busch MP. New strategies for blood donor screening for hepatitis B virus: nucleic acid testing versus immunoassay methods. Molecular diagnosis & therapy [Internet]. 2006;10:77–91. Available from: http://europepmc.org/abstract/MED/16669606
Xi D, Luo X, Ning Q. Detection of HBV and HCV coinfection by TEM with Au nanoparticle gene probes. J Huazhong Univ Sci Technolog Med Sci [Internet]. 2007;27:532–4. Available from: http://europepmc.org/abstract/MED/18060628
Shevtsov M, Zhao L, Protzer U, van de Klundert MAA. Applicability of Metal Nanoparticles in the Detection and Monitoring of Hepatitis B Virus Infection. Viruses [Internet]. 2017;9:E193. Available from: https://europepmc.org/articles/PMC5537685
Martiskainen I, Talha SM, Vuorenpää K, Salminen T, Juntunen E, Chattopadhyay S, et al. Upconverting nanoparticle reporter-based highly sensitive rapid lateral flow immunoassay for hepatitis B virus surface antigen. Anal Bioanal Chem [Internet]. 2021;413:967–78. Available from: http://europepmc.org/abstract/MED/33230700
Gong Y, Zheng Y, Jin B, You M, Wang J, Li X, et al. A portable and universal upconversion nanoparticle-based lateral flow assay platform for point-of-care testing. Talanta [Internet]. 2019;201:126–33. Available from: https://www.sciencedirect.com/science/article/pii/S003991401930373X
Zhao N, Liu JX, Li D, Sun DX. [Loop-mediated isothermal amplification for visual detection of hepatitis B virus]. Zhonghua Gan Zang Bing Za Zhi [Internet]. 2016;24:406–11. Available from: http://europepmc.org/abstract/MED/27465942
Xiao Y, Thompson AJ, Howell J. Point-of-Care Tests for Hepatitis B: An Overview. Cells [Internet]. Multidisciplinary Digital Publishing Institute; 2020 [cited 2023 Feb 25];9:2233. Available from: https://www.mdpi.com/2073-4409/9/10/2233
Akram A, Islam SMR, Munshi SU, Tabassum S. Detection of Hepatitis B Virus DNA among Chronic and potential Occult HBV patients in resource-limited settings by Loop-Mediated Isothermal Amplification assay. J Viral Hepat [Internet]. 2018;25:1306–11. Available from: http://europepmc.org/abstract/MED/29768691
Nyan D-C, Ulitzky LE, Cehan N, Williamson P, Winkelman V, Rios M, et al. Rapid detection of hepatitis B virus in blood plasma by a specific and sensitive loop-mediated isothermal amplification assay. Clin Infect Dis [Internet]. 2014;59:16–23. Available from: http://europepmc.org/abstract/MED/24704724
moslemi E, Shahhosseiny MH, Javadi G, Praivar K, Sattari TN, Amini HK. Loop mediated isothermal amplification (LAMP) for rapid detection of HBV in Iran. 2009.
Chen H-W, Belinskaya T, Zhang Z, Ching W-M. Simple Detection of Hepatitis B Virus in Using Loop-Mediated Isothermal Amplification Method. Mil Med [Internet]. 2019;184:e275–80. Available from: http://europepmc.org/abstract/MED/30690497
Quoc NB, Phuong NDN, Chau NNB, Linh DTP. Closed tube loop-mediated isothermal amplification assay for rapid detection of hepatitis B virus in human blood. Heliyon [Internet]. 2018;4:e00561. Available from: https://europepmc.org/articles/PMC5857714
Cai T, Lou G, Yang J, Xu D, Meng Z. Development and evaluation of real-time loop-mediated isothermal amplification for hepatitis B virus DNA quantification: a new tool for HBV management. J Clin Virol. 2008;41 4:270–6.
New england biolabs inc. NEB LAMP Primer Design Tool version 1.3.0. https://lamp.neb.com/#!/. 2023.
Satoh K, Iwata-Takakura A, Yoshikawa A, Gotanda Y, Tanaka T, Yamaguchi T, et al. A new method of concentrating hepatitis B virus (HBV) DNA and HBV surface antigen: an application of the method to the detection of occult HBV infection. Vox Sang [Internet]. 2008;95:174–80. Available from: http://europepmc.org/abstract/MED/19121181
Jani I v, Peter TF. Nucleic Acid Point-of-Care Testing to Improve Diagnostic Preparedness. Clinical Infectious Diseases [Internet]. Oxford Academic; 2022 [cited 2023 Feb 25];75:723–8. Available from: https://academic.oup.com/cid/article/75/4/723/6500010
Hu J, Xiao K, Jin B, Zheng X, Ji F, Bai D. Paper-based point-of-care test with xeno nucleic acid probes. Biotechnol Bioeng [Internet]. 2019;116:2764–77. Available from: http://europepmc.org/abstract/MED/31282991
Maity SN, Poonati R, Punati RD, Mallepaddi P, Marothi Y, Mallepaddi PC. Development of sensitive and specific loop-mediated isothermal amplification combined with lateral flow device for the rapid detection of hepatitis B virus infection. Brazilian Journal of Microbiology [Internet]. 2022;53:615–23. Available from: https://doi.org/10.1007/s42770-022-00690-9
Land KJ, Boeras DI, Chen X-S, Ramsay AR, Peeling RW. REASSURED diagnostics to inform disease control strategies, strengthen health systems and improve patient outcomes. Nat Microbiol [Internet]. 2019;4:46–54. Available from: http://europepmc.org/abstract/MED/30546093
Vanhomwegen J, Kwasiborski A, Diop A, Boizeau L, Hoinard D, Vray M, et al. Development and clinical validation of loop-mediated isothermal amplification (LAMP) assay to diagnose high HBV DNA levels in resource-limited settings. Clin Microbiol Infect. 2021;
Shirato K. Detecting amplicons of loop-mediated isothermal amplification. Microbiol Immunol [Internet]. 2019;63:407–12. Available from: http://europepmc.org/abstract/MED/31342547
Zhou Y, Wu Y, Ding L, Huang X, Xiong Y. Point-of-care COVID-19 diagnostics powered by lateral flow assay. Trends Analyt Chem [Internet]. 2021;145:116452. Available from: http://europepmc.org/abstract/MED/34629572

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Optimization of a rapid and sensitive nucleic acid lateral flow biosensor for hepatitis B virus detection

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Journal Publication

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Abstract

Figures

Introduction

Material And Methods

Reagents, chemicals and machines

Clinical Sample Collection And Sample Preparation

Primer Design

Loop-mediated Isothermal Amplification (Lamp)

Visualization Of Products Of Loop-mediated Isothermal Amplification

Lateral flow test

Evaluation Of Performance Metrics

Results

Optimal reaction conditions

Performance Metric Analysis

Sensitivity analysis and Detection limit

Discussion

Conclusion

Declarations

References

Status:

Journal Publication

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