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Nanoparticle-Based Lateral Flow Biosensors Integrated With Loop-Mediated Isothermal Amplification for the Rapid and Visual Diagnosis of Hepatitis B Virus in Clinical Application.

ABSTRACT: Hepatitis B virus (HBV) infection remains one of the major public health issues worldwide. Developing a rapid, sensitive, specific, easy-to-operate, and cost-saving approach for the diagnosis of HBV is essential for its therapy and prevention. Here, we first devised a novel approach, termed "loop-mediated isothermal amplification integrated with a nanoparticle-based lateral flow biosensor (LAMP-LFB)," for the detection of HBV in clinical application. The results indicated that a set of LAMP primers based on the S gene were valid for the establishment of HBV-LAMP-LFB. The optimal HBV-LAMP can be carried out at a constant temperature of 65°C for 40 min. The whole detection process, including HBV genomic DNA preparation (∼10 min), LAMP (40 min), and LFB reading (within 2 min), can be accomplished within 60 min. The limit of detection of the HBV-LAMP-LFB assay was 7.5 IU per test. The specificity of this assay was one hundred percent, and there was no cross-reactivity with other pathogens. Hence, these results indicated that the HBV-LAMP-LFB assay established in the current study is a sensitive, rapid, specific, visual, simple, and cost-saving method for the screening of HBV agents. More importantly, the HBV-LAMP-LFB has remarkable potential to develop a point-of-care testing in clinical application, especially in resource-scarce regions.

SUBMITTER: Chen X

PROVIDER: S-EPMC8477041 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus that has caused the outbreak of coronavirus disease 2019 (COVID-19) all over the world. In the absence of appropriate antiviral drugs or vaccines, developing a simple, rapid, and reliable assay for SARS-CoV-2 is necessary for the prevention and control of the COVID-19 transmission.MethodsA novel molecular diagnosis technique, named multiplex reverse transcription loop-mediated isothermal amplification, that has been linked to a nanoparticle-based lateral flow biosensor (mRT-LAMP-LFB) was applied to detect SARS-CoV-2 based on the SARS-CoV-2 RdRp and N genes, and the mRT-LAMP products were analyzed using nanoparticle-based lateral flow biosensor. The mRT-LAMP-LFB amplification conditions, including the target RNA concentration, amplification temperature, and time were optimized. The sensitivity and specificity of the mRT-LAMP-LFB method were tested in the current study, and the mRT-LAMP-LFB assay was applied to detect the SARS-CoV-2 virus from clinical samples and artificial sputum samples.ResultsThe SARS-CoV-2 specific primers based on the RdRp and N genes were valid for the establishment of mRT-LAMP-LFB assay to detect the SARS-CoV-2 virus. The multiple-RT-LAMP amplification condition was optimized at 63°C for 30 min. The full process, including reaction preparation, viral RNA extraction, RT-LAMP, and product identification, could be achieved in 80 min. The limit of detection (LoD) of the mRT-LAMP-LFB technology was 20 copies per reaction. The specificity of mRT-LAMP-LFB detection was 100%, and no cross-reactions to other respiratory pathogens were observed.ConclusionThe mRT-LAMP-LFB technique developed in the current study is a simple, rapid, and reliable method with great specificity and sensitivity when it comes to identifying SARS-CoV-2 virus for prevention and control of the COVID-19 disease, especially in resource-constrained regions of the world.

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Nanoparticle-Based Lateral Flow Biosensors Integrated With Loop-Mediated Isothermal Amplification for the Rapid and Visual Diagnosis of Hepatitis B Virus in Clinical Application.

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