Development of sensitive and specific loop-mediated isothermal amplification combined with lateral flow device for the rapid detection of hepatitis B virus infection

Maity, Soumendra Nath; Poonati, Revathi; Punati, Rudrama Devi; Marothi, Yogyata; Mallepaddi, Prudhvi Chand

doi:10.1007/s42770-022-00690-9

Cited by 3 publications

(2 citation statements)

References 21 publications

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“…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 10 2 − 10 1 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.…”

Section: Discussionsupporting

confidence: 89%

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

Husseini,

Baydar

2023

Mol Biol Rep

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Background and ObjectiveRapid 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 ow biosensor for convenient HBV nucleic acid detection at the point-of-care. MethodsClinical serum samples with HBV-positive real-time PCR assays were used for the experiments. Spin column nucleic acid puri cation and serum heat treatment were used for sample preparation. A 250 bp fragment of the HBV polymerase gene was ampli ed with 3 pairs of speci c 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 uorometric analysis, white turbidity, and lateral ow assay. Ready-to-use Milenia HybriDetect1 strips were used to visualize the LAMP amplicons.Subsequently, 10-fold serial dilutions of a secondary standard containing 10 8 IU/ml viral load were used to evaluate lateral ow biosensor performance. ResultsThe LAMP reaction was optimized at 67 •C, and appreciable turbidity was achieved after 30 minutes of incubation. Templates from 10 8 IU/ml to 10 1 IU/ml exhibited distinct test bands when spin column approach was used as the puri cation method. However, the minimum viral load detectable by lateral ow was 10 3 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 10 1 IU/ml only in 66.6% of cases. ConclusionThe 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 ampli cation without DNA puri cation is excluded and may lead to poor performance of the approach.

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Section: Discussionsupporting

confidence: 89%

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

Husseini,

Baydar

2023

Mol Biol Rep

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“…Moreover, this device is compatible with microfluidic platforms and can be adapted for high throughput point-of-care diagnosis of HBV. In addition, Chen et al 80 and Maity et al 81 reported a LAMP based lateral flow device for the rapid detection of HBV infection. The entire detection process can be completed within 60 minutes, with a detection limit of 7.5 IU per test and a specificity of 100%.…”

Section: Loop-mediated Isothermal Amplification (Lamp)mentioning

confidence: 99%

Advances in isothermal nucleic acid amplification methods for hepatitis B virus detection

Li,

Song,

et al. 2023

Analyst

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

Husseini

Baydar

2023

Preprint

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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.

show abstract

Development of sensitive and specific loop-mediated isothermal amplification combined with lateral flow device for the rapid detection of hepatitis B virus infection

Cited by 3 publications

References 21 publications

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

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

Advances in isothermal nucleic acid amplification methods for hepatitis B virus detection

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

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