Rapid detection of hepatitis B virus in blood plasma by a specific and sensitive loop-mediated isothermal amplification assay.
BACKGROUND
Hepatitis B virus (HBV) is an important blood-borne pathogen that causes hepatic inflammation and can lead to liver cirrhosis and hepatocellular carcinoma. Conventional methods of HBV detection are time consuming and require highly trained personnel and elaborate equipment. This report describes the development of a rapid, simple, specific, and sensitive loop-mediated isothermal amplification assay (LAMP) for detection of HBV genotypes A, B, C, D, E, and F in blood samples.


METHODS
HBV standard plasma panels and clinical donor plasma specimens were used for the development and validation of the LAMP assay. Amplification was performed at 60°C for 60 minutes using extracted DNA or heat-treated plasma specimens without DNA extraction. The assay was evaluated for its ability to detect various HBV genotypes and for its sensitivity, specificity, and time-point of detection.


RESULTS
The LAMP assay detected HBV genotypes A-F and demonstrated a sensitivity of 10-100 IU per reaction of HBV DNA. The assay also detected 69 of 75 (92%) HBV-positive donor plasma specimens tested and demonstrated a specificity of 100%.


CONCLUSIONS
These results demonstrate that our HBV-LAMP assay is rapid, sensitive and specific, and capable of detecting the major HBV genotypes. This assay could be used in clinical point-of-care settings, mainly in endemic and resource-limited environments for HBV diagnostics, donor screening, epidemiological studies, and therapeutic monitoring of patients undergoing antiviral treatment.
Laboratory of Emerging Pathogens, Office of Blood Research and Review, Division of Emerging and Transfusion-Transmitted Diseases, Center for Biologics Evaluation and Research, US Food and Drug Administration, Bethesda, Maryland.
Dougbeh-Chris
DC
Nyan
Laboratory of Emerging Pathogens, Office of Blood Research and Review, Division of Emerging and Transfusion-Transmitted Diseases, Center for Biologics Evaluation and Research, US Food and Drug Administration, Bethesda, Maryland.
Laura E
LE
Ulitzky
Laboratory of Emerging Pathogens, Office of Blood Research and Review, Division of Emerging and Transfusion-Transmitted Diseases, Center for Biologics Evaluation and Research, US Food and Drug Administration, Bethesda, Maryland.
Nicoleta
N
Cehan
Creative Testing Solutions, Tempe, Arizona.
Phillip
P
Williamson
Creative Testing Solutions, Tempe, Arizona.
Valerie
V
Winkelman
Laboratory of Emerging Pathogens, Office of Blood Research and Review, Division of Emerging and Transfusion-Transmitted Diseases, Center for Biologics Evaluation and Research, US Food and Drug Administration, Bethesda, Maryland.
Maria
M
Rios
Laboratory of Emerging Pathogens, Office of Blood Research and Review, Division of Emerging and Transfusion-Transmitted Diseases, Center for Biologics Evaluation and Research, US Food and Drug Administration, Bethesda, Maryland.
Deborah R
DR
Taylor
D005838:Genotype; D006509:Hepatitis B / Q000175:diagnosis* / Q000821:virology*; D006515:Hepatitis B virus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D010949:Plasma / Q000821:virology*; D019095:Point-of-Care Systems; D012680:Sensitivity and Specificity; D013696:Temperature; D013997:Time Factors
hepatitis B virus; international units per reaction; loop-mediated isothermal amplification; nucleotide; rapid detection