Rapid detection and quantitation of hepatitis B virus DNA by real-time PCR using a new fluorescent (FRET) detection system.
BACKGROUND
The diagnosis of hepatitis B virus (HBV) has until recently been based on traditional serologic methods targeting viral antigens and antibodies to viral proteins. The development of molecular methods allowing for the quantitation of HBV DNA is proving clinically valuable for monitoring therapy and detecting early treatment failures.


OBJECTIVE
Here we report a new real-time (LightCycler) quantitative PCR for the detection of HBV DNA based on sequence specific hybridisation probes (designed in-house), targeting the HBV surface antigen.


METHODS
The assay was evaluated using a 10-fold dilution series of standard HBV DNA [Eurohep standard reference 1, genotype A, HBsAg subtype adw with a unitage of 10(6) WHO. i.u./ml] and 89 clinical serum samples. The performance was measured against a quantified standard HBV DNA working reagent (NIBSC code 98/780) and the sensitivity compared with our conventional thermal-block PCR.


CONCLUSIONS
Real-time PCR detected HBV DNA in 45% (40/89) and thermal-block PCR in 16% (14/75) of clinical samples. Results for 26 samples were below the detection limit of the thermal-block PCR but could be quantified by real-time (LightCycler) PCR. The LightCycler assay was at least 5 logs more sensitive than thermal-block PCR and could detect HBV in a linear range between 5 and 10(7) i.u. per reaction. The broad generic nature of the PCR primers coupled with the enhanced sensitivity and specificity of the fluorescent hybridisation probes makes this assay potentially valuable for both routine diagnostic and epidemiological work.
Aliyu
Sani Hussein
SH
Health Protection Agency, Clinical Microbiology and Public Health Laboratory Addenbrookes Hospital, Box 236, Hills Road, Cambridge CB2 2QW, UK. sani.aliyu@addenbrookes.nhs.uk
Aliyu
Muktar Hassan
MH
Salihu
Hamisu M
HM
Parmar
Surendra
S
Jalal
Hamid
H
Curran
Martin David
MD
D001483:Base Sequence; D003199:Computer Systems; D017931:DNA Primers; D004279:DNA, Viral / Q000032:analysis* / Q000235:genetics* / Q000302:isolation & purification; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D006515:Hepatitis B virus / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D019562:Viral Load
D017931:DNA Primers; D004279:DNA, Viral; D012367:RNA, Viral