Multiplex detection of 60 hepatitis B virus variants by maldi-tof mass spectrometry.
BACKGROUND
Variations in the hepatitis B virus (HBV) genome may develop spontaneously or under selective pressure from antiviral therapy. Such variations may confer drug resistance or affect virus replication capacity, resulting in failure of antiviral therapy.


METHODS
A duplex PCR was used to amplify the region of the reverse transcriptase gene, the precore promoter, and the basal core promoter of the HBV genome. Four multiplex primer-extension reactions were used to interrogate 60 frequently observed HBV variants during antiviral therapy. Automated MALDI-TOF mass spectrometry (MS) was used for mutation detection. Capillary sequencing was used to confirm the MS results.


RESULTS
The limit of quantification was 1000 HBV copies/mL for multiplex detection of HBV variants. Fifty-three variants (88.3%) were analyzed successfully in at least 90% of the sera from 88 treatment-naive patients and 80 patients with virologic breakthrough. MS was able to detect twice as many minor variants as direct sequencing while achieving close to full automation. MS and direct sequencing showed only 0.1% discordance in variant calls.


CONCLUSIONS
This platform based on multiplex primer extension and MALDI-TOF MS was able to detect 60 HBV variants in 4 multiplex reactions with accuracy and low detection limits.
Luan
Ju
J
Stanley Ho Centre for Emerging Infectious Diseases and Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong SAR.
Yuan
Jing
J
Li
Xiaohe
X
Jin
Shengnan
S
Yu
Ling
L
Liao
Mingfeng
M
Zhang
Hongmei
H
Xu
Cheng
C
He
Qing
Q
Wen
Bin
B
Zhong
Xunhua
X
Chen
Xinchun
X
Chan
Henry L Y
HL
Sung
Joseph J Y
JJ
Zhou
Boping
B
Ding
Chunming
C
D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000032:analysis* / Q000235:genetics* / Q000302:isolation & purification; D024882:Drug Resistance, Viral; D014644:Genetic Variation; D016679:Genome, Viral; D006515:Hepatitis B virus / Q000235:genetics* / Q000302:isolation & purification*; D006801:Humans; D009154:Mutation; D016133:Polymerase Chain Reaction; D016415:Sequence Alignment; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000191:economics / Q000379:methods*
D017931:DNA Primers; D004279:DNA, Viral