Simultaneous detection of two major lamivudine-resistant mutants using competitively differentiated-PCR.
Rapid, specific and sensitive methods without advanced equipment are required urgently in developing countries in order to detect or monitor lamivudine-resistant mutants routinely before or in the course of the therapy. A protocol is described for the detection of two major YMDD mutations simultaneously through modifying a previous competitively differentiated-PCR (CD-PCR) by revising the strategy, increasing the number of competitively differentiated primers, increasing the number of labeled haptens, optimizing the amplification system and analyzing its products by enzyme immunoassay. Special care was taken to promote the sensitivity, specificity and the ability of the protocol to detect mutation in mixture of the mutants and wild strain. YMDD mutants in clinical serum samples were detected simultaneously, specifically and rapidly only with assistance of the equipment used widely in highly prevalent areas of hepatitis B virus infection.
Peng
Xiao Mou
XM
Department of Infectious Diseases, The Third Affiliated Hospital, Sun Yat-Sen University, 600 Tianhe Road, Guangzhou 510630, Guangdong Province, PR China. xiaomoupeng@hotmail.com
Gu
Lin
L
Huang
Yang Su
YS
Ma
Hui Hui
HH
Xie
Qi Feng
QF
Li
Gang
G
Gao
Zhi Liang
ZL
D000998:Antiviral Agents / Q000494:pharmacology*; D017931:DNA Primers; D004279:DNA, Viral / Q000032:analysis / Q000302:isolation & purification; D024882:Drug Resistance, Viral / Q000235:genetics*; D006509:Hepatitis B / Q000821:virology; D006515:Hepatitis B virus / Q000187:drug effects* / Q000235:genetics; D006801:Humans; D019259:Lamivudine / Q000494:pharmacology*; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology*
D000998:Antiviral Agents; D017931:DNA Primers; D004279:DNA, Viral; D018894:Reverse Transcriptase Inhibitors; D019259:Lamivudine