The polymerase chain reaction(PCR) assay offers a promising alternative to tissue culture for the rapid and sensitive detection of cytomegalovirus infection. However, high levels of background amplification imposes limitation on this assay.
Developing
appropriate primer set is one of the key-point to overcome this limitation. This study was performed to develop novel sets of primer pairs with which the polymerase chain reaction assay would detect human cytomegalovirus(HCMV) genes in clinical
samples
with higher sensitivity and specificity.
Four sets of primer pairs were used to amplify sequences from four genes of HCMV including DNA polymerase gene(pol1, pol2), immediate early gene(IE1, IE2), gene encoding late antigen of gp64(LA1, LA2) and the gene encoding phosphoprotein 67(pp1,
pp2)
which was cloned from (gt11 expression library using HCMV-specific monoclonal antibody of MCMVA51 as a probe. Seventeen CMV clinical isolates, herpes simplex virus type 1 strain McIntyre, herpes simplex virus type 2 strain G, varicella-zoster
virus
strain Ellen and Epstein-Barr virus strain B95-8 were examined. PCR assay using pol1 and pol2 showed low sensitivity(15/17). Amplification using IE1 and IE2 produced high levels of background bands. PCR reaction using LA1 and LA2 also showed
several
nonspecific bands. On the other hand, PCR with pp1 and pp2 showed specific products without any background amplification.
By eliminating the difficulty in interpretation of borderline positive, the novel primer set developed in this study made PCR detection of HCMV DNA more sensitive.
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