Identification of a highly conserved region in the human cytomegalovirus glycoprotein H gene and design of molecular diagnostic methods targeting the region.
Genomic polymorphism of human cytomegalovirus (HCMV) leads to difficulties in the design of molecular diagnostic systems; therefore, a suitable target region was determined in the glycoprotein H (gH) gene, which has been reported to be the most conserved gene. A highly conserved region was identified from codon 1,282 to 1,988 of the gH gene by alignment of 23 nucleotide sequences (14 registered in the DNA Data Bank of Japan and 9 sequenced in this laboratory). Diagnostic methods based on nested PCR, real-time PCR and loop-mediated isothermal temperature amplification (LAMP) were designed for this region. Primers and a probe for nested and real-time PCR were designed for the completely conserved sequences in all HCMV strains. However, a few mismatched nucleotides could not be excluded from the LAMP primers due to the need for eight primer-binding sites in a 200bp-region. The sensitivities of the nested PCR, real-time PCR and LAMP reactions were 5, 10 and 100 copies/tube, respectively. An analysis of clinical specimens showed that both nested and real-time PCR detected HCMV with greater sensitivity than did a pp65 antigenemia assay and were expected to minimize the incidence of false-negative results, whereas the sensitivity of the LAMP reaction was comparable with that of the antigenemia assay.
Fukushima
Eiko
E
Department of Microbiology, Fukushima Medical University, Fukushima, Japan.
Ishibashi
Kei
K
Kaneko
Hisatoshi
H
Nishimura
Hidekazu
H
Inoue
Naoki
N
Tokumoto
Tadahiko
T
Tanabe
Kazunari
K
Ishioka
Ken
K
Ogawa
Hiroshi
H
Suzutani
Tatsuo
T
D002460:Cell Line; D003587:Cytomegalovirus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D003586:Cytomegalovirus Infections / Q000175:diagnosis* / Q000821:virology; D017931:DNA Primers; D004279:DNA, Viral / Q000032:analysis / Q000097:blood; D005347:Fibroblasts / Q000821:virology; D006801:Humans; D016030:Kidney Transplantation / Q000009:adverse effects*; D025202:Molecular Diagnostic Techniques; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D014759:Viral Envelope Proteins / Q000235:genetics*
D017931:DNA Primers; D004279:DNA, Viral; D014759:Viral Envelope Proteins; C499358:glycoprotein H, Human cytomegalovirus