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 codon1282 to 1988 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.