Specific detection and identification of herpes B virus by a PCR-microplate hybridization assay

Abstract

Herpes B virus DNA was specifically amplified by PCR, targeting the regions that did not cross-react with herpes simplex virus (HSV). The amplified products, which were shown to be highly genetic polymorphisms among herpes B virus isolates, were identified by microplate hybridization with probes generated by PCR. The products immobilized in microplate wells were hybridized with the biotin-labeled probes derived from the SMHV strain of herpes B virus. The amplified products derived from the SMHV and E2490 strains of herpes B virus were identified by microplate hybridization. PCR products amplified from the trigeminal ganglia of seropositive cynomolgus macaques were identified as herpes B virus DNA. The utility of the PCR-microplate hybridization assay for genetic detection and identification of the polymorphic region of herpes B virus was determined.

FIG. 1.

Target regions of PCR and primers used in the present study. Arrows on the genetic map indicate the locations of each primer. Open boxes indicate open reading frames of the 3′ side of the US4 gene, the complete US5 gene, and the 5′ side of the US6 gene. Nucleotide positions in this figure are cited as the E2490 strain sequence numbers deposited in the GenBank database (accession no. AF083210).

FIG. 2.

Specific detection of herpes B virus by gene amplification of the A, C, and E regions (A, B, and C, respectively). (D) Amplification of HSV and CMV with each specific primer set (primer pairs KM1-KM2 and CM3-CM2, respectively). HSV-1 isolates are in lanes 1 to 3 (strains K8, K200, and 198, respectively); HSV-2 isolates are in lanes 4 to 6, (strains 79-29, 27, and 111, respectively); HCMV isolates are in lanes 7 to 10 (strains Towne, AD169, KH, and OK-1, respectively); and SCMV isolates are in lanes 11 and 12 (strains 68-1 and 1090K, respectively). Lanes B and M show SMHV/pBV-DNA and DNA standard size markers (DNA-Molecular Weight Marker V; Roche Diagnostics), respectively.

FIG. 3.

Agarose gel electrophoresis of the amplified products derived from the herpes B virus strains. The gene amplification was performed to yield the C-region products with the primer pair HB2A-HB2B. The amplified products derived from the seropositive monkey specimens are in lanes 1 to 4, the E2490 strain is in lane 5, and SMHV/pBV-DNA is in lane 6. The PCR reactant of the negative control is in lane 7. A difference in band size can be seen between the PCR products generated from the E2490 strain or SMHV/pBV-DNA and that from the seropositive monkeys (specimens 3 and 4 for lanes 3 and 4, respectively).

FIG. 4.

Dilution curves from serial dilutions of the amplified product of the C regions. FU values were obtained by hybridization with probes from the A (squares) and C (triangles) regions at 56°C (solid symbols) and 42°C (open symbols). Dilution curves obtained by using the probes from the A and C regions are shown by dotted and solid lines, respectively.

FIG. 5.

Dilution curves of herpes B virus amplified products (E2490 strain and SMHV/pBV-DNA) and the HSV-1 HF strain obtained by PCR- microplate hybridization. Microplate hybridization with probes from the C regions was performed at 56°C (right) and 42°C (left). FU values are shown for E2490 (□), SMHV/pBV-DNA (▵), HSV HF (×), and negative control (⧫).

FIG. 6.

Dilution curves of herpes B virus amplified products of seropositive monkey specimens (specimens 1 to 4) and SMHV/pBV-DNA obtained by PCR-microplate hybridization. Microplate hybridization with probes from the C regions was performed at 56°C (right) and 42°C (left). FU values are shown for seropositive specimens (▪, •, □, and ○ for specimens 1 to 4, respectively), SMHV/pBV-DNA (▵), and a negative control (⧫).