Detection of orthopoxvirus DNA by real-time PCR and identification of variola virus DNA by melting analysis

Abstract

Although variola virus was eradicated by the World Health Organization vaccination program in the 1970s, the diagnosis of smallpox infection has attracted great interest in the context of a possible deliberate release of variola virus in bioterrorist attacks. Obviously, fast and reliable diagnostic tools are required to detect variola virus and to distinguish it from orthopoxviruses that have identical morphological characteristics, including vaccinia virus. The advent of real-time PCR for the clinical diagnosis of viral infections has facilitated the detection of minute amounts of viral nucleic acids in a fast, safe, and precise manner, including the option to quantify and to genotype the target reliably. In this study a complete set of four hybridization probe-based real-time PCR assays for the specific detection of orthopoxvirus DNA is presented. Melting analysis following PCR enables the identification of variola virus by the PCR product's characteristic melting temperature, permitting the discrimination of variola virus from other orthopoxviruses. In addition, an assay for the specific amplification of variola virus DNA is presented. All assays can be performed simultaneously in the same cycler, and results of a PCR run are obtained in less than 1 h. The application of more than one assay for the same organism significantly contributes to the diagnostic reliability, reducing the risk of false-negative results due to unknown sequence variations. In conclusion, the assays presented will improve the speed and reliability of orthopoxvirus diagnostics and variola virus identification.

FIG. 1.

Calibration curves of the four LightCycler assays. PCRs for each individual assay were carried out with serial 10-fold dilutions of the corresponding quantified plasmid. C_T values were plotted against initial plasmid copy number (copy #). The correlation of C_T value and plasmid copy number is shown in the box.

FIG. 2.

Melting analysis of assay rpo18 of variola virus. Melting curves of amplified DNA from 12 different orthopoxviruses (as listed in Table 1), covering camelpox, cowpox, monkeypox, ectromelia, and vaccinia virus, compared to 50 and 500 copies of the variola virus-resembling plasmid pVar-rpo18. Only the variola virus-resembling plasmid showed a melting point of 63°C (peak A), attributed to perfectly matching hybridization probes. Due to mismatches in the hybridization probe-binding region, DNA of all other orthopoxviruses show a decreased T_m of 59°C (peak B).

FIG. 3.

Melting analysis of the VETF assay. (a) Melting curves of amplified DNA from 12 different orthopoxviruses (as listed in Table 1), covering camelpox, cowpox, monkeypox, ectromelia, and vaccinia virus, compared to 50 and 500 copies of the variola virus-resembling plasmid pVar-VETF. Besides the characteristic melting peak for the variola virus-resembling plasmid pVar-VETF at 63°C (peak A), all non-variola virus orthopoxviruses fall into the four distinct peaks—peaks B, C, D, and E—as described in the text. (b) Alignment of the VETF sequence corresponding to the two LightCycler hybridization probes VETF Var181 Sen and VETF Var181 Anc. Subsequent to LightCycler PCR, amplicons were sequenced and aligned with the variola virus-specific sequence. Only nucleotide differences are shown. Identical nucleotides are indicated as dots. Sequences for the hybridization probes are boxed.

FIG. 3.

Melting analysis of the VETF assay. (a) Melting curves of amplified DNA from 12 different orthopoxviruses (as listed in Table 1), covering camelpox, cowpox, monkeypox, ectromelia, and vaccinia virus, compared to 50 and 500 copies of the variola virus-resembling plasmid pVar-VETF. Besides the characteristic melting peak for the variola virus-resembling plasmid pVar-VETF at 63°C (peak A), all non-variola virus orthopoxviruses fall into the four distinct peaks—peaks B, C, D, and E—as described in the text. (b) Alignment of the VETF sequence corresponding to the two LightCycler hybridization probes VETF Var181 Sen and VETF Var181 Anc. Subsequent to LightCycler PCR, amplicons were sequenced and aligned with the variola virus-specific sequence. Only nucleotide differences are shown. Identical nucleotides are indicated as dots. Sequences for the hybridization probes are boxed.

FIG. 4.

Melting analysis of the A13L VAR and A13L CAM assays. Shown are melting curves of amplified DNA from 12 different non-variola virus orthopoxviruses (as listed in Table 1) amplified with assay A13L CAM compared to melting curves of 50 and 500 copies of the artificial variola virus A13L sequence amplified with the A13L VAR assay. Melting curve analysis shows characteristic peaks for variola virus at 65°C (peak A) and two additional peaks (peaks B and C) for the non-variola virus orthopoxviruses as described in the text.