Development of real-time PCR assay for specific detection of cowpox virus

Abstract

Background: The number of recorded human cowpox cases are recently increasing. The symptoms caused by cowpox virus (CPXV) in a number of human cases are close to the symptoms characteristic of the orthopoxviral human infections caused by monkeypox or smallpox (variola) viruses. Any rapid and reliable real-time PCR method for distinguishing cowpox from smallpox and monkeypox is yet absent.

Objectives: The aim of this study was to develop a quick and reliable real-time TaqMan PCR assay for specific detection of cowpox virus and to determine the sensitivity and specificity of this method.

Study design: Based on aligned nucleotide sequences of orthopoxviruses, we found a virus-specific region in the CPXV genome and selected the oligonucleotide primers and hybridization probe within this region. The specificity of the developed method was tested using a panel of various orthopoxvirus (OPV) DNAs. The sensitivity was determined using the recombinant plasmid carrying a fragment of CPXV DNA and genomic DNA of the CPXV strain GRI-90.

Results: The analytical specificity of this method was determined using DNAs of 17 strains of four OPV species pathogenic for humans and amounted to 100%. The method allows 6 copies of plasmid DNA and 20 copies of CPXV DNA in the reaction mixture to be detected.

Conclusion: A quick and reliable TaqMan PCR assay providing for a highly sensitive and specific detection of CPXV DNA was developed.

Fig. 1

Comparison of the ORF D11L sequence of CPXV strain GRI-90 with the corresponding regions of the strains Germany 91-3 and Brighton Red. The identical nucleotides in the compared sequences of virus genomes relative to the CPXV Brighton Red sequence are denoted with dots and the deletions, with dashes. The positions of nucleotides in the analyzed DNA segment are shown to the left and right. The sequences of the oligonucleotide primers CPXV_D11L_upper and CPXV_D11L_lower and hybridization probe CPXV_D11L_probe for fluorescence detection of CPXV DNA are boldfaced.

Fig. 2

The dependences of fluorescence signal on the number of real-time PCR cycles recorded by an iQ 5 (BioRad) using the oligonucleotide primers and probe calculated for the region of CPXV GRI-90 ORF D11L. The groups of curves reflect the shift in the threshold cycle depending on the concentration of recombinant plasmid; Cycle, the number of real-time PCR cycle, and RFU, the value of fluorescence signal.