Real-time PCR assay for the detection of tanapox virus and yaba-like disease virus

Abstract

The yatapoxvirus genus contains three members: tanapox virus (TPV), yaba-like disease virus (YLDV) and yaba monkey tumor virus (YMTV), two of which (TPV and YLDV) may infect humans. However, only a very small number of patients have been diagnosed with TPV outside Africa. Given the increased international travel and the similarity of clinical signs during the early stages of a TPV/YLDV infection as compared to diseases caused by agents of potential biological warfare, such as smallpox, monkeypox, tularemia and anthrax, the rapid and reliable recognition of a TPV/YLDV infection is crucial. A real-time PCR assay using TaqManchemistry was developed in order to identify unambiguously TPV/YLDV. Primers and probe targeting a 101bp region of the PstI L fragment of TPV, initial optimisations steps were carried out with YLDV DNA as template. Using probit regression analysis, the lower limit of detection was calculated to be ca. 8 copies per assay. A total of five TPV strains, one YDLV strain and scab-derived DNA from a patient with a TPV infection yielded specific amplification, whereas the DNA of YMTV was not amplified. Various viral and bacterial pathogens (n=29) associated with rash-causing illnesses were not detected using this assay.

Fig. 1

Detection of tanapox virus and yaba-like disease virus using the IT-TaqMan^® PCR. Amplification plots show the results from five tanapox viruses (TNV#81-I-92, TNV#81-I-124, TNV#81-I-231, TNV#81-I-228 and TNVWI-38), one yaba-like disease virus and a scab-derived DNA preparation from a patient (PM/99). Plasmid preparations containing ca. 680 copies/μl (plasmid high copy) and 6.8 copies/μl (plasmid low copy) served as positive controls. Yaba monkey tumor virus (YMTV) and the non-template control (NTC) did not show any amplification.

Fig. 2

Sensitivity and linearity of the IT-TaqMan^® PCR. Ten-fold serial dilutions of yaba-like disease virus DNA (6.8e + 006 − 6.8 copies/μl) were eight-fold amplified. The mean C_t-values were plotted against the copy number.

Fig. 3

Predicted proportion of positive amplification results versus the input concentration of purified plasmid DNA as determined by probit regression analysis. The concentration at which 95% of results are expected to be positive was calculated to be ca. 8 copies/μl.