Detection of human orthopoxvirus infections and differentiation of smallpox virus with real‐time PCR

Abstract

We developed a real-time PCR protocol to detect orthopoxviruses (OPVs) from different clinical specimens and to separate variola virus from other OPVs. In our protocol, we used automated nucleic acid extraction system together with realtime PCR to create a simple, safe and fast procedure to obtain an initial result. The sensitivity was better by using designed hybridization probes as compared to SYBR green I for detection. The detection limit ranged from 13 to 1,300 copies per 20 ml reaction volume depending on the sample type. The PCR detected all OPVs pathogenic to human (variola, cowpox, monkeypox, vaccinia) as well as camelpox and ectromelia viruses. Amplification of variola virus sequences could be distinguished from other OPVs by melting curve analysis. We also demonstrated the applicability of the assay in human cases of cowpox and vaccinia virus infections.

Lymphocytic choriomeningitis, Ljungan and orthopoxvirus seroconversions in patients hospitalized due to acute Puumala hantavirus infection

“…In an attempt to identify virus species in OPV-seroconverted samples, we used previously described methods [35]. For arenaviruses, one-step RT-PCR was carried out using Invitrogen SuperScript ® III One-Step RT-PCR System with Platinum ® Taq DNA Polymerase (Invitrogen, Carlsbad, CA) and RiViGene primers: two forward primers: LVL3359D Yplus(5-AGAATCAGTGAAAGGGAAAGCAAY) and LVL3359G Yplus (5 -AGAATTAGTGAAAGGGAGAGTAAT); and two reverse primers: LVL3754D Rminus (5 -CACATCATTGGTCCCCATTTACTGTGR) and …”

Detection of pseudocowpox virus in water buffalo (Bubalus bubalis) with vesicular disease in the state of São Paulo, Brazil, in 2016

“…However, in this work we also found the virus in serum. The presence of viral DNA in serum also occurs with other poxviruses such as the VACV (Putkuri et al 2009). Molecular tests may be an important tool to detect animals without symptoms since such animals also transmit this zoonotic agent (Yaegashi et al 2016).…”

Variola: Smallpox

“…A theoretical evaluation of the assay 12 [11] No (0/110) Yes VETF 12 [11] No (0/110) Yes A13L 12 [11] No (0/110) Yes 14 kda 46 [12] No (0/190) Yes HA Artificial construct [13] No ( design was performed by using a BLAST search to check the specificity of primers and detection probes. A practical evaluation of those assays that met theoretical demands was done by testing up to 110 nonvariola poxviruses (Table 11.2).…”

“…In 2009 Putkuri et al [13] published a LightCycler assay using generic OPV primers to amplify part of the HA gene. Identification of VARV is achieved by FMCA.…”

“…Considering the broadened melting peaks due to the long hybridization probes applied, this small difference in T m of 2 • C may lead to difficulties in the correct genotyping of VARV. Due to this publication being so recent [13], we have not yet performed experimental analysis of this assay with the poxvirus test panel.…”