Use of Reverse Transcription-Polymerase Chain Reaction to Determine the Stability of Rabies Virus Genome in Brains Kept at Room Temperature

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Abstract
In tropical and subtropical climates, the shipment of animal brains for rabies diagnosis may be a problem because brain specimens sometimes arrive decomposed at the diagnostic laboratory. In this situation, reverse transcription-polymerase chain reaction (RT-PCR) may serve as a potential solution because of its high sensitivity. However, little is known about the stability of rabies viral RNA in decomposed brain tissue. To determine the stability of rabies virus genomic RNA in brain samples, 72 mice were inoculated with the challenge virus strain-11 of rabies virus. After incubation period, mice were euthanized to obtain their brains. These were categorized in 2 different groups. In the first group, 36 brains were kept at room temperature (25–27°C) immediately after euthanasia. In the second group, the other 36 inoculated brains were frozen at −70°C and later maintained at room temperature. In both groups, RT-PCR was performed at days 0, 1, 2, 3, 4, 7, 10, 12, 16, 18, 23, and 26 by using primers previously described in the literature and a primer set specifically designed for a Mexican variant of vampire-bat rabies. Reverse-transcriptase PCR experiments were performed in 3 different inoculated brains, in which the direct fluorescent antibody (DFA) test was previously conducted to detect rabies viral antigen in the brains kept at room temperature and in the frozen brains. The DFA test resulted positive in both groups up to day 7. In brain samples stored at ambient temperature (25–27°C), the intensity of the RT-PCR band started to diminish by day 12; however, rabies virus genome could be successfully amplified by RT-PCR up to 23 days. These results indicate that brain samples kept at ambient temperature (up to 27°C) may reach a reference laboratory in an adequate state for rabies diagnosis by RT-PCR.