A simple and rapid protocol for real-time PCR detection of monkeypox virus (all clades including Ib) v1

Monkeypox virus is re-emerging. It is causing outbreaks again in many countries, especially in African countries such as Congo, and poses a threat to other countries as immunity from the smallpox vaccine is not present in the entire world population. This is why the World Health Organization has declared a state of emergency because its clade Ib is spreading worldwide. It is essential to test various samples, especially skin lesions, for this virus and perform differential diagnosis, e.g. cowpox virus, varicella zoster virus and Mycobacterium leprae, which cause similar symptoms. We show the protocol for monitoring this virus for research studies with our real-time PCR that detects all clades including recent clade Ib. The user can analyze its old pathological samples too. Developing a highly specific test for monkey pox virus is a challenge as most of tests are likely to cross react with very closely related pathogens like cow pox virus, varicella zoster virus, variola etc, so they can deliver false positive results. Even many tests available in the literature cannot properly detect this virus, as they target the region affected by mutations. Such tests give false negative results. The user must take care of this point.