Optimisation of RT-PCR for detection of enteroviruses.
Enteroviruses are members of Enterovirus genus of Picornaviridae family. On the basis of their pathogenesis and host range, most human enteroviruses are classified into one of three groups (Coxsackie's viruses, echoviruses and polioviruses). Some unclassified human enteroviruses may cause bronchitis (type 68), acute hemorrhagic conjunctivitis (type 70), meningitis and paralysis (types 70 and 71) and hepatitis (type 72 or hepatitis A virus). Enteroviruses can be propagate in primary cultures of human monkey kidney cells and in some cell lines such as HeLa, Vero and WI-38. Virions are small (22-30 nm diameters) containing ss RNA, monopartite and have icosahedra symmetry. The fast, high sensitive and specific detection of enteroviruses today is achieved by using of PCR (Polymerase chain reaction) method. But, PCR is so much more than just mixing reagents in a tube and running a thermal cycler. In each laboratory with PCR facilities, it is necessary to find optimal PCR conditions (performing of PCR optimization experiments). In this paper we presented results of PCR optimization for enteroviruses by using of poliovirus type 1(Sabin). Optimal obtained PCR parameters were: 2,5 mM MgCl2, dNTPs dilution 10(-1) and annealing temperature 50 degrees C, after 30 amplification cycles in Perkin Elmer 2400 thermal cycler.
Subasic
Djemo
D
Institute for Clinical Immunology, Department of Molecular Genetics, Clinical Centre University of Sarajevo.
Karamehic
Jasenko
J
D004770:Enterovirus / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*