Meyer H, Pfeffer M, Rziha H J
Institute of Microbiology, Federal Armed Forces Medical Academy, München, Germany.
J Gen Virol. 1994 Aug;75 ( Pt 8):1975-81. doi: 10.1099/0022-1317-75-8-1975.
A PCR protocol was established that not only allows the detection of, but also the differentiation of species of the genus Orthopoxvirus. This assay was accomplished by the selection of oligonucleotides located within the gene that encodes the A-type inclusion protein of cowpox virus. The primer pair flanked a region exhibiting distinct and specific DNA deletions in the corresponding sequences of vaccinia, mousepox, monkeypox and camelpox virus. For this reason, PCR resulted in DNA fragments of different sizes. The presented PCR protocol, combined with BglII restriction digests, allowed the unequivocal assignment of 42 orthopoxvirus (OPV) strains and isolates to the correct OPV species. The resulting classification corresponded exactly with known biological data for the OPV strains investigated. Furthermore, 13 out of 22 cowpox virus isolates could be subtyped by the presence or absence of a small BglII fragment. DNA sequencing showed that the lack of this BglII fragment was caused by a deletion of 72 nucleotides.
建立了一种聚合酶链反应（PCR）方法，该方法不仅能检测正痘病毒属的病毒，还能区分该属的不同病毒种类。此检测方法是通过选择位于编码牛痘病毒A型包涵体蛋白基因内的寡核苷酸来实现的。引物对侧翼的区域在痘苗病毒、鼠痘病毒、猴痘病毒和骆驼痘病毒的相应序列中呈现出独特且特定的DNA缺失。因此，PCR产生了不同大小的DNA片段。所提出的PCR方法与BglII限制性酶切相结合，能够明确地将42株正痘病毒（OPV）菌株和分离株归为正确的OPV种类。所得分类结果与所研究的OPV菌株的已知生物学数据完全相符。此外，22株牛痘病毒分离株中有13株可根据是否存在一个小的BglII片段进行亚型分类。DNA测序表明，该BglII片段的缺失是由72个核苷酸的缺失导致的。
