Fan H H, Kleven S H, Jackwood M W, Johansson K E, Pettersson B, Levisohn S
Department of Avian Medicine, College of Veterinary Medicine, University of Georgia, Athens 30602-4875, USA.
Avian Dis. 1995 Apr-Jun;39(2):398-407.
Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis were used to detect and differentiate four pathogenic species (Mycoplasma gallisepticum, M. iowae, M. meleagridis, and M. synoviae) and ten nonpathogenic species of avian mycoplasma. A sequence of 1026 base pairs within the gene for 16S ribosomal RNA (16S rRNA) from avian mycoplasmas was successfully amplified by PCR with oligonucleotide primers (M16SPCR5' and M16SPCR3') common to all avian mycoplasmas tested. Restriction endonucleases (REs) with unique restriction sites, selected by computer-assisted analysis of known sequences of the amplified segment of avian mycoplasma, were then used to digest the PCR products. After electrophoresis of the resulting RE fragments, the RFLP patterns were compared. Combinations of up to six REs (HpaI, HhaI, HaeIII, HphI, FokI, and NlaIV) produced unique RFLP patterns by which the 14 species of avian mycoplasmas could be differentiated. The newly classified avian species M. imitans was also investigated by this method; M. imitans and M. gallisepticum gave identical RFLP patterns with the REs used in this study. The results obtained by the PCR and RFLP analysis were in agreement with current methods for species identification of avian mycoplasmas.
采用聚合酶链反应（PCR）和限制性片段长度多态性（RFLP）分析来检测和区分四种致病性禽支原体（鸡毒支原体、火鸡支原体、滑液支原体和衣阿华支原体）以及十种非致病性禽支原体。使用针对所有受试禽支原体的寡核苷酸引物（M16SPCR5'和M16SPCR3'），通过PCR成功扩增了禽支原体16S核糖体RNA（16S rRNA）基因内1026个碱基对的序列。然后，通过计算机辅助分析禽支原体扩增片段的已知序列，选择具有独特限制性位点的限制性内切酶（REs）来消化PCR产物。对所得的RE片段进行电泳后，比较RFLP图谱。多达六种REs（HpaI、HhaI、HaeIII、HphI、FokI和NlaIV）的组合产生了独特的RFLP图谱，据此可区分14种禽支原体。还通过该方法对新分类的禽支原体物种模仿支原体进行了研究；在本研究中使用的REs条件下，模仿支原体和鸡毒支原体产生了相同的RFLP图谱。PCR和RFLP分析获得的结果与当前禽支原体物种鉴定方法一致。
