Tan Ching Giap, Ideris Aini, Omar Abdul R, Yii Chen Pei, Kleven Stanley H
Department of Veterinary Clinical studies, Universiti Putra Malaysia.
Onderstepoort J Vet Res. 2014 Sep 2;81(1):e1-e7. doi: 10.4102/ojvr.v81i1.708.
The present study was based on the reverse transcription polymerase chain reaction (RT-PCR) of the 16S ribosomal nucleic acid (rRNA) of Mycoplasma for detection of viable Mycoplasma gallisepticum. To determine the stability of M. gallisepticum 16S rRNA in vitro, three inactivation methods were used and the suspensions were stored at different temperatures. The 16S rRNA of M. gallisepticum was detected up to approximately 20-25 h at 37 °C, 22-25 h at 16 °C, and 23-27 h at 4 °C. The test, therefore, could detect viable or recently dead M. gallisepticum (< 20 h). The RT-PCR method was applied during an in vivo study of drug efficacy under experimental conditions, where commercial broiler-breeder eggs were inoculated with M. gallisepticum into the yolk. Hatched chicks that had been inoculated in ovo were treated with Macrolide 1. The method was then applied in a flock of day 0 chicks with naturally acquired vertical transmission of M. gallisepticum, treated with Macrolide 2. Swabs of the respiratory tract were obtained for PCR and RT-PCR evaluations to determine the viability of M. gallisepticum. This study proved that the combination of both PCR and RT-PCR enables detection and differentiation of viable from non-viable M. gallisepticum.
本研究基于对支原体16S核糖体核酸（rRNA）进行逆转录聚合酶链反应（RT-PCR），以检测鸡毒支原体的存活情况。为了确定鸡毒支原体16S rRNA在体外的稳定性，使用了三种灭活方法，并将悬浮液保存在不同温度下。鸡毒支原体的16S rRNA在37℃下可检测到约20 - 25小时，在16℃下可检测到22 - 25小时，在4℃下可检测到23 - 27小时。因此，该测试可以检测存活的或最近死亡的（<20小时）鸡毒支原体。RT-PCR方法应用于实验条件下药物疗效的体内研究，将商业肉种鸡卵接种鸡毒支原体到卵黄中。在卵内接种的孵化雏鸡用大环内酯1进行治疗。然后该方法应用于一群自然感染鸡毒支原体垂直传播的0日龄雏鸡，用大环内酯2进行治疗。采集呼吸道拭子进行PCR和RT-PCR评估，以确定鸡毒支原体的存活情况。本研究证明，PCR和RT-PCR相结合能够检测并区分存活与非存活的鸡毒支原体。
