建立实时荧光定量PCR方法监测运输应激对肉牛3种瘤胃细菌的影响

旨在建立一种有效的定量方法监测肉牛在运输前后瘤胃内3种主要细菌的变化,本试验选用8头体质量相近的健康夏南牛,在运输前、运输中6h、运输后1、3、7、15d时间段采集瘤胃液。分别以黄化瘤胃球菌、嗜淀粉瘤胃杆菌、脂解厌氧弧杆菌的16SrRNA序列设计引物,运用Real-time PCR技术研究运输应激对肉牛以上3种瘤胃微生物的影响。结果显示,与运输前相比,黄化瘤胃球菌在运输中6h、运后1d浓度显著升高（P〈0.05）。嗜淀粉瘤胃杆菌在运后3d浓度升高到运输前的13.86倍,随后逐渐降低,15d时为运输前8.42倍。脂解厌氧弧杆菌在运输后3d数量出现极显著的降低（P〈0.05）,在7、15d逐渐恢复。结果表明,运输应激对黄化瘤胃球菌影响不大;脂解厌氧弧杆菌在运输应激后出现显著的降低,运输后半月内逐渐恢复正常水平。嗜淀粉瘤胃杆菌则呈现明显的上升趋势。采用Real-time PCR方法对瘤胃细菌进行定量可以快速有效的反映出运输应激对菌的数量变化趋势。

Establish ment of a Real-Time PCR for monitoring on the 3 bacterial population in the rumen of beef cattle with transport stress

The aim of our study is to establish an effective quantitative method to monitor effects of transport stress on 3 rumen bacteria in beef cattle. Eight healthy Xia-nan cattle similar weight were choosed,respectively rumen fluid was collected before transportation; transportation for 6 h and 1,3,7,15 d after transportation. Based on the 16S rRNA gene sequences of Ruminococcus fla- vefaciens, Ruminobacter amylophilus, anaerovibrio lipolytica, the primers were designed. Using real time fluorescence quantitative PCR assay analyzed effects of transport stress on 3 rumen bacteria in cattle. The results showed that, compared with that before transport, the concentration of Ruminococcus flavefaciens was increased significantly（P〈0.05） 6 h at transportation and 1 d af- ter transportation, the concentration of Ruminobacter amylophilus increased by la. 86 times at 3 d after transport,and then gradually decreased,with anincrease of 8.42 times at 15 d after transport and Anaerovibrio lipolytica at 3 d after transportation was reduce significantly（P~0.05）, then gradually restored at 7 d and 15 d. The results indicated that transport stress had little effect on Ruminococcus flavefaciens, while Ruminobacter amylophilus increased significantly, anaerovibrio lipolytica was reduce significantly,then returned to normal levels after 15 d. And the use of realtime PCR method for quantification of rumen bacteria can quickly and effectively reflects the effect of transport stress on the bacteria in rumen.