Development of a real-time PCR assay for monitoring anaerobic fungal and cellulolytic bacterial populations within the rumen
Development of a real-time PCR assay for monitoring anaerobic fungal and cellulolytic bacterial populations within the rumen
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DOI：
10.1111/j.1574-6941.2006.00190.x
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发表时间：
2006-12-01
影响因子：
4.2
通讯作者：
McSweeney, Christopher S.
中科院分区：
文献类型：
--
作者：
Denman, Stuart E.;McSweeney, Christopher S.
传统的计数和鉴定瘤胃内微生物种群的方法既耗时又繁琐。涉及培养和显微镜的方法也可能是不确定的，特别是在研究厌氧瘤胃真菌时。介绍了一种针对瘤胃真菌、黄色瘤胃球菌和琥珀酸纤维杆菌的实时荧光定量聚合酶链式反应SYBR Green方法，包括设计和验证。对多中心和单中心真菌分离物的DNA和粗蛋白质含量与真菌生物量的关系进行了跨真菌生长阶段的调查，以帮助生成标准曲线。所使用的引物组被发现是靶标特异性的，没有可检测到的交叉反应。随后，在一项研究中使用了实时荧光定量聚合酶链式反应来检测牛瘤胃内的这些种群。从投喂后0~12h，厌氧真菌靶标增加3.6倍。结果还表明，在摄食后0至12小时内，产琥珀酸假单胞菌的靶标数量增加了5.4倍，而黄色葡萄球菌则大致保持了一致的水平。这是第一份实时聚合酶链式反应检测瘤胃厌氧真菌数量的报告。
Traditional methods for enumerating and identifying microbial populations within the rumen can be time consuming and cumbersome. Methods that involve culturing and microscopy can also be inconclusive, particularly when studying anaerobic rumen fungi. A real-time PCR SYBR Green assay, using PCR primers to target total rumen fungi and the cellulolytic bacteria Ruminococcus flavefaciens and Fibrobacter succinogenes, is described, including design and validation. The DNA and crude protein contents with respect to the fungal biomass of both polycentric and monocentric fungal isolates were investigated across the fungal growth stages to aid in standard curve generation. The primer sets used were found to be target specific with no detectable cross-reactivity. Subsequently, the real-time PCR assay was employed in a study to detect these populations within cattle rumen. The anaerobic fungal target was observed to increase 3.6-fold from 0 to 12 h after feeding. The results also indicated a 5.4-fold increase in F. succinogenes target between 0 and 12 h after feeding, whereas R. flavefaciens was observed to maintain more or less consistent levels. This is the first report of a real-time PCR assay to estimate the rumen anaerobic fungal population.