Comparative study of a new quantitative real-time PCR targeting the xylulose-5-phosphate/fructose-6-phosphate phosphoketolase bifidobacterial gene (xfp) in faecal samples with two fluorescence in situ hybridization methods
Comparative study of a new quantitative real-time PCR targeting the xylulose-5-phosphate/fructose-6-phosphate phosphoketolase bifidobacterial gene (xfp) in faecal samples with two fluorescence in situ hybridization methods
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DOI：
10.1111/j.1365-2672.2009.04408.x
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发表时间：
2010-01-01
影响因子：
4
通讯作者：
Le Blay, G.
中科院分区：
文献类型：
--
作者：
Cleusix, V.;Lacroix, C.;Le Blay, G.
目的：建立一种新的定量多重实时PCR（qPCR）方法，对粪便中双歧杆菌进行检测和计数，并与荧光原位杂交（FISH）方法进行比较。通过使用靶向来自结构域细菌的16 S rRNA基因的通用Eub引物靶向双歧杆菌木酮糖-5-磷酸/果糖-6-磷酸转酮酶基因（xfp）和总细菌。qPCR测定显示出高灵敏度和特异性以及约2个中心点5 × 103个双歧杆菌细菌细胞/克粪便的低检测限。将qPCR结果与FISH结合显微镜或流式细胞术（FCM）进行比较。三种方法测得的成人粪便中的双歧杆菌计数平均值之间无统计学差异。总细菌计数平均值较高的FISH方法加上显微镜分析相比，FISH与FCM，而总细胞数估计qPCR是两个FISH methods.Conclusions之间的中间：新的qPCR检测被证明是灵敏，快速和准确的计数bibirusin faec.Significance和Impact的研究：这种方法是一个有价值的替代其他分子方法检测粪便bibirusin细菌，特别是当它们的计数低于检测限的FISH方法。
Aims:To detect and enumerate bifidobacteria in faeces with a new quantitative multiplex real-time PCR (qPCR) method and to compare the results obtained with fluorescence in situ hybridization (FISH) methods.Methods and Results:A multiplex qPCR assay was developed, which enabled the enumeration of Bifidobacterium spp. by targeting the bifidobacterial xylulose-5-phosphate/fructose-6-phosphate phosphoketolase gene (xfp) and total bacteria using universal Eub-primers targeting 16S rRNA gene from the domain bacteria. The qPCR assay showed high sensitivity and specificity and a low detection limit of about 2 center dot 5 x 103 bifidobacterial cells per gram of faeces. The qPCR results were compared with FISH combined with microscopy or flow cytometry (FCM). No statistical differences among bifidobacterial counts averages measured in adult faeces with the three methods were observed. Total bacterial count averages were higher with the FISH method coupled with microscopic analyses compared to FISH with FCM, whereas total cell numbers estimated by qPCR were intermediate between the two FISH methods.Conclusions:The new qPCR assay was shown to be sensitive, rapid and accurate for enumerating bifidobacteria in faeces.Significance and Impact of the Study:This method is a valuable alternative for other molecular methods for detecting faecal bifidobacteria, especially when their counts are below the detection limit of the FISH methods.