应用real-time PCR测定幼兔肠道内双歧杆菌和乳杆菌的变化

Quantification of bifidobacteria and lactobacilli in feces of PN rabbits by real-time PCR

目的：通过研究PN过程中肠道双歧杆菌和乳杆菌的变化，探讨PN导致肠屏障功能障碍的机制．方法：将新西兰种白兔16R，分成对照组（正常兔食饲养）和PN组（幼兔完全肠外营养）．10d后自直肠取出新鲜粪便，抽提所有细菌DNA，常规PCR检测合成的双歧杆菌和乳杆菌属引物的特异性．应用青春双歧杆菌和植物乳杆菌作外标准品，梯度稀释制作标准曲线，作灵敏度测定．应用SYBRGreenIReal-timePCR法，定量检测粪便中双歧杆菌和乳杆菌含量．结果：琼脂糖凝胶电泳显示两种引物特异性良好，灵敏度达到10^2个菌．溶解曲线显示单一峰形，说明没有引物二聚体形成．粪便细菌定量结果显示，PN组幼兔湿粪（0．05g）中双歧杆菌和乳杆菌含量显著低于对照组（4．62±0．24，4．29±0．49vs5．84±0．92．5．144±1．07；P〈0．01）．结论：Real-timePCR法能准确迅速地对幼兔粪便双歧杆菌和乳杆菌进行定量检测．长期肠外营养导致的肠屏障障碍可能与肠内双歧杆菌和乳杆菌的数量减少有关．

AIM： To quantitatively detect bifidobacteria and lactobacilli in feces of Parenteral nutrition （PN） rabbits by real-time quantitative PCR.METHODS： Sixteen New Zealand white rab- bits weighing 200-250 g were divided into two experimental groups： control group and PN group. On day 10, we obtained fresh feces from the rectum. Whole bacterial DNA was extracted from the feces, genus-specific primers of bifido- bacterium and lactobacillus were synthesized, and the specificities of the primers were evaluated by conventional PCR.RESULTS： The detection limit of the method used in this study was found to be 100 per PCR.Standard curves obtained for the strains bifido- bacterium adolescentis and lactobacillus plant were in good agreement. These two kinds of genus- specific primers were proved to be species- specific by agarose gel electrophoresis. The pop- ulation of bifidobacteria and lactobacilli in the wet feces （0.05 g） of PN rabbits was significantly lower than that in the control group （4.62 ± 0.24 and 4.29 ± 0.49 vs 5.84 ± 0.92 and 5.14 ± 1.07; P 〈 0.01）.CONCLUSION： The real-time PCR procedure described here is a specific, accurate, rapid, and easy method for the quantification of bifidobac- teria and lactobacilli in feces of infant rabbits. Dysfunction of＇the intestinal barrier induced by long-time PN-fed may be related to the decreas- ing number of bifidobacteria and lactobacilli in the intestine.