应用实时荧光定量PCR法检测仔猪肠道内乳酸杆菌和双歧杆菌
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摘要
应用实时荧光定量PCR法对1月龄仔猪粪便中所含的乳酸杆菌和双歧杆菌进行定量检测,揭示肠道相关菌群改变在仔猪腹泻发病中的作用及意义,研究分别设计乳酸杆菌和双歧杆菌的特异性引物和探针,收集仔猪出现腹泻症状的粪便样本30份及正常对照样本30份,提取细菌基因组DNA,应用实时荧光定量PCR检测细菌的数量。结果表明,仔猪出现腹泻症状的粪便组乳酸杆菌和双歧杆菌的数量较正常对照组明显减少,差异有统计学意义。表明肠道菌群与腹泻的发生有一定的关系。
To detect the amount of Lactobacillus and Bifidobacteria in 1-mouth-piglets' fecal specimens using fluorescent real-time PCR, the feces of piglets with diarrhea(n=30)and healthy(n=30)were collected. A set of species-specific primeirs and probe for Lactobacillus and Bifidobacteria were designed. Bacterial genome DNA was estracted from fecal samples,was quantified by fluorescent real-time PCR to analyze bacterial amounts. The result showed that the level of Lactobacillus and Bifidobacteria in diarrhea of piglets were significantly lower than those of healthy control group, the difference was statistically significant. These results indicate that the occurrence of diarrhea may be related to intestinal microflora.
To detect the amount of Lactobacillus and Bifidobacteria in 1-mouth-piglets' fecal specimens using fluorescent real-time PCR, the feces of piglets with diarrhea(n=30)and healthy(n=30)were collected. A set of species-specific primeirs and probe for Lactobacillus and Bifidobacteria were designed. Bacterial genome DNA was estracted from fecal samples,was quantified by fluorescent real-time PCR to analyze bacterial amounts. The result showed that the level of Lactobacillus and Bifidobacteria in diarrhea of piglets were significantly lower than those of healthy control group, the difference was statistically significant. These results indicate that the occurrence of diarrhea may be related to intestinal microflora.
引文
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[20]蒋曼,姚萍,杨涛,等.实时荧光定量PCR法研究溃疡性结肠炎患者肠道双歧杆菌属、柔嫩梭菌属及拟杆菌属量的变化[J].中国微生态学杂志,2013,25(11):1245-1249,1254.
[21]张吉鹍.乳酸菌及其在养猪生产中的应用[J].猪业科学,2018,35(11):86-90.
[22]DONG P,YANG Y,WANG W P.The role of intestinal bifidobacteria on immune system development in young rats[J].Early Human Development,2010,86(1):51-58.
[23]刘国荣,任桂美,李雪,等.动物双歧杆菌细菌素bifidocin A群体感应合成调控行为分析[J].食品科学,2018,39(12):161-166.
[24]赵胜娟.实时荧光定量PCR法检测双歧杆菌对小鼠肠道菌群影响的研究[D].乌鲁木齐:新疆农业大学,2008.
[2]尹清强,李小飞,常娟,等.微生态制剂对哺乳和断奶仔猪生产性能的影响及作用机理研究[J].动物营养学报,2011,23(4):622-630.
[3]刘平,李宗军,许爱清.胃肠道微生态系统及其功能研究[J].中国微生态学杂志,2010,22(3):277-278,281.
[4]PORUK K E,GAY D Z,BROWN K,et al.The clinical utility of CA19-9 in pancreatic adenocarcinoma:diagnostic and prognostic updates[J].Current Molecular Medicine,2013,13(3):340-351.
[5]CHEN Y,YANG F,LU H,et al.Characterization of fecal microbial communities in patients with liver cirrhosis[J].Hepatology,2011,54(2):562-572.
[6]ECKBURG P B,BIK E M,BERNSTEIN C N,et al.Diversity of the human intestinal microbial flora[J].Science,2005,308:1635-1638.
[7]章文明.仔猪源高粘附性乳酸杆菌的分离鉴定、肠道定植及其粘附相关表面蛋白功能分析[D].杭州:浙江大学,2014.
[8]赵丹,杨禹宸,杨志峰,等.酸奶中双歧杆菌浓度的快速分子检测[J].山西农业科学,2018,46(5):756-758.
[9]方磊涵,毕玉霞,王振,等.绞股蓝多糖对断奶仔猪肠道微生物区系的影响[J].河南农业科学,2017,46(12):122-125,138.
[10]董晓丽,张乃锋,周盟,等.复合菌制剂对断奶仔猪生长性能、粪便微生物和血清指标的影响[J].动物营养学报,2013,25(6):1285-1292.
[11]张勇,郭亮,郭上铭,等.乳酸菌在犊牛生产中的应用研究进展[J].中国奶牛,2018(10):9-12.
[12]黄瑞煜.一起猪流行性腹泻病毒病的诊治报告[J].福建畜牧兽医,2012,34(5):76-77.
[13]冯华,刘运超,陈玉梅,等.猪圆环病毒2型SYBRGreen Real-time qPCR检测方法的建立[J].华北农学报,2018,33(4):98-103.
[14]MACK D R,MICHAEL S,WEI S,et al.Probiotics inhibit enteropathogenic Escherichia coli adherence in vitro by inducing intestinal mucin gene expression[J].The American Journal of Physiology,1999,276:941-950.
[15]刁其玉,屠焰,齐广海.益生菌(素)的研究及其在饲料中的应用[J].饲料工业,2002(10):1-4.
[16]DEITCH E A,XU D,QI L,et al.Bacterial translocation from the gut impairs systemic immunity[J].Surgery,1991,109:269-276.
[17]WENK C.Growth promoter alternatives after the ban on antibiotics[J].Pig News and Information,2003,24(1):11-16.
[18]TANABE S,KINUTA Y,SAITO Y.Bifidobacterium infantis suppresses proinflammatory interleukin-17 production in murine splenocytes and dextran sodium sulfate-induced intestinal inflammation[J].International Journal of Molecular Medicine,2008,22(2):181-185.
[19]邢爽,冯京海.乳酸杆菌对肠道上皮紧密连接蛋白的调控[J].动物营养学报,2019,31(4):1540-1546.
[20]蒋曼,姚萍,杨涛,等.实时荧光定量PCR法研究溃疡性结肠炎患者肠道双歧杆菌属、柔嫩梭菌属及拟杆菌属量的变化[J].中国微生态学杂志,2013,25(11):1245-1249,1254.
[21]张吉鹍.乳酸菌及其在养猪生产中的应用[J].猪业科学,2018,35(11):86-90.
[22]DONG P,YANG Y,WANG W P.The role of intestinal bifidobacteria on immune system development in young rats[J].Early Human Development,2010,86(1):51-58.
[23]刘国荣,任桂美,李雪,等.动物双歧杆菌细菌素bifidocin A群体感应合成调控行为分析[J].食品科学,2018,39(12):161-166.
[24]赵胜娟.实时荧光定量PCR法检测双歧杆菌对小鼠肠道菌群影响的研究[D].乌鲁木齐:新疆农业大学,2008.