摘要
为建立一种能同时快速检测多种奶牛乳腺炎致病菌的多重PCR检测方法,本研究以化脓隐秘杆菌ISR基因、无乳链球菌cfb基因、大肠杆菌phoA基因、副乳房链球菌23S rRNA基因和金黄色葡萄球菌nuc基因为靶基因,设计并合成了5对特异性引物,通过方阵法对多重PCR反应体系及反应条件优化,建立了一种能快速检测5种病原菌的多重PCR方法。对各菌种随机组合后利用该多重PCR方法检测,结果显示,该方法能同时快速检测奶牛乳腺炎乳样中5种主要病原菌,而其他病原菌则呈阴性结果,具有较强特异性。分别将化脓隐秘杆菌、无乳链球菌、大肠杆菌、副乳房链球菌、金黄色葡萄球菌菌液10倍倍比稀释后,利用建立的多重PCR方法进行检测,确定该方法敏感性,结果显示,该方法对5种病原菌最低检测浓度分别为:金黄色葡萄球菌6.25×10^(4)cfu/mL,大肠杆菌2.95×10^(6)cfu/mL,化脓隐秘杆菌2.95×10^(5)cfu/mL,副乳房链球菌4.3×10^(5)cfu/mL,无乳链球菌3.15×10^(5)cfu/mL。对临床送检及人工模拟的乳样检测结果显示该方法具有较好符合率及灵敏度。本研究首次建立了对包括副乳房链球菌在内的奶牛乳腺炎致病菌多重PCR检测方法,为快速检测奶牛乳腺炎病原菌提供了可行的技术手段。
To establish a multiplex PCR method for rapid and simultaneous detection of major pathogenic bacteria of bovine mastitis,five pairs of specific primers were designed and synthesized with the gene ISR of Trueperella pyogenes,the gene cfb of Streptococcus agalactiae,the gene PhoA of Escherichia coli,the gene 23S rRNA of Streptococcus parauberis and the gene nuc of Staphylococcus aureus as characteristic target genes.Through optimizing reaction conditions with square experiment,multiplex PCR was successfully established a rapid real-time 5-fold PCR assay for detecting five pathogenic bacteria.Then the specificity was determined by the random combination of strains and the sensitivity was determined by gradient dilution of the concentration of the target bacteria.The results showed that this method could simultaneously detect five kinds of main pathogenic bacteria in milk samples of bovine mastitis.At the same time,other pathogens were negative,indicating this method has strong specificity.The minimum detection concentrations of the five pathogens were 6.25×10^(4)cfu/mL for Staphylococcus aureus,2.95×10^(6)cfu/mL for Escherichia coli,2.95×10^(6)cfu/mL for Trueperella pyogenes,4.3×10^(5)cfu/mL for Streptococcus parauberis,and 3.15×10^(5)cfu/mL for Streptococcus agalactiae.The multiplex PCR method was used to test clinical and artificial simulation milk samples,showing it has a good coincidence rate and sensitivity.In this study,a multiplex PCR method for the detection of Streptococcus parauberis was established for the first time.It has important guiding significance for the rapid diagnosis of pathogenic bacteria of bovine mastitis and improving the therapeutic effect of bovine mastitis.
作者
申纪饶
王丹
李新圃
杨峰
武小虎
丁学智
王胜义
严作廷
王旭荣
李宏胜
SHEN Ji-rao;WANG Dan;LI Xin-pu;YANG Feng;WU Xiao-hu;DING Xue-zhi;WANG Sheng-yi;YAN Zuo-ting;WANG Xu-rong;LI Hong-sheng(Key Laboratory of Veterinary Medicine Development of the Ministry of Agriculture,Gansu Key Laboratory of New Veterinary Medicine Engineering,Lanzhou Institute of Husbandry and Pharmaceutical Sciences of CAAS,Lanzhou 730050,China)
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2021年第5期507-511,533,共6页
Chinese Journal of Preventive Veterinary Medicine
基金
国家重点研发项目(2017YFD0502200)
国家自然科学基金(31802232)
中国农业科学院奶牛疾病研究创新工程项目(CAAS-ASTIP-2014-LIHPS-03)。
作者简介
共同第一作者:申纪饶(1996-),女,重庆巴南人,硕士研究生,主要从事奶牛乳房炎病原微生物研究;共同第一作者:王丹(1991-),女,河南郑州人,硕士研究生,主要从事奶牛乳房炎致病菌研究;通信作者:李宏胜,E-mail:lihsheng@sina.com。
