牛支原体等温扩增冻干试剂盒研究

发布时间：2018-06-17 16:48

  本文选题：牛支原体 + 等温扩增　； 参考：《中国农业科学》2016年16期

【摘要】：【目的】牛支原体(Mycoplasma bovis)是导致牛多种疾病综合征的病原体之一,在世界范围广泛流行。为了有效监测此病在中国的流行情况,迫切需要敏感、便捷的诊断试剂产品。【方法】通过构建含有牛支原体uvrC基因片段的重组质粒并转化TOP10感受态细胞,获得重组大肠杆菌rP-uvrC。重组大肠杆菌大量表达并提取重组质粒后,获得质粒浓度为10~4拷贝/μL的溶液,作为质控用阳性对照品。根据文献报道的浓度配制甜菜碱溶液和显色液(主成份为SYBR Green I和HNB),分别作为冻干品溶解用溶液和等温扩增产物显色溶液。在已建立的牛支原体环介导等温扩增(loop-mediated isothermal amplification,LAMP)检测技术的基础上配制等温扩增试剂,并通过考察等温扩增反应情况在常用的8种冻干疫苗耐热保护剂中选择对等温扩增反应没有影响的3种冻干保护剂,每种保护剂分别选用3种不同的浓度,共设计27种保护剂组方,通过观察冻干品物理性状选择最佳的一组保护剂配制冻干用等温扩增试剂,放到冻干机中测定共晶点,并优化一次干燥升温时间、干燥时间和二次干燥时间。通过对冻干制品物理性状的检验、真空度的检测以及残余水分含量的测定,筛选出一条适合等温扩增试剂的冻干曲线,并以此制备等温扩增试剂冻干品。取一定量等温扩增试剂冻干品、甜菜碱溶液、阳性对照品溶液和显色液,组装成试剂盒。利用6个浓度梯度(10~0-10~5个拷贝)重组质粒溶液检测试剂盒的敏感性,利用浓度为10~4 CCU·mL~(-1)的PG-45株、HB-1株、SD-2株牛支原体菌液、10~8CCU·mL~(-1)的牛鼻支原体和无乳支原体菌液、10~8CFU·mL~(-1)的多杀性巴氏杆菌和结核分枝杆菌,检测试剂盒的特异性。另外,将等温扩增冻干试剂盒分别置于不同温度下保存,检测其稳定性。【结果】8种冻干保护剂中只有海藻糖、甘露醇和牛血清白蛋白不影响等温扩增反应,在此基础上优选的冻干保护剂配方为5%海藻糖+1.25%甘露醇+1.25%牛血清白蛋白,等温扩增试剂共晶点为-16℃,一次干燥的升温时间3h,一次干燥时间6h,二次干燥时间4 h。组装后的试剂盒最低可检测到10个拷贝数的重组质粒,检验PG45株、HB-1株以及SD-2株牛支原体均为阳性,检验牛鼻支原体、无乳支原体、多杀性巴氏杆菌和结核分枝杆菌均为阴性。试剂盒在20℃保存6个月、37℃保存10 d后,敏感性仍为10个拷贝数,与第0天相同,推测4℃保存有效期为24个月左右。【结论】笔者研制的等温扩增冻干试剂盒敏感性高、特异性好、稳定性好、操作便捷,适合基层兽医现场检测使用。
[Abstract]:Objective: Mycoplasma bovisis is one of the pathogens leading to many disease syndromes in cattle. In order to effectively monitor the prevalence of this disease in China, sensitive and convenient diagnostic reagent products are urgently needed. [methods] Recombinant Escherichia coli rP-uvrC was obtained by constructing recombinant plasmid containing UvrC gene fragment of Mycoplasma bovis and transforming TOP10 competent cells. After the recombinant Escherichia coli was expressed in large quantities and the recombinant plasmid was extracted, the solution containing 10 ~ 4 copies / 渭 L of the plasmid was obtained, which was used as a positive reference substance for quality control. According to the reported concentration, betaine solution and chromogenic solution (the main components are SYBR Green I and HNBX) were prepared and dissolved as lyophilization products, respectively, and chromogenic solution of isothermal amplification products. The isothermal amplification reagent was prepared on the basis of the established loop-mediated isothermal amplification technique for the detection of Mycoplasma bovis. Through the investigation of isothermal amplification reaction, three kinds of lyophilized protectants which had no effect on the isothermal amplification reaction were selected among 8 kinds of commonly used lyophilized vaccine heat-resistant protectants, and three different concentrations were selected for each kind of protectants. A total of 27 kinds of protective agents were designed. By observing the physical properties of freeze-dried products, the optimum group of protective agents were selected to prepare the isothermal amplification reagent for freeze-drying, and the eutectic point was determined in the freeze-drying machine, and the time of the first drying heating was optimized. Drying time and secondary drying time. A freeze-drying curve suitable for isothermal amplification reagent was selected by testing physical properties, vacuum and residual moisture content of freeze-dried products, and the freeze-dried products of isothermal amplification reagents were prepared. A certain amount of isothermal amplification reagent, lyophilized substance, betaine solution, positive reference substance solution and chromogenic solution were used to assemble the kit. The sensitivity of the kit was detected by using 6 concentration gradient (10 ~ 10 ~ 5 copies) recombinant plasmid solution. PG-45 strain HB-1 and strain SD-2 were used to detect the specificity of Mycoplasma bovis and Mycoplasma rhinorrhoeae and Mycoplasma lactoplasticum (108CFU mLLP-1) and Mycobacterium tuberculosis (Mycobacterium tuberculosis) at the concentration of 10 ~ 4 CCU / mL ~ (-1) and 10 ~ (8) CCU / mL ~ (-1), respectively, and the specificity of the test kit was determined by using PG-45 strain and Mycoplasma tuberculosis strain SD-2 (10 ~ (8) CCU / mL ~ (-1). In addition, the isothermal amplification lyophilized kit was stored at different temperatures to detect its stability. [results] only trehalose was the only one of the 8 kinds of lyophilized protectants, mannitol and bovine serum albumin did not affect the isothermal amplification reaction. On this basis, the optimized lyophilization protectant formula is 5% trehalose 1.25% mannitol 1.25% bovine serum albumin, the eutectic point of isothermal amplification reagent is -16 鈩，

本文编号：2031720