本文選題：牛支原體 + 等溫?cái)U(kuò)增��； 參考：《中國農(nóng)業(yè)科學(xué)》2016年16期

【摘要】：【目的】牛支原體(Mycoplasma bovis)是導(dǎo)致牛多種疾病綜合征的病原體之一,在世界范圍廣泛流行。為了有效監(jiān)測此病在中國的流行情況,迫切需要敏感、便捷的診斷試劑產(chǎn)品�！痉椒ā客ㄟ^構(gòu)建含有牛支原體uvrC基因片段的重組質(zhì)粒并轉(zhuǎn)化TOP10感受態(tài)細(xì)胞,獲得重組大腸桿菌rP-uvrC。重組大腸桿菌大量表達(dá)并提取重組質(zhì)粒后,獲得質(zhì)粒濃度為10~4拷貝/μL的溶液,作為質(zhì)控用陽性對照品。根據(jù)文獻(xiàn)報(bào)道的濃度配制甜菜堿溶液和顯色液(主成份為SYBR Green I和HNB),分別作為凍干品溶解用溶液和等溫?cái)U(kuò)增產(chǎn)物顯色溶液。在已建立的牛支原體環(huán)介導(dǎo)等溫?cái)U(kuò)增(loop-mediated isothermal amplification,LAMP)檢測技術(shù)的基礎(chǔ)上配制等溫?cái)U(kuò)增試劑,并通過考察等溫?cái)U(kuò)增反應(yīng)情況在常用的8種凍干疫苗耐熱保護(hù)劑中選擇對等溫?cái)U(kuò)增反應(yīng)沒有影響的3種凍干保護(hù)劑,每種保護(hù)劑分別選用3種不同的濃度,共設(shè)計(jì)27種保護(hù)劑組方,通過觀察凍干品物理性狀選擇最佳的一組保護(hù)劑配制凍干用等溫?cái)U(kuò)增試劑,放到凍干機(jī)中測定共晶點(diǎn),并優(yōu)化一次干燥升溫時(shí)間、干燥時(shí)間和二次干燥時(shí)間。通過對凍干制品物理性狀的檢驗(yàn)、真空度的檢測以及殘余水分含量的測定,篩選出一條適合等溫?cái)U(kuò)增試劑的凍干曲線,并以此制備等溫?cái)U(kuò)增試劑凍干品。取一定量等溫?cái)U(kuò)增試劑凍干品、甜菜堿溶液、陽性對照品溶液和顯色液,組裝成試劑盒。利用6個(gè)濃度梯度(10~0-10~5個(gè)拷貝)重組質(zhì)粒溶液檢測試劑盒的敏感性,利用濃度為10~4 CCU·mL~(-1)的PG-45株、HB-1株、SD-2株牛支原體菌液、10~8CCU·mL~(-1)的牛鼻支原體和無乳支原體菌液、10~8CFU·mL~(-1)的多殺性巴氏桿菌和結(jié)核分枝桿菌,檢測試劑盒的特異性。另外,將等溫?cái)U(kuò)增凍干試劑盒分別置于不同溫度下保存,檢測其穩(wěn)定性。【結(jié)果】8種凍干保護(hù)劑中只有海藻糖、甘露醇和牛血清白蛋白不影響等溫?cái)U(kuò)增反應(yīng),在此基礎(chǔ)上優(yōu)選的凍干保護(hù)劑配方為5%海藻糖+1.25%甘露醇+1.25%牛血清白蛋白,等溫?cái)U(kuò)增試劑共晶點(diǎn)為-16℃,一次干燥的升溫時(shí)間3h,一次干燥時(shí)間6h,二次干燥時(shí)間4 h。組裝后的試劑盒最低可檢測到10個(gè)拷貝數(shù)的重組質(zhì)粒,檢驗(yàn)PG45株、HB-1株以及SD-2株牛支原體均為陽性,檢驗(yàn)牛鼻支原體、無乳支原體、多殺性巴氏桿菌和結(jié)核分枝桿菌均為陰性。試劑盒在20℃保存6個(gè)月、37℃保存10 d后,敏感性仍為10個(gè)拷貝數(shù),與第0天相同,推測4℃保存有效期為24個(gè)月左右。【結(jié)論】筆者研制的等溫?cái)U(kuò)增凍干試劑盒敏感性高、特異性好、穩(wěn)定性好、操作便捷,適合基層獸醫(yī)現(xiàn)場檢測使用。
[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，

本文編號：2031720