摘要
本研究采用改良Thiaucourt’s培养基培养绵羊肺炎支原体(Mycoplasma ovipleuneniae,MO)临床分离株SC01,收集不同时间的培养物,分别用颜色变化单位(CCU)法、浊度法及实时荧光定量PCR(Real-time PCR)法进行定量,并绘制MO生长动态曲线。CCU检测结果显示,接种后0～4h为SC01的迟缓期,4～16h为对数生长期,lg CCU/mL最高达到10.7000±0.4700,16～20h为稳定期,20h以后进入衰亡期;浊度法定量检测结果显示,SC01培养物经过5倍浓缩后的D420nm随着培养时间延长逐渐上升,2h时为0.0028±0.0002,4～12hD420nm增加较缓慢,而12～24h增加最快,24h最高达到0.8609±0.0045,以后则趋于稳定;Real-time PCR法检测结果显示,SC01的拷贝数随着培养时间延长平稳上升,2h时lg拷贝/mL为4.5889±0.0048,在30h时lg拷贝/mL达到7.6165±0.1089。相关性分析结果表明,在迟缓期和对数生长期,CCU法与Real-time PCR法、CCU法与浊度法、Real-time PCR法与浊度法对MO的定量检测结果高度相关,相关系数分别为0.967、0.720和0.849,而Real-time PCR法和浊度法对MO的定量检测结果则在2～30h内均高度相关,相关系数为0.912。经对检测所需时间及精确度等方面综合比较,本研究认为Real-time PCR法具有快速、准确及易标准化等特点,可以取代CCU法和浊度法用于MO的定量。
The Mycoplasma ovipneumoniae（MO） SC01 isolates were cultured in the modified Thiaucourt’s medium and collected in different times.The growth curve of MO was determined by the color change unit（CCU）,nephelornetry and Real-time PCR.The results of CCU showed 0 to 4 h was the lag phase,4 to 16 h was logarithmic phase and the most of lg CCU/mL was 10.7000±0.4700,16 to 20 h was the stationary phase,and 20 h later was decline phase of SC01 isolates.The results of nephelornetry method showed that D420 nm of five folds concentration of SC01 cultures gradually increased with the culture time,following the 0.0028±0.0002 in 2 h,slowly increased in 4 to 12 h,fast increased in 12 to 24 h,the 0.8609±0.0045 of highest level in 24 h and then stabilizaed.The results of Real-time PCR showed that the copy of SC01 isolates steadily increased in 2 to 30 h that the lg copy/mL was 4.5889±0.0048 in 2 h and the most of lg copy/mL was 7.6165±0.1089 in 30 h.Furthermore,the correlation analysis exhibited that,in lag phase and logarithmic phase,the CCU had a high correlation with the Real-time PCR and nephelornetry,and Real-time PCR also existed a high correlation with nephelornetry,following that the correlation coefficients were 0.967,0.720 and 0.849,respectively.During 2 to 30 h,there was a high correlation between the Real-time PCR method and nephelornetry and the correlation coefficients was 0.912.Based on the above results of the time and accuracy of three detection methods,the study indicated that the Real-time PCR method was fast,accurate and easy to standardization and could replace for the CCU and nephelornetry methods.
出处
《中国畜牧兽医》
CAS
北大核心
2013年第3期177-180,共4页
China Animal Husbandry & Veterinary Medicine
基金
国家"863计划"项目(2012AA101304)
四川省科技创新产业链示范工程项目--山羊现代产业链关键技术集成研究与产业化示范(2011NZ0003)
