解淀粉芽孢杆菌TF28活菌定量PMA-qPCR技术

Quantification detection of Bacillus amyloliquefaciens TF28by PMA-qPCR

[目的]建立解淀粉芽孢杆菌TF28特异性活菌分子定量技术。[方法]基于TF28特有基因设计qPCR引物,建立菌株特异性qPCR技术;优化PMA处理条件,建立PMA-qPCR定量TF28活菌技术,对其特异性、灵敏性与可靠性进行检测。[结果]建立的qPCR技术对菌株TF28具有特异性。优化的PMA处理条件为:PMA终浓度150μmol/L、暗培养10 min、光照20 min,在此条件下,可封闭10^6cfu/mL以下死菌基因组DNA;建立的PMA-qPCR技术特异性强,灵敏度高,最低检测限10^2cfu/mL;线性关系好,R^2=0. 997;在菌浓度10^3cfu/mL^10^7cfu/mL范围内重复性好,CT值变异系数小于2%,与平板活菌计数比较,差异不显著。[结论]建立的PMA-qPCR技术对TF28具有特异性,能够对菌浓度10^3cfu/mL^10^7cfu/mL活菌进行定量。

[Objective]To establish a strain-specific quantification method for monitoring the viable cell ofBacillus amyloliquefaciensTF28.[Method]Based on a strain-specific gene,the primers for qPCR were designed and the strain-specific qPCR technology was established. PMA treatment condition was optimized and PMA-qPCR was established for quantifying viable cell of TF28. The specificity,sensitivity and reliability of PMA-qPCR were detected.[Result]The established qPCR is specific to strain TF28. The optimized PMA treatment conditions were final concentration of PMA 150 μm,dark culture for 10 minutes and illumination for 20 minutes. 106 cfu/mL dead bacterial genomic DNA was blocked up under the optimal PMA condition. The PMA-qPCR has strong specificity and high sensitivity with a minimum detection limit of 102 cfu mL and a good linear relationship(R^2= 0. 997). In the range of 10^3 cfu/mL-10^7 cfu/mL,the repeatability is good,and the coefficient of variation of CTvalue is less than 2%. Compared with the viable count,the difference is not significant.[Conclusion]PMA-qPCR technology was specific to TF28,and can be used for quantifying viable cell of TF28 with a concentration of 10^3 cfu mL-10^7 cfu/mL.