实时荧光定量PCR法定量微生物的条件及在鱼露和虾油微生物检测中的应用研究

摘要

文中研究了以实时荧光定量PCR法(FQ-PCR)定量细菌和真菌数量的条件,通过比较多对引物的扩增效果,确定分别采用16S rDNA细菌通用引物63F-335R和18S rDNA真菌通用引物0817F-1196R扩增细菌和真菌,并确定退火温度均为60℃.采用多种细菌和真菌对上述条件进行验证,结果显示,无论单一菌种或混合菌种,在上述条件下扩增效率E均在90％～120％范围内,所得标准曲线决定系数R2 ＞0.98,符合定量要求.用此方法定量鱼露和虾油的微生物,结果显示鱼露发酵过程中细菌数量在3.16 ×104 ～3.16 ×105个/mL范围内变动,数量远高于真菌数,而且该法测得的细菌和真菌数比平板计数法高1～2个数量级.%In this daper,the conditions of determining baterial and fangal populations by real-time fluorescert quantitative PCR (FQ-PCR) method were investigated.By comparing the amplification effects of several sets of primers,the bacterial 16S rDNA universal primer 63F-335R and the fungal 18S rDNA universal primer 0817F-1196R were respectively selected as the primers for FQ-PCR detection of bacterial and fungal populations.And the annealing temperatures were set to be 60 ℃.The selected FQ-PCR primers and the annealing temperatures were validated using many strains of bacteria and fungi.The results showed that for any single or mixed strains,the amplification efficiencies were within 90％-120％ and the determination coefficients of the standard curves were greater than 0.98,which satisfied the requirements of quantification.The bacterial and fungal populations in fish sauce and shrimp sauce weredetected by FQ-PCR under above conditions.The results revealed that during fermentation of fish sauce,the counts ofbacterial population varied within the range of 3.16 × 104-3.16 × 105 cells/ml,which were far higher than those ofthe fungal population.Besides,the counts of bacterial and fungal populations in fish sauce and shrimp sauce deter-mined by the FQ-PCR method were 1-2 orders of magnitude higher than those determined by plate count method.