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【目的】比较分析基于反转录实时荧光定量PCR(Real-time Reverse-Transcriptase PCR,Real-time RTPCR)技术建立的3种标准曲线定量样品中活副溶血性弧菌数量的差异性。【方法】体外转录合成带有目的片段(tlh基因)的RNA标准品,提取纯培养副溶血性弧菌(108CFU/mL)RNA样品,提取接种于虾上副溶血性弧菌(106CFU/g)RNA样品,将3种RNA样品分别反转录得到cDNA,10倍梯度稀释,进行Real-time PCR扩增,建立标准曲线A、B、C(其中标准曲线A和C为本研究首次建立)。用标准曲线A、B、C分别定量6个样品(2个纯培养副溶血性弧菌样品、2个人工污染煮熟南美白对虾样品和2个人工污染生南美白对虾样品)中活副溶血性弧菌数量,并与传统涂布计数法进行比较,分析定量结果的差异性。【结果】3种标准曲线均具有较好的线性关系(R2>0.99);3种标准曲线定量6个样品中活副溶血性弧菌结果均显著(p<0.05)低于涂布定量结果,与涂布定量结果的相对误差大小顺序为:标准曲线A(30.0%)>标准曲线C(18.8%)>标准曲线B(6.9%);标准曲线A对6个样品定量结果与标准曲线B、C定量结果的差值平均值分别为-2.25Lg CFU/mL和-0.75 Lg CFU/mL,相对误差平均值为48.2%和15.9%,标准曲线B与C对6个样品定量结果的差值在(1.47-1.53)Lg CFU/mL之间,相对误差在19.0%-23.8%之间。【结论】标准曲线B可以广泛应用于Real-time RT-PCR准确定量样品中微生物数量。
【Objective】 The purpose of this study is to compare the quantitative analysis of Vibrio parahaemolyticus among three standard curves based on Real-time Reverse-Transcriptase PCR (Real-time RT-PCR). 【Method】 RNA samples with the target fragment (tlh gene) were synthesized and transcribed in vitro. RNA samples of purified V. parahaemolyticus (108 CFU / mL) were extracted and extracted for V. parahaemolyticus (106 CFU / g) The RNA samples were reverse transcribed from each of the three RNA samples to obtain a 10-fold gradient of the cDNA. Real-time PCR amplification was performed to establish standard curves A, B and C (standard curves A and C were first established in this study). Six samples (2 samples of pure Vibrio parahaemolyticus, 2 samples of Artemisia capsici and 2 samples of Artemisia propylaus) were quantified using standard curves A, B and C respectively. The number of Vibrio spp., Compared with the traditional coating counting method, the quantitative analysis of the differences. 【Result】 The results showed that the three standard curves all had good linear relationship (R2> 0.99). The three standard curves showed that the Vibrio parahaemolyticus was significantly lower (p <0.05) The relative error of the quantitative results with the order of coating: the standard curve A (30.0%)> C (18.8%)> B (6.9%); standard curve A quantitative analysis of 6 samples and the standard curve B, The average value of the difference of C quantitative results were -2.25Lg CFU / mL and -0.75 Lg CFU / mL respectively, the relative error was 48.2% and 15.9% (1.47-1.53) Lg CFU / mL, the relative error was between 19.0% -23.8%. 【Conclusion】 The standard curve B can be widely used in the accurate quantification of the number of microorganisms in the samples by Real-time RT-PCR.