基于反转录实时荧光定量PCR的3种标准曲线定量活副溶血性弧菌的差值分析

Difference of three standard curves of real-time reversetranscriptase PCR in viable Vibrio parahaemolyticus quantification

摘要:

摘要:【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(108 CFU/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－2.25 Lg 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。

Abstract:

Abstract:［Objective］We evaluated the difference of three standard curves in quantifying viable Vibrio parahaemolyticus in samples by real-time reverse-transcriptase PCR (Real-time RT-PCR).［Methods］ The standard curve A was established by 10-fold diluted cDNA. The cDNA was reverse transcripted after RNA synthesized in vitro．The standard curve B and C were established by 10-fold diluted cDNA．The cDNA was synthesized after RNA isolated from Vibrio parahaemolyticus in pure cultures (108 CFU/mL) and shrimp samples (106 CFU/g)(Standard curve A and C were proposed for the first time). Three standard curves were performed to quantitatively detect V．parahaemolyticus in six samples，respectively (Two pure cultured V．parahaemolyticus samples，two artificially contaminated cooked Litopenaeus vannamei samples and two artificially contaminated Litopenaeus vannamei samples)．Then we evaluated the quantitative results of standard curve and the plate counting results and then analysed the differences.［Results］The three standard curves all show a strong linear relationship between the fractional cycle number and V．parahaemolyticus concentration (R2＞0.99);The quantitative results of Real-time PCR were significantly (p＜0.05) lower than the results of plate counting．The relative errors compared with the results of plate counting ranked standard curve A (30.0%)＞standard curve C (18.8%)＞ standard curve B (6.9%); The average differences between standard curve A and standard curve B and C were－2.25 Lg CFU/mL and－0.75 Lg CFU/mL，respectively，and the mean relative errors were 48.2% and 15.9%，respectively; The average difference between standard curve B and C was among ( 1.47－1.53) Lg CFU/mL and the average relative errors were among 19.0%－23.8%.［Conclusion］Standard curve B could be applied to Realtime RT-PCR when quantify the number of viable microorganisms in samples.