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

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.