title: [Difference of three standard curves of real-time reverse-transcriptase PCR in viable Vibrio parahaemolyticus quantification].
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 (10(8) CFU/mL) and shrimp samples (10(6) 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%.
  
  
  CONCLUSIONS
  Standard curve B could be applied to Real-time RT-PCR when quantify the number of viable microorganisms in samples.
authors.forename: Mengtong
authors.initials: M
authors.lastname: Jin
authors.forename: Wenshuo
authors.initials: W
authors.lastname: Sun
authors.forename: Qin
authors.initials: Q
authors.lastname: Li
authors.forename: Xiaohong
authors.initials: X
authors.lastname: Sun
authors.forename: Yingjie
authors.initials: Y
authors.lastname: Pan
authors.forename: Yong
authors.initials: Y
authors.lastname: Zhao
mesh_terms: D000818:Animals; D004269:DNA, Bacterial / Q000032:analysis; D005506:Food Contamination / Q000032:analysis; D033561:Penaeidae / Q000382:microbiology*; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods* / Q000592:standards*; D012758:Shellfish / Q000032:analysis / Q000382:microbiology*; D014736:Vibrio parahaemolyticus / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D004269:DNA, Bacterial; D012194:RNA-Directed DNA Polymerase