Application of quantitative reverse-transcription PCR (qRT-PCR) for the determination of the total viable count (TVC) on meat samples

作者：Dolan A^*; Burgess C M; Fanning S; Duffy G

来源：Journal of Applied Microbiology, 2010, 109(1): 91-98.

DOI：10.1111/j.1365-2672.2009.04636.x

• food
• PCR
• quantitative
• total viable count

Aim:
To apply a quantitative reverse-transcription PCR (qRT-PCR) method to determine the total viable count (TVC) on meat samples.
Methods and Results:
Using two sets of primers to target the ribonuclease-P (RNase P) RNA transcripts of Gram-positive and Gram-negative bacteria, standard curves were generated using the LightCycler 2.0 instrument (Roche Diagnostics). RNA standards were extracted from known cell numbers and subsequently converted to cDNA for the construction of standard curves for quantification of the TVC of beef carcass swabs (n = 60) and beef (n = 30), chicken (n = 50) and pork (n = 49) pieces. A high correlation between the standard plate count method and the qRT-PCR was observed for beef swabs (R2 = 0 center dot 93) and beef pieces (R2 = 0 center dot 82). The correlation coefficient for chicken pieces and pork pieces were R2 = 0 center dot 34 and 0 center dot 55, respectively. Using beef pieces (n = 13), an interlaboratory study was conducted and each participating laboratory (n = 3) found a reasonable degree of agreement between the cultural method and the PCR method.
Conclusions:
The qRT-PCR assay used in this study can enumerate the total bacteria on beef samples with a high degree of accuracy.
Significance and Impact of the Study:
The qRT-PCR method may have the potential to be applied to various sample types as an alternative rapid method for determining TVCs; however, further validation would be required.

• 出版日期2010-7