Molecular targets and methods for formulation screening and preservative efficacy testing

摘要

Methods and kits are disclosed for distinguishing viable from nonviable microbial cells. The methods and kits are useful in the screening of cell culture formulations and the testing of preservative efficacy. The methods involve the amplification and quantitation of microbe-specific DNA from precursor rRNA or Elongation Factor 3 mRNA in treated versus nontreated test samples using the reverse transcription polymerase chain reaction.

主权项

1. A method for identifying and quantifying viable microbial cells in one or more samples containing a microorganism of interest, comprising:
(a) amplification of species-specific DNA from pre-rRNA-containing samples using a reverse transcription-polymerase chain reaction, said amplification step comprising:
using a first primer complementary to a pre-rRNA region of said microorganism of interest;using a second primer complementary to a mature rRNA region of said microorganism of interest; andperforming multiple cycles of amplification using said first primer and said second primer to yield detectable levels of amplified species-specific DNA; and (b) quantitation of said amplified species-specific DNA, said quantitation step comprising:
using a fluorescently labeled hybridizing probe complementary to said mature rRNA region of said microorganism of interest;using said first primer complementary to said pre-rRNA region of said microorganism of interest;using said second primer complementary to said mature rRNA region of said microorganism of interest; andperforming multiple cycles of amplification using said fluorescently labeled hybridizing probe and said first primer and said second primer to yield increasing levels of fluorescence signal above fluorescence background; wherein said amplification of said species-specific DNA indicates the presence of said viable microbial cells in said one or more samples, wherein at least one of said first primer, said second primer or said probe comprise nucleic acids complementary to the mRNA of the Elongation Factor 3 gene, and wherein said quantitation of said amplified species-specific DNA provides a relative measurement of the amount of said viable microbial cells in said one or more samples.