Effects of Primers and Taq Polymerase on Randomly Amplified Polymorphic DNA Analysis for Typing Listeria monocytogenes From the Environment of a Shrimp Processing Plant

Abstract

Ninety-nine randomly selected isolates of Listeria monocytogenes from several processing environment locations, in a shrimp processing plant, obtained during a 5-month sampling period were subjected to randomly amplified polymorphic DNA (RAPD) analysis with the use of four primers. Preliminary studies indicated that the number of DNA bands and their intensity differed greatly with respect to the commercial source of the Taq polymerase used with individual isolates. Eighteen composite RAPD types were discerned with the use of the four primers. Among these 18 composite RAPD types, type 1 comprised 14 indistinguishable isolates, and type 9 comprised 49 indistinguishable isolates. These results indicate that the shrimp processing plant was dominated by these 2 RAPD types that comprised 63.6% of the 99 randomly selected isolates.

Key Words:

• L. monocytogenes
• PCR
• RAPD type
• shrimp
• processing environment
• Taq polymerase
• discrimination index
• random primers

ACKNOWLEDGEMENTS

This study was supported by Seafood Safety Special Research Grants 2000–34423–9131 and 2001–34423–10313 from the U.S.D.A. and by Hatch project 836 from the University of Massachusetts, Amherst, Agricultural Experiment Station.

Notes

^aProprietary concentration, and therefore concentration not known.

^bIdentity is proprietary and not known. 5x concentrated stock solution.

^cFinal concentration of MgCl₂ in all PCR reactions was 2.5 mM.