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Page No. : 99-103

ABSTRACT

A PCR based method was standardized for rapid detection of spollage of buffalo liver using a set of forward and reverse primers coding for conserved region of 23S rRNA gene present in Pseudomonas fragi. A template was prepared from buffalo liver stored at 4±1°C for 4 days and PCR assay was repeated on day 0, 2 and 4 of storage with initial denaturation at 95°C, 30 cycles of denaturation at 92°C, annealing at 56°C and extension at 74°C. Results showed that, when PCR was performed on day 0 on serially diluted (10 fold) fresh liver homogenate, amplification of 207 bp sequence of DNA of bacterial flora was observed in the dilutions of upto 10-4 levels. Whereas on day 4, PCR amplification was observed up to level of 10-6 bacteria in the liver indicating Increase in number of bacteria during storage. Conventional standard plate count, done simultaneously, also revealed similar counts. However, decrease in intensity of band or narrowing of band was observed with increasing dilution and decreasing number of bacteria per ml dilution. These results suggested that standardized PCR procedure could be effectively used for detecting meat spoilage bacteria and to evaluate the shelf life of liver.

Key Words: Denaturation, polymerase chain reaction, Pseudomonas fragi