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Rapid and sensitive detection of Escherichia coli in milk by 16S rRNA gene targeted PCR

Indian Agricultural Research Journals

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Title Rapid and sensitive detection of Escherichia coli in milk by 16S rRNA gene targeted PCR
 
Creator GAUTAM, SANJEEV K
KUMAR, S SURESH
BATISH, V K
GROVER, SUNITA
MOHANTY, A K
 
Subject E. coli, Milk; PCR; 16S rRNA
 
Description The present study was conducted to optimize PCR amplification targeted against Escherichia coli DNA for detection of 14 different pathogenic and non pathogenic strains of this organism in milk. 16S rRNA gene is present as multiple copy gene, thus targeting this gene enhances the sensitivity of the assay by 1000–5000 times. The template DNA from broth cultures and milk (spiked as well as natural) was extracted using a modified method of Pospiech and Neumann followed by agarose gel electrophoresis of genomic DNA. The PCR parameters were optimized using different combinations of 16S rRNA gene based primers, namely, 16E1, 16E2 and 16E3. The PCR conditions optimized included initial denaturation at 95°C/5 min followed by 35 cycles each of denaturation, at 94° C/30 sec; annealing at 60°C/30 sec and extension at 72°C/30 sec followed by final extension at 72°C/2 min. Using all the primers mentioned above, the PCR amplified a 584 bp product that was specific for E. coli. The lowest level of DNA that could be detected by PCR assay was 1 ng and 10 cells of E. coli in broth. E. coli could be recovered from almost all the raw milk samples and their number ranged from 0.17×104 to 8.1×104 cfu/ml and 1.7 ×104 to 87 × 104 cfu/ml before and after enrichment, respectively.
 
Publisher Directorate of Knowledge Management in Agriculture
 
Contributor —
 
Date 2012-02-06
 
Type info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
—
 
Format application/pdf
 
Identifier http://epubs.icar.org.in/ejournal/index.php/IJAnS/article/view/15269
 
Source The Indian Journal of Animal Sciences; Vol 82, No 2 (2012)
0367-8318
 
Language eng
 
Relation http://epubs.icar.org.in/ejournal/index.php/IJAnS/article/view/15269/7655
 
Rights Copyright (c) 2014 The Indian Journal of Animal Sciences