Development of specific and rapid detection of bacterial pathogens in dairy products by PCR

Abstract

A simple and specific method for direct detection of bovine mastitis pathogens (Streptococcus agalactiae (GBS), Staphylococcus aureus and Escherichia coli) in milk products, bacterial samples from milk and isolated bacterial DNA was developed. The method is based on polymerase chain reaction (PCR) using sequence-specific primers only for GBS and species-specific primers derived from 16S and 23S rRNA for all chosen species. The presence of the gene of surface immunogenic protein (Sip) in bovine GBS isolates, described previously only in human GBS isolates was confirmed. The GBS detection was performed with the sequence coding for surface immunogenic protein from GBS human isolates designated as Sip specific sequence (SSS); this sequence was selected for specific primer design. The sequence is unique for GBS and was designed from a consensus of all known sip genes. The specific identification was shown on a collection of 75 GBS bovine isolates from different localities in Slovakia. All isolates were positive to SSS, 16S and 23S rRNA sequence. The 16S and 23S rRNA PCR detection was also performed with S. aureus and E. coli isolates and specific PCR products were also detected. The detection limit of this assay for milk products was 6 CFU/microL (i.e. 6000 CFU/mL) for GBS and E. coli, and 16 CFU/microL for S. aureus. This rapid, sensitive and specific diagnostic method can be performed within hours and represents an innovative diagnostic tool for the detection of milk pathogens in dairy products.

Multiplex PCR for detection of Escherichia coli O157:H7 in foods

“…Two cells were enough for the amplification of PCR products with primer pair EAE, and even one single cell with primer pair HLYA or H7. The observation of detection limit for species-specific Ecoli primer pair was the same as stated previously by Chotár et al (2006).…”

“…All oligonucleotide primers were synthesized by Microsynth (Switzerland). Briefly, PCR assay was carried out directly from E. coli O157:H7 cells prepared according to modified procedure described previously by Chotár et al (2006). Modification concerned the treatment of the overnight culture before washing steps.…”

“…No PCR products were generated with these primers by using negative control strains E. coli BL21 and DH5α. Amplification of 663 bp PCR product was observed when species-specific primer pair (Ecoli1 and Ecoli2; Chotár et al 2006) was used with E. coli O157:H7 as well as E. coli BL21 or DH5α (Fig. 1).…”

Characterization of the microbial community in a lotic environment to assess the effect of pollution on nitrifying and potentially pathogenic bacteria

“…The DNA used was the same as that used for the nitrifying bacteria analyses, and PCR amplifications were carried out as previously described by (Amann et al, 1990;Trebesius et al, 2000;Watanabe et al, 2001;Fukushima et al, 2003;Qin et al, 2003;Aranda et al, 2004;Rinttilä et al, 2004;Chotár et al, 2006). The negative control was the PCR reaction containing all the components needed to perform this analysis, except DNA.…”

Incidence of Staphylococci and Streptococci During Winter in Mastitic Milk of Sahiwal Cow and Murrah Buffaloes

“…Molecular identification of pathogens is being adopted by clinical sector, as the techniques are quite quick, easy and reproducible. Several PCR based methods have been reported for detection of mastitis pathogens [12,13]. Among various PCR based assay, multiplex is quiet useful for in vitro detection of more than one pathogen within a single tube reaction.…”