title: Highly Specific Culture-Independent Detection of YGNGV Motif-Containing Pediocin-Producing Strains.
abstract: A novel method based on (1) initial microbiological screening and (2) a highly specific PCR is described for selection of strains expressing YGNGV motif-containing pediocin. Initial screening is carried out using spot on the lawn assay for selection of acid-free, hydrogen peroxide (H2O2)-free and secreted heat-stable inhibitory activity producing strains. This is followed by highly specific PCR for amplification of 406-bp fragment using forward primer: 5'-tggccaatatcattggtggt-3' targeting signal peptide sequence of pediocin structural gene and reverse primer: 5'-ctactaacgcttggctggca-3' encoding N-terminus of immunity gene. The assay was validated with Pediococcus pentosaceus NCDC273 and Pediococcus acidilactici NCDC252 using (1) digestion of amplified 406-bp fragment with HindIII restriction enzyme-producing two restriction fragments of expected sizes (227 and 179 bp), (2) nucleotide sequencing of 406-bp fragment from both strains found these pediocins identical to pediocin PA-1/AcH and (3) identification of both pediocins as pediocin PA-1 at protein level using RP-HPLC. The assay was used for screening six strains (3 pediococci, 2 lactobacilli and an Enterococcus faecium) producing acid-free, hydrogen peroxide (H2O2)-free and secreted heat-stable inhibitory activity. This resulted in the detection of three new strains (P. pentosaceus NCDC35, E. faecium NCDC124 and Lactobacillus plantarum NCDC20) producing YGNGV motif-containing pediocins.
authors.affiliation: Division of Animal Biochemistry, National Dairy Research Institute, Karnal, 132001, Haryana, India.
authors.forename: S K
authors.initials: SK
authors.lastname: Sood
authors.affiliation: Division of Animal Biochemistry, National Dairy Research Institute, Karnal, 132001, Haryana, India.
authors.forename: B
authors.initials: B
authors.lastname: Vijay Simha
authors.affiliation: Division of Animal Biochemistry, National Dairy Research Institute, Karnal, 132001, Haryana, India. rash4787@gmail.com.
authors.forename: Rashmi
authors.initials: R
authors.lastname: Kumariya
authors.affiliation: Division of Animal Biochemistry, National Dairy Research Institute, Karnal, 132001, Haryana, India.
authors.forename: Anita Kumari
authors.initials: AK
authors.lastname: Garsa
authors.affiliation: Division of Animal Biochemistry, National Dairy Research Institute, Karnal, 132001, Haryana, India.
authors.forename: Jitender
authors.initials: J
authors.lastname: Mehla
authors.affiliation: Division of Animal Biochemistry, National Dairy Research Institute, Karnal, 132001, Haryana, India.
authors.forename: Sunita
authors.initials: S
authors.lastname: Meena
authors.affiliation: Division of Animal Biochemistry, National Dairy Research Institute, Karnal, 132001, Haryana, India.
authors.forename: Puja
authors.initials: P
authors.lastname: Lather
keywords: Enterococcus; Lactic acid bacteria; Lactobacillus; PCR-based detection; Pediocin; Pediococcus; YGNGV motif