Development of solution phase hybridisation PCR-ELISA for the detection and quantification of Enterococcus faecalis and Pediococcus pentosaceus in Nurmi-type cultures.
Nurmi-type cultures (NTCs), derived from the fermentation of caecal contents of specifically pathogen-free (SPF) birds, have been used successfully to control salmonella colonisation in chicks. These cultures are undefined in nature and, consequently, it is difficult to obtain approval from regulatory agencies for their use as direct fed microbials (DFMs) for poultry. Progress towards the generation of effective defined probiotics requires further knowledge of the composition of these cultures. As such, species-specific, culture-independent quantification methodologies need to be developed to elucidate the concentration of specific bacterial constituents of NTCs. Quantification of specific bacterial species in such ill-defined complex cultures using conventional culturing methods is inaccurate due to low levels of sensitivity and reproducibility, in addition to slow turnaround times. Furthermore, these methods lack selectivity due to the nature of the accompanying microflora. This study describes the development of a rapid, sensitive, reliable, reproducible, and species-specific culture-independent, solution phase hybridisation PCR-ELISA procedure for the detection and quantification of Enterococcus faecalis and Pediococcus pentosaceus in NTCs. In this technique, biotin-labelled primers were designed to amplify a species-specific fragment of a marker gene of known copy number, in both species. Resulting amplicons were hybridised with a dinitrophenol (DNP)-labelled oligonucleotide probe in solution and were subsequently captured on a streptavidin-coated microtitre plate. The degree of binding was determined by the addition of IgG (anti-DNP)-horseradish peroxidase conjugate, which was subsequently visualised using a chromogenic substrate, tetramethylbenzidine. This novel quantitative method was capable of detecting E. faecalis and P. pentosaceus at levels as low as 5 CFU per PCR reaction.
Waters
Sinéad M
SM
Alltech Ireland, Sarney, Summerhill Road, Dunboyne, Co. Meath, Ireland. swaters@alltech.com
Doyle
Sean
S
Murphy
Richard A
RA
Power
Ronan F G
RF
D000818:Animals; D001431:Bacteriological Techniques; D001483:Base Sequence; D002432:Cecum / Q000382:microbiology; D002645:Chickens; D004269:DNA, Bacterial / Q000235:genetics; D013293:Enterococcus faecalis / Q000235:genetics* / Q000302:isolation & purification; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000706:statistics & numerical data; D010376:Pediococcus / Q000235:genetics* / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D019936:Probiotics; D015203:Reproducibility of Results; D013045:Species Specificity; D013047:Specific Pathogen-Free Organisms
D004269:DNA, Bacterial