Real-time PCR detection of Enterococcus faecalis associated with amyloid arthropathy.
OBJECTIVE
To develop a RT-PCR method for detection of the multilocus sequence type 82 of Enterococcus faecalis associated with amyloid arthropathy (AA) in layers.


RESULTS
Bacteria were selected from lesions including AA in layers. The primers were designed based on the phosphate ATP binding cassette transporter (pstS) and xanthine phosphoribosyltransferase (xpt) genes and first tested against three isolates with known base pairs at the specific sites. Subsequently, 12 isolates were selected from our collection by one researcher, and RT-PCR was performed blinded. The sequence type (ST) was then confirmed by multilocus sequence analysis. Two single-nucleotide polymorphisms in the pstS and xpt genes allowed an unambiguous identification of ST82. As an alternative to DNA extraction, a boiling method for release of DNA from cells was used.


CONCLUSIONS
The real-time PCR targeting ST82 enables rapid screening of Ent. faecalis cultured from suspect cases with results available after a few hours, much faster than multilocus sequence typing and pulse field gel electrophoresis.


CONCLUSIONS
The new method allows a rapid screening of isolates with results available after only few hours. This RT-PCR method could be a useful tool for molecular epidemiological studies on the spread of arthropathic and amyloidogenic Ent. faecalis within and between birds more efficiently.
Petersen
A
A
Department of Veterinary Disease Biology, Faculty of Life Sciences, University of Copenhagen, Copenhagen, Denmark.
Bisgaard
M
M
Christensen
H
H
D000686:Amyloidosis / Q000150:complications* / Q000382:microbiology; D001170:Arthritis, Infectious / Q000150:complications / Q000175:diagnosis* / Q000382:microbiology; D001426:Bacterial Proteins / Q000235:genetics; D001431:Bacteriological Techniques / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D013293:Enterococcus faecalis / Q000235:genetics / Q000302:isolation & purification*; D016908:Gram-Positive Bacterial Infections / Q000150:complications / Q000175:diagnosis* / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide
D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial