Real-time PCR detection of Enterococcus faecalis  associated with amyloid arthropathy

TY - JOUR

T1 - Real-time PCR detection of Enterococcus faecalis  associated with amyloid arthropathy

AU - Petersen, Andreas Henrik H

AU - Bisgaard, Magne

AU - Christensen, Henrik

PY - 2010/7

Y1 - 2010/7

N2 - Aims: To develop a RT-PCR method for detection of the multilocus sequence type 82 of Enterococcus faecalis associated with amyloid arthropathy (AA) in layers. Methods and 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. Significance and Impact of the Study: 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.

AB - Aims: To develop a RT-PCR method for detection of the multilocus sequence type 82 of Enterococcus faecalis associated with amyloid arthropathy (AA) in layers. Methods and 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. Significance and Impact of the Study: 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.

KW - Former LIFE faculty

KW - clone

KW - layer chicken

KW - MLST

KW - ST82

U2 - 10.1111/j.1472-765x.2010.02861.x

DO - 10.1111/j.1472-765x.2010.02861.x

M3 - Journal article

SN - 0370-1778

VL - 51

SP - 61

EP - 64

JO - Proceedings of the Society for Applied Bacteriology

JF - Proceedings of the Society for Applied Bacteriology

IS - 1

ER -