Recommendations for the detection of Leptospira in urine by PCR.
In the present study PCR was applied to detect leptospires in human urine. Several approaches for sample processing were evaluated to optimize the detection of leptospires in urine mixed with this bacterium. Furthermore, some changes in the composition of the reaction mix were studied. No amplification was observed in acidic urine, therefore neutralization of the sample immediately after collection is strongly recommended. PBS gave better results than Tris or NaOH as neutralizing reagents. Freezing and thawing of samples before processing yielded negative results. Elimination of epithelial cells, leukocytes and crystals by centrifugation at 3,000 rpm at room temperature increased sensitivity. In addition, both the washing step after collecting leptospires by centrifugation and the inclusion of 0.1% bovine serum albumin in the reaction mix minimized the interference of other inhibitory compounds. These modifications were useful to improve the detection of Leptospira in urine by PCR.
Lucchesi
Paula M A
PM
Laboratorio de Inmunoquímica y Biotecnología, Facultad de Ciencias Veterinarias, Universidad Nacional del Centro de la Provincia de Buenos Aires, Tandil, Argentina. paulaluc@vet.unicen.edu.ar
Arroyo
Guillermo H
GH
Etcheverría
Analía I
AI
Parma
Alberto E
AE
Seijo
Alfredo C
AC
D000818:Animals; D002417:Cattle; D006801:Humans; D007202:Indicators and Reagents; D007919:Leptospira / Q000302:isolation & purification*; D007922:Leptospirosis / Q000652:urine*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013048:Specimen Handling / Q000379:methods* / Q000592:standards
D007202:Indicators and Reagents