An immunomagnetic separation-PCR method for detection of pathogenic Leptospira in biological fluids.
Abstract Leptospirosis is a zoonotic disease that occurs worldwide and is caused by pathogenic bacteria of the genus Leptospira. Clinical manifestations of leptospirosis are similar to other febrile illnesses and this fact frequently retards the beginning of antibiotic therapy. Thus, early and accurate diagnosis is a prerequisite for proper treatment of leptospirosis. Antigen and DNA-based detection tests offer potential advantage over tests based on antibody detection for early diagnosis of leptospirosis since antibodies only reach detectable levels several days after the onset of the infection. This work describes a method for detection of pathogenic Leptospira that associates an immunoseparation step with a PCR assay and uses an internal amplification control (IAC) to ensure accuracy of the test. The immunoseparation was performed with protein A-magnetic beads in house coated with an MAb specific for LipL32, the major outer membrane protein of pathogenic Leptospira; PCR was performed using lipL32 specific primers. The IMS-PCR method enhanced detection of Leptospira in experimentally contaminated human sera and urine when compared to PCR performed alone. IMS-PCR was able to detect 10(2) Leptospira cells per mL of human sera and urine, corresponding to 25 genomic copies per PCR reaction.
Fernandes
Cláudia Pinho Hartleben
CP
Centro de Biotecnologia, Universidade Federal de Pelotas, Pelotas, Brazil.
Seixas
Fabiana Kömmling
FK
Coutinho
Mariana Loner
ML
Vasconcellos
Flávia Aleixo
FA
Moreira
Angela Nunes
AN
Conceição
Fabricio Rochedo
FR
Dellagostin
Odir Antônio
OA
Aleixo
José Antonio Guimarães
JA
D000911:Antibodies, Monoclonal / Q000276:immunology; D000942:Antigens, Bacterial / Q000276:immunology; D001826:Body Fluids / Q000382:microbiology; D004269:DNA, Bacterial / Q000097:blood* / Q000652:urine; D006801:Humans; D018189:Immunomagnetic Separation / Q000379:methods; D007921:Leptospira interrogans / Q000235:genetics / Q000302:isolation & purification*
D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D004269:DNA, Bacterial