title: Real-time PCR method for the detection of the gene encoding surface lipoprotein LipL32 of pathogenic Leptospira: use in the laboratory diagnosis of the acute form of leptospirosis.
abstract: BACKGROUND
  The aims of this work were to replace the obsolete PCR method for the laboratory diagnosis of the acute form of leptospirosis using the G1, G2 and B64 I, B64 II primers, and to improve the PCR detection time.
  
  
  METHODS
  We introduced a real-time PCR method for the detection of the gene encoding the surface lipoprotein LipL32 of pathogenic Leptospira into our laboratory diagnosis of the acute form of leptospirosis. The positive and negative analytical specificities of the real-time PCR method were both equal to 100%; the detection limit was determined to be 1-5 genome copies/1 ml of liquid biological material. The method was further validated on 230 laboratory strains of leptospires.
  
  
  RESULTS
  All laboratory strains of pathogenic Leptospira were evaluated as LipL32-positive and all non-pathogenic strains as LipL32-negative. In addition, 455 biological materials (253 plasma, 121 urine, 72 cerebrospinal fluid (CSF), 7 bronchoalveolar lavage, and 2 sputum) from 295 patients with suspected leptospirosis were examined. From this set of patients, 9 were evaluated to be LipL32-positive, from 15 positive biological materials (10 urine, 4 blood plasma, and 1 CSF).
  
  
  CONCLUSIONS
  This real-time PCR method for the detection of the gene encoding the surface lipoprotein LipL32 is a reliable, sensitive, and rapid method for the detection of the acute form of leptospirosis.
authors.affiliation: Department of Clinical Microbiology, Charles University in Prague, Faculty of Medicine in Hradec Kralove and University Hospital Hradec Kralove, Hradec Kralove, Czech Republic.
authors.forename: Zuzana
authors.initials: Z
authors.lastname: Cermakova
authors.forename: Petra
authors.initials: P
authors.lastname: Kucerova
authors.forename: Lenka
authors.initials: L
authors.lastname: Pliskova
authors.forename: Pavla
authors.initials: P
authors.lastname: Kubickova
mesh_terms: D000328:Adult; D000368:Aged; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics*; D001431:Bacteriological Techniques / Q000379:methods*; D019411:Clinical Laboratory Techniques / Q000379:methods; D005260:Female; D006801:Humans; D007919:Leptospira / Q000235:genetics / Q000302:isolation & purification*; D007922:Leptospirosis / Q000175:diagnosis* / Q000382:microbiology; D008074:Lipoproteins / Q000235:genetics*; D008297:Male; D008875:Middle Aged; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013997:Time Factors; D055815:Young Adult
chemical_list: D001425:Bacterial Outer Membrane Proteins; C406589:LipL32 protein, Leptospira; D008074:Lipoproteins