A rapid and quantitative method for the detection of Leptospira species in human leptospirosis.
Prompt laboratory diagnosis of leptospirosis infection facilitates patient management and initiation of therapy. A cost effective real-time PCR assay using SYBR Green I was developed for detection of pathogenic leptospires in serum specimens. Specific PCR products were obtained only with DNA of pathogenic Leptospira genomospecies. LightCycler PCR ability to distinguish between species was possible using melting curves, providing an approach for identification with a specific Tm assigned to a single species or set of species. Assay sensitivity was approximately 50 leptospires/ml, corresponding to one to two genome copies in a PCR mixture. Fifty-one patients who had clinical symptoms consistent with leptospirosis were tested both with a previously described rrs amplification and our real-time assay. Our LFB1 real-time assay confirmed the diagnosis for 25 patients (49%, 25/51) and revealed an estimated density of 8.0x10(1)-3.9x10(4) leptospires/ml of blood. The total assay time for 12 clinical samples from sample to data analysis was less than 3 h. These data illustrate the potential of our LFB1 real-time assay for the rapid detection of leptospires in serum samples and their subsequent quantification in a single run.
Merien
Fabrice
F
Laboratoire de recherche en bactériologie, Institut Pasteur de Nouvelle-Calédonie, BP 61 98845 Noumea, New Caledonia. fmerien@pasteur.nc
Portnoi
Denis
D
Bourhy
Pascale
P
Charavay
Françoise
F
Berlioz-Arthaud
Alain
A
Baranton
Guy
G
D000293:Adolescent; D000328:Adult; D000368:Aged; D052160:Benzothiazoles; D002648:Child; D017931:DNA Primers; D004269:DNA, Bacterial / Q000097:blood* / Q000302:isolation & purification; D003959:Diamines; D005260:Female; D006801:Humans; D007919:Leptospira / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D007922:Leptospirosis / Q000175:diagnosis* / Q000382:microbiology*; D008297:Male; D008875:Middle Aged; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines; D012680:Sensitivity and Specificity; D013997:Time Factors
D052160:Benzothiazoles; D017931:DNA Primers; D004269:DNA, Bacterial; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I