Identification of pathogenic Leptospira genospecies by continuous monitoring of fluorogenic hybridization probes during rapid-cycle PCR

Abstract

Partial sequences of 23S rRNA gene PCR products from 23 strains of 6 pathogenic Leptospira genospecies and from 8 strains of the saprophytic Leptospira biflexa were determined. Sequence analyses enabled Leptospira genus-specific amplification primers and pathogen-specific fluorogenic adjacent hybridization probes to be designed and synthesized. A PCR protocol was developed in which changes in fluorescence emission resulting from specific annealing of fluorogenic adjacent hybridization probes to the target DNA were continuously monitored. Nine strains of the pathogenic Leptospira genospecies could be differentiated from Leptonema illini, Escherichia coli, and eight strains of Leptospira biflexa. The PCR method was rapid, requiring 18 min for the completion of 45 cycles. It was also simple and flexible, as DNA templates prepared by four different methods, including the simple boiling method, could be used without adverse effects. Two hundred copies of target, equivalent to 100 cells, could be detected.

Real-time PCR in the microbiology laboratory

“…Likewise, the study of new and emerging viruses has been ideally complemented by the use of homogeneous real-time PCR assays as tools to demonstrate and strengthen epidemiological links between unique viral sequences and the [177,234,240,253,254,266,267,280,284,292,294] Solid tissues [172,198,238,[256][257][258]271,288,295] Cerebrospinal fluid [136,190,192,194,248] Peripheral blood mononuclear cells [183] Bone marrow [120] Whole blood [179,291] Plasma [118] Serum [171,172,180,279,287] Swabs [192,259,296] Bronchoalveolar lavage [243,246] Amniotic fluid [286] Saliva and sputum [158,233] Faeces [264,285] Urine [12,…”

“…However, specific bacterial species are more frequently the focus for real-time PCR assays, especially when long culture times can be replaced by rapid and specific gene detection. Leptospira genospecies, Mycobacterium and Propionibacterium spp., Chlamydia spp., Legionella pneumophila and Listeria monocytogenes have all been detected and in some cases quantitated with the use of real-time PCR assays [41,[234][235][236][237][238][239][240][241][242][243][244][245][246].…”

Possible linkages between lignite aquifers, pathogenic microbes, and renal pelvic cancer in northwestern Louisiana, USA

“…Cells were harvested from the filtrates by centrifugation at 15,000 g for 15 min, resuspended in 100 ll of phosphate buffered saline, and stored at -70°C. Following DNA extraction, PCR was performed using pathogenic leptospire primers and conditions described in Gravekamp et al (1993) and Woo et al (1997). Purified Leptospira DNA obtained from the Centers for Disease Control and Prevention (CDC) reference laboratory was used as the PCR positive control.…”

Advances in Real‐Time PCR: Application to Clinical Laboratory Diagnostics

“…[123][124][125][126], Propionibacterium spp. [127], Borrelia burgdorferi [128], and Leptospira genospecies [129]. DeGraves et al [5] established an eYcient real-time PCR platform to detect single Chlamydia spp.…”