Simultaneous Detection and Differentiation of Pathogenic and Nonpathogenic Leptospira spp. by Multiplex Real-Time PCR (TaqMan) Assay

Bedir, Orhan; Kılıç, Abdullah; Atabek, Erdinc; Kuşkucu, Ahmet Mert; Turhan, Vedat; Başustaoğlu, Ahmet

doi:10.33073/pjm-2010-026

Cited by 33 publications

(23 citation statements)

References 25 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…An additional PCR primer pair, 23S -P1 and 23S -P2 16 , was applied to water isolates yielding positive 16S and negative LipL32 results for differentiating L. biflexa isolates from those of other saprophytic Leptospira species. In amplifications, 0.8 uM of 23S -P1 and 23S -P2 primers were used with the same reaction conditions as those of 16S PCR, except for the annealing temperature, which was set at 54 °C.…”

Section: Methodsmentioning

confidence: 99%

Characterization of Leptospira isolates from humans and the environment in Uruguay

Meny

Menéndez

Quintero

et al. 2017

Rev. Inst. Med. trop. S. Paulo

View full text Add to dashboard Cite

Laboratory diagnosis of human leptospirosis usually relies on indirect methods exploring specific immune response. Isolation and identification of the involved strains are cumbersome, but can provide biological resources for pathogenic studies and relevant information for guiding prevention and control measures. The aim of the research we are hereby reporting was the characterization of Leptospira isolates obtained from humans and the environment in Uruguay. Blood cultures were performed from early samples of 302 Uruguayan patients, mainly rural workers, and from 36 water samples taken from their living or working environments. Eight human isolates and seven environmental isolates were obtained and analyzed by end point Polymerase Chain Reaction (PCR), Multilocus Variable Number of Tandem Repeat Analysis (MLVA) and other molecular methods. Human isolates corresponded to several serogroups and serovars of Leptospira interrogans and Leptospira kirschneri species, probably reflecting the infection with similar involved Leptospira species and serovars of an extended animal reservoir in rural settings of the country, mostly dedicated to meat and dairy production. Culture-positive patients were older than usually affected workers, and presented signs and symptoms of severe illness. A high organic and circulating bacterial burden may explain an easier positive result from these workers’ samples. Environmental isolates were mainly identified as Leptospira biflexa strains, with a single L. meyeri isolate of uncertain significance.

show abstract

Section: Methodsmentioning

confidence: 99%

Characterization of Leptospira isolates from humans and the environment in Uruguay

Meny

Menéndez

Quintero

et al. 2017

Rev. Inst. Med. trop. S. Paulo

View full text Add to dashboard Cite

show abstract

“…The melting curve analysis was used for species level genotyping of pathogenic Leptospira by targeting variable-number tandem repeats (Naze et al 2015). The earlier studies have developed methods for differentiation of pathogenic and nonpathogenic Leptospira based on multiplex real-time PCR and MALDI TOF (Bedir et al 2010;Xiao et al 2015). The above-mentioned studies were performed in clinical samples focused on identification of pathogen.…”

Section: Development Of Realampmentioning

confidence: 99%

Development of real-time loop-mediated isothermal amplification (RealAmp) method for sensitive and rapid detection of pathogenic and nonpathogenicLeptospira

Monica

Rathinasabapathi

Ramya

2019

Lett Appl Microbiol

View full text Add to dashboard Cite

Leptospirosis is a zoonotic disease that is spread through water contaminated with the spirochete Leptospira. In our study, RealAmp (real‐time loop‐mediated isothermal amplification) was developed to distinguish between pathogenic and nonpathogenic Leptospira spp. Melt curve analysis of RealAmp showed two distinct melt curves for pathogenic and nonpathogenic Leptospira spp with a Tm value of 90 ± 2 and 88 ± 2°C. The sensitivity of the developed method is as low as 1 pg μl−1 of Leptospira DNA. Specificity was achieved to distinguish Leptospira species using the marker genes (lipL32 and lipL21). Different environmental water samples collected from cattle shelter, pisciculture and stagnant water were tested using this RealAmp method. Pisciculture water samples were found to be highly contaminated with both pathogenic and nonpathogenic Leptospira spp (50%), followed by cattle shelter and stagnant water samples (15%). Significance and Impact of the Study The occurrence of pathogenic Leptospira in the environment is a threat to humans and animals. Molecular‐based differentiation of the pathogenic and nonpathogenic Leptospria is essential for environmental monitoring; however, the currently available detection methods fail to distinguish between these species. We report here a real‐time loop‐mediated isothermal amplification‐based method that differentiates between the pathogenic and nonpathogenic Leptospira based on the melt curve analysis of the marker genes lipL32 and lipL21. The method was successfully tested with a variety of environmental water samples to study the prevalence of Leptospira in the environment.

show abstract

“…Several qPCR assays have been described; some of them amplify particular sequences of genes, which universally present in bacteria, such as rrs (16S rRNA) (8), gyrB (9) and secY (6), or genes that are restricted to the pathogenic serovars of Leptospira, e.g., lipL32 (10,11), ligA (12), ligB (12), and lfb1 (6), or genes found in non-pathogenic strains such as 23 S rRNA (12).…”

Section: Introductionmentioning

confidence: 99%

First Real-Time PCR in Morocco for Human Leptospirosis Using TaqMan Probes Targeting the LipL32 Gene

Al-orry

Arahou

Hassikou

et al. 2019

Arch Clin Infect Dis

View full text Add to dashboard Cite

Background: Currently, qPCR has been used as a rapid diagnostic method for human leptospirosis. Previous studies have indicated that qPCR has high sensitivity in the early days of the illness. Objectives: The aim of this study was to evaluate qPCR as a diagnostic method for human leptospirosis in the National Institute of Hygiene, Rabat, Morocco. Methods: From 2004 to 2016, 67 sera related to 67 patients with clinical signs mimic to leptospirosis were sent to the laboratory of Bacteriology for routine diagnosis and confirmation. SAT, ELISA IgM, ELISA IgG, and qPCR were used for the diagnosis. Results: High positivity was observed by SAT (88.24%), ELISA IgM (58.82%), and real-time PCR (17.64%), in sequence. No negative results by serological tests had positive results by real-time PCR. Forty-six patients were males (68.68%) and 21 were females (31.34%). The high incidence observed was from Sidi Qacem (40%). Conclusions: SAT and ELISA IgM are useful for the diagnosis of human leptospirosis in Morocco and they can provide prompt and low-cost diagnosis, especially when resources are limited.

show abstract

Simultaneous Detection and Differentiation of Pathogenic and Nonpathogenic Leptospira spp. by Multiplex Real-Time PCR (TaqMan) Assay

Cited by 33 publications

References 25 publications

Characterization of Leptospira isolates from humans and the environment in Uruguay

Characterization of Leptospira isolates from humans and the environment in Uruguay

Development of real-time loop-mediated isothermal amplification (RealAmp) method for sensitive and rapid detection of pathogenic and nonpathogenicLeptospira

First Real-Time PCR in Morocco for Human Leptospirosis Using TaqMan Probes Targeting the LipL32 Gene

Contact Info

Product

Resources

About