An Immunomagnetic Separation-PCR Method for Detection of Pathogenic<i>Leptospira</i>in Biological Fluids

Fernandes, Cláudia Pinho Hartleben; Seixas, Fabiana Kömmling; Coutinho, Mariana Loner; Vasconcellos, Flávia Aleixo; Moreira, Ângela Nunes; Conceição, Fabrício Rochedo; Dellagostin, Odir A.; Aleixo, José Antônio Guimarães

doi:10.1089/hyb.2008.0029

Cited by 17 publications

(19 citation statements)

References 37 publications

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“…Recent studies report the immunomagnetic separation (IMS) technique prior to PCR assay as an approach to reduce the effect of inhibitory substances present in biological fluids and food samples (2, 3, 4, 6, 7, 9, 14, 15). For diagnosis of leptospirosis, the IMS-PCR approach was reported to detect Leptospira spp.…”

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confidence: 99%

“…For diagnosis of leptospirosis, the IMS-PCR approach was reported to detect Leptospira spp. in bovine urine (15) and in human biological fluids (6). In order to improve PCR sensitivity and specificity, we developed a novel IMS-PCR approach by using both magnetic beads in house coated with a monoclonal antibody (mAb) and specific PCR primers for pathogenic Leptospira spp.…”

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confidence: 99%

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Diagnosis of canine leptospirosis using an immunomagnetic separation-PCR method

Monte¹,

Jorge²,

Luiz³

et al. 2012

Braz. J. Microbiol.

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confidence: 99%

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confidence: 99%

Diagnosis of canine leptospirosis using an immunomagnetic separation-PCR method

Monte¹,

Jorge²,

Luiz³

et al. 2012

Braz. J. Microbiol.

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“…The most popular variant of the selective isolation of bacteria is immunomagnetic separation, which exploits bacteriaspecific antibodies immobilized on magnetic particles [9][10][11][12][13][14]. After the incubation with the sample, the particles with the captured bacteria can be collected with a magnet, washed, and resuspended in a desired volume of liquid for further analysis.…”

Section: Introductionmentioning

confidence: 99%

Application of immobilized synthetic anti-lipopolysaccharide peptides for the isolation and detection of bacteria

Sandetskaya

Engelmann

Brandenburg

et al. 2015

Eur J Clin Microbiol Infect Dis

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The molecular detection of microorganisms in liquid samples generally requires their enrichment or isolation. The aim of our study was to evaluate the capture and pre-concentration of bacteria by immobilized particular cationic antimicrobial peptides, called synthetic anti-lipopolysaccharide peptides (SALP). For the proof-of-concept and screening of different SALP, the peptides were covalently immobilized on glass slides, and the binding of bacteria was confirmed by microscopic examination of the slides or their scanning, in case of fluorescent bacterial cells. The most efficient SALP was further tethered to magnetic beads. SALP beads were used for the magnetic capture of Escherichia coli in liquid samples. The efficiency of this strategy was evaluated using polymerase chain reaction (PCR). Covalently immobilized SALP were capable of capturing bacteria in liquid samples. However, PCR was hampered by the unspecific binding of DNA to the positively charged peptide. We developed a method for DNA recovery by the enzymatic digestion of the peptide, which allowed for a successful PCR, though the method had its own adverse impact on the detection and, thus, did not allow for the reliable quantitative analysis of the pathogen enrichment. Immobilized SALP can be used as capture molecules for bacteria in liquid samples and can be recommended for the design of the assays or decontamination of the fluids. For the accurate subsequent detection of bacteria, DNA-independent methods should be used.

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“…Besides high sensitivity, a great advantage of leptospiral antigen and whole cell detection methods is the use of different types of samples, like urine, Immuno-magnetic separation of Leptospira 9 serum or other body fluids (Yan et al 1998). The preenrichment method known as immuno-magnetic separation (IMS) has been effectively used to isolate Leptospira prior to its detection by using fluorescence spectroscopy, ELISA or PCR (Taylor et al 1996;Yan et al 1998;Fernandes et al 2008). The highly effective combination of IMS and PCR (IMS-PCR) seems to become an increasingly reliable technique for leptospiral antigen detection in urine and sera (Fernandes et al 2008).…”

Section: Introductionmentioning

confidence: 99%

“…The preenrichment method known as immuno-magnetic separation (IMS) has been effectively used to isolate Leptospira prior to its detection by using fluorescence spectroscopy, ELISA or PCR (Taylor et al 1996;Yan et al 1998;Fernandes et al 2008). The highly effective combination of IMS and PCR (IMS-PCR) seems to become an increasingly reliable technique for leptospiral antigen detection in urine and sera (Fernandes et al 2008). Previous efforts to culture and detect environment-derived leptospiral organisms have been inconsistent, while IMS coupled detection methods provide a great potential to be more successful (Alexander et al 1975;Henry et al 1978;Ganoza et al 2006).…”

Section: Introductionmentioning

confidence: 99%

Evaluation of zero-length cross-linking procedure for immuno-magnetic separation of Leptospira

et al. 2010

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Leptospirosis constitutes a major health problem in tropical and subtropical countries and is caused by pathogenic Leptospira. Immuno-magnetic separation (IMS) is considered to be an effective pre-enrichment method to isolate Leptospira from liquid specimen. We applied an inexpensive and simple IMS protocol using zero-length cross-linkers to immobilize polyclonal anti-leptospiral antibodies onto magnetic particles. The IMS-system has been optimized and evaluated by the assessment of the capture efficiency (CE). Main parameters that influence the conjugation procedure were optimized, including the amount of protein per milligram of magnetic particles, the pH and ionic strength of the conjugation buffer. The bead-bound leptospiral fraction was identified by using acridine orange fluorescence dye. The highest value for CE occurred when using high molar phosphate saline buffer at a pH around the isoelectric point of the antibodies. Finally, up to 3×10 8 leptospiral cells per mL could have been captured with approximately 50 µg of antibody-labelled particles. Strong particle agglutination could be observed during incubation for leptospiral concentrations in the range of 10 7 -10 8 cells per mL. Despite covalent binding, we show that the physical adsorption parameters pH and ionic strength of the conjugation buffer greatly affect the entire immobilization process with regard to the CE, thus being able to increase the reactivity of the particles. We therefore conclude that a well-adjusted conjugation buffer for the used chemistry could possibly replace expensive and more complicated antibody immobilization methods.

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An Immunomagnetic Separation-PCR Method for Detection of PathogenicLeptospirain Biological Fluids

Cited by 17 publications

References 37 publications

Diagnosis of canine leptospirosis using an immunomagnetic separation-PCR method

Diagnosis of canine leptospirosis using an immunomagnetic separation-PCR method

Application of immobilized synthetic anti-lipopolysaccharide peptides for the isolation and detection of bacteria

Evaluation of zero-length cross-linking procedure for immuno-magnetic separation of Leptospira

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