Leishmania (Viannia) braziliensis: New primers for identification using polymerase chain reaction

Marcussi, Viviane Mathias; Marcussi, Lílian Mathias; Barbosa-Tessmann, Ione Parra; Lonardoni, Maria Valdrinez Campana; Silveira, Thaís Gomes Verzignassi

doi:10.1016/j.exppara.2008.08.005

Cited by 16 publications

(19 citation statements)

References 21 publications

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“…21 However, these latter authors used the DNA hybridization method, which results in higher sensitivity and therefore efficiency of the primer. Recently, Marcussi 20 developed a pair of primers (LBF1-LBR1) capable of amplifying a specific sequence of the kDNA minicircle of L. (V.) braziliensis. Data obtained in the present study using this pair showed that it was capable of detecting 64 pg of DNA, showing greater sensitivity in DNA detection compared to the limit of 4 ng reported by Marcussi.…”

Section: Discussionmentioning

confidence: 99%

“…19 LBF1 (5'-AAA TTC GCG TTT TTT GGC CTC CCC G-3') and LBR1 (5'-GCA TAA ACT AGA GAC GGA ACA GAG-3'), 20 and 13A (5'-GTG GGG GAG GGG CGT TCT-3') and 13B (5'-ATT TTA CAC CAA CCC CCA GTT-3'). 21 The reaction medium (25 µL) contained 1 µM of each of the primers (Invitrogen®, Brazil), 0.2 mM dNTP (Invitrogen®, USA), 1 U Taq DNA polymerase (Invitrogen®, USA), 1.5 mM MgCl 2 , 1X enzyme buffer, and 2 µL of the extracted DNA.…”

Section: Polymerase Chain Reaction (Pcr)mentioning

confidence: 99%

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Comparison of different primes for PCR-based diagnosis of cutaneous leishmaniasis

Oliveira¹,

Lonardoni²,

Teodoro³

et al. 2011

Braz J Infect Dis

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Objective: The objective of this study was to analyze different primers that are commonly used in epidemiological studies for the detection of Leishmania DNA by PCR, and to compare them to the conventional direct parasite search for American cutaneous leishmaniasis (ACL) diagnosis. Material and methods: Five pairs of primers, four of them derived from Leishmania kDNA sequences (MP3H-MP1L; B1-B2; LBF1-LBR1; 13A-13B), and one derived from the SL RNA (mini-exon) gene repeat (LU5A-LB3C), reported previously, were used. Results: The MP3H-MP1L primers were the best at amplifying the DNA, detecting 2 fg of Leishmania spp. DNA. The 13A-13B primers presented the worst performance, detecting 512 x 10 3 fg of DNA. Conclusion: The wide variation in the analytical sensitivity of the primers used in the PCR, and the significant differences from the conventional method of ACL diagnosis found in this study, emphasize the importance of standardizing the PCR technique, analyzing sensitivity, and selecting suitable oligonucleotide primers.

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Section: Discussionmentioning

confidence: 99%

Section: Polymerase Chain Reaction (Pcr)mentioning

confidence: 99%

Comparison of different primes for PCR-based diagnosis of cutaneous leishmaniasis

Oliveira¹,

Lonardoni²,

Teodoro³

et al. 2011

Braz J Infect Dis

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“…The tCL also showed better sensitivity than reported by Bruijn and Barker [26], for the uniplex B1/B2 PCR, which is widely used in research laboratories in Brazil. Marcussi and coworkers [27] presented the LBF1/LBR1 primers for identification of L. braziliensis with a detection limit of 50 ng of DNA in the reaction, which is in contrast with the one obtained with our tCL assay (10 pg).…”

Section: Discussionmentioning

confidence: 64%

Inclusion of Quality Controls on Leishmaniases Molecular Tests to Increase Diagnostic Accuracy in Research and Reference Laboratories

Gonçalves-de-Albuquerque

Silva

Trajano-Silva

et al. 2014

Mol Biotechnol

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Early detection of leishmaniases and prompt institution of treatment are paramount for individuals and communities affected by these diseases. To overcome the remaining limitations inherent to molecular methods currently used and to ensure the accuracy of results in leishmaniases diagnosis, two triplex polymerase chain reaction (PCR) assays with quality controls for the reactions were developed. Validity indicators were assessed in 186 dog blood samples from endemic areas in Brazil. The level of agreement between the new tools and their singleplex protocols was assessed by kappa analysis. The triplex PCR for visceral leishmaniasis showed sensitivity (S) = 78.68 %, specificity (E) = 85.29 %, and efficiency (e) = 81.05 %. The cutaneous leishmaniasis protocol showed S = 97.29 %, E = 79.16 %, and e = 90.16 %. Both protocols showed good agreement with gold standards. These new tools enable, in a single reaction, the diagnosis of the diseases and the evaluation of the sample quality and DNA extraction process, thus reducing the cost of reagents and avoiding the eventual need for collecting a second sample.

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“…Studies describing techniques based on PCR for identification of the species from Viannia subgenus using DNA of clinical samples have been reported such as the Polymorphism-Specific PCR (PS-PCR) 26 , G6PD PCR 8 , LBF1-LBR1 PCR 27 . Despite the ability to identify the Leishmania' s species, the use of these techniques as routine may not be validated since they require high concentration of total DNA 27 and the employment of several primers for each species 8 26 .…”

Section: Discussionmentioning

confidence: 99%

APPLICABILITY OF kDNA-PCR FOR ROUTINE DIAGNOSIS OF AMERICAN TEGUMENTARY LEISHMANIASIS IN A TERTIARY REFERENCE HOSPITAL

Satow

Yamashiro-Kanashiro

Rocha

et al. 2013

Rev. Inst. Med. trop. S. Paulo

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SUMMARYThis study evaluated the applicability of kDNA-PCR as a prospective routine diagnosis method for American tegumentary leishmaniasis (ATL) in patients from the Instituto de Infectologia Emílio Ribas (IIER), a reference center for infectious diseases in São Paulo - SP, Brazil. The kDNA-PCR method detected Leishmania DNA in 87.5% (112/128) of the clinically suspected ATL patients, while the traditional methods demonstrated the following percentages of positivity: 62.8% (49/78) for the Montenegro skin test, 61.8% (47/76) for direct investigation, and 19.3% (22/114) for in vitro culture. The molecular method was able to confirm the disease in samples considered negative or inconclusive by traditional laboratory methods, contributing to the final clinical diagnosis and therapy of ATL in this hospital. Thus, we strongly recommend the inclusion of kDNA-PCR amplification as an alternative diagnostic method for ATL, suggesting a new algorithm routine to be followed to help the diagnosis and treatment of ATL in IIER.

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Leishmania (Viannia) braziliensis: New primers for identification using polymerase chain reaction

Cited by 16 publications

References 21 publications

Comparison of different primes for PCR-based diagnosis of cutaneous leishmaniasis

Comparison of different primes for PCR-based diagnosis of cutaneous leishmaniasis

Inclusion of Quality Controls on Leishmaniases Molecular Tests to Increase Diagnostic Accuracy in Research and Reference Laboratories

APPLICABILITY OF kDNA-PCR FOR ROUTINE DIAGNOSIS OF AMERICAN TEGUMENTARY LEISHMANIASIS IN A TERTIARY REFERENCE HOSPITAL

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