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

Abstract

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.

Evaluating the specificity of primers employed for PCR-based diagnostics of Leishmaniases using multiple alignment analysis

“…Interestingly, the primer pairs MP3H-MP1L and B1-B2 demonstrated alignments against multiple strains of Leishmania (such as L. braziliensis complex. Although low sensitivity of the primer pair LU5A-LB3C was observed in amplification reactions by Oliveira et al (2011), the findings described herein demonstrated that this primer pair showed high specificity in comparison to other primer pairs, revealing alignments exclusively against sequences of Leishmania (Table 1). In addition, Harris et al (1998) and Gomes et al (2007) also confirm that primers derived from the mini-exon gene, such as oligonucleotides LU5A and LB3C, represent successful tools to detect and characterize DNA in PCR-based diagnosis of distinct species of Leishmania.…”

“…The findings reported in this study emphasize differentiated degrees of specificity of distinct primer pair sequences employed for molecular Leishmaniasis detection by using comparative search and alignment analyses. The performance of these primer pairs has been tested under experimental approaches, demonstrating their variable sensitivity in PCR-based diagnostics for Leishmania (Oliveira, Lonardoni, Teodoro, Silveira, 2011). According to Oliveira et al (2011), the primers MP3H-MP1L and B1-B2 showed good sensitivity with amplified DNA fragments for strains of the MP3H-MP1L, 13A-13B, B1-B2 and LU5A-LB3C are 70bp, 120bp, 750bp and 146-149bp, respectively.…”

“…Four pairs of primers were selected randomly for comparative analyses of multiple alignments: MP3H (5'-GAACGGGGTTTCTGTATGC-3') and MP1L (5'-TACTCCCCGACATGCCTCTG-3') (Lopez, Inga, Cangalaya, Echevarria, Llanos-Cuentas et al, 1993); B1 (5'-GGGGTTGGTGTAATATAGTGG-3') and B2 (5'-CTAATTG TGCACGGGGAGG-3') (de Bruijn & Barker, 1992); LU5A (5'-TTTATTGGTATGCGAAACTTC-3') and LB3C (5'-CGT(C/G)CCGAACCCCGTGTC-3') (Harris, Kropp, Belli, Rodriguez, Agabian, 1998); 13A (5'-GTGGGG GAGGGGCGTTCT-3') and 13B (5'-ATTTTACACCAAC CCCCAGTT-3') (Rodgers, Popper, Wirth, 1990). These primers were chosen since they have been routinely employed successfully in the molecular diagnosis of Leishmaniasis (Oliveira, Lonardoni, Teodoro, Silveira, 2011).…”

Polymerase chain reaction–miniexon: a promising diagnostic method for mucocutaneous leishmaniasis

“…Clinical features and treatment efficacy vary in leishmaniasis according to species. Identification through classical methods is difficult and time consuming, and molecular biology techniques using targets such as the miniexon gene have been used and allow both diagnosis and species identification …”

“…Identification through classical methods is difficult and time consuming, and molecular biology techniques using targets such as the miniexon gene have been used and allow both diagnosis and species identification. 11,12,19,20 The aim of this study was to validate PCR with miniexon gene as a diagnostic test for mucocutaneous leishmaniasis using fresh or paraffin-embedded mucosal biopsies comparing it to a composite reference standard used previously by our group and to evaluate performance for species identification.…”