Researches and Results | Molecular Biology

Molecular Biology

• PCR-REA of gyrB gene
• PCR-REA of inhA gene
• PCR-sequencing of IS900

PCR-REA of gyrB gene Single Nucleotide Polymorphisms (SNPs) have been widely used to study genomic diversity in a broad range of microorganisms. The analysis of the gyrA and gyrB genes in M. a. paratuberculosis Type I, II and III yielded Type-specific single nucleotide polymorphisms. Based on these results, a REA has been developped to discriminate Types I and III isolates, targeting the SNP located at position 1626 bp of the gene gyrB. The restriction enzymatic reaction with enzyme Hpy188 III lead to a four band pattern (551, 153, 112 and 80 bp) for Types I and II isolates and three bands (551, 192 and 153 bp) for isolates of Type III.

Polymorphisms in gyrA and gyrB genes among Mycobacterium avium subspecies paratuberculosis Types I, II and III. Castellanos E., Aranaz A., Romero B., de Juan L., Alvarez J., Bezos J., Rodríguez S., Stevenson K., Mateos A., Domínguez L. Journal Clinical Microbiology, 45: 3439-3442. 2007. PCR-REA of gyrB gene PCR-REA of inhA gene The analysis of the sequences of the enoyl-(acyl carrier protein) reductase (inh-A) gene in M. a. paratuberculosis Type I, II and III revealed two SNP at positions 224 and 228 bp. A REA has been developed with the restriction enzyme SinI targeting the Type III specific SNP located at 224 bp, which enable the differentiation of the strains into two different banding patterns. A two-band pattern in the case of Type I and II isolates (699 bp and 65 bp), whereas a three-banding pattern was obtained in the Type III strains (477 bp, 222 bp and 65 bp).

Use of Single Nucleotide Polymorphisms in inhA gene to classify Mycobacterium avium subespecies paratuberculosis into Types I, II and III. Castellanos E., Álvarez J., Aranaz A., Romero B., de Juan L., Bezos J., Rodríguez S., Stevenson K., Mateos M., Domínguez L. Póster. 9th International Colloquium on Paratuberculosis. Tsukuba (Japón), 29 octubre - 2 noviembre de 2007. PCR-REA of inhA gene PCR-sequencing of IS900 The IS900 sequence has been used in different molecular techniques such as multiplex PCR and restriction fragment length polymorphism and hybridization to IS900 (IS900-RFLP), leading to the identification of individual strains and classification of M. a. paratuberculosis into strain types. In this case the analysis of the 5’ end of the IS900 sequences revealed conserved polymorphisms that are M .a. paratuberculosis-Type specific. Every Type I ovine isolate showed a SNP at position 216 with a G instead of an A, with no other sequence modifications. Type II isolates gave a complete homology to M. a. paratuberculosis K-10, Type II reference strain. However, Type III isolates showed double peaks of the same size for some of the isolates tested at positions 169 bp (T/C) and 216 bp (G/A) or a single T peak and a G.

Single nucleotide polymorphisms in the IS900 sequence of Mycobacterium avium subspecies paratuberculosis are strain type-specific. Castellanos E., Aranaz A., de Juan L., Álvarez J., Rodríguez S., Romero B., Bezos J., Stevenson K., Mateos A., Domínguez L. Journal of Clinical Microbiology, 47(7)2260-2264. 2009. PCR-sequencing of IS900 ADIAVET® PCR kit and Adiapure® kit A commercially available Map-DNA extraction kit, designed specifically to extract Map DNA from milk- Adiapure® has been developed for MAP detection in milk samples. Adiapure® milk used magnetic beads technology in order to concentrate the bacteria from raw milk and cheese. The Adiapure® extraction combined with PCR detection have been evaluated in a ring trail and give consistent detection sensitivities down to 2cellsml-1

A commercial realtime PCR kits designed as Adiavet® Paratb has also been developed. The Adiavet® Paratb provided a new protocol for faecal extraction. 3 to 10g faecal samples can be treated with an Adiafilter® centri-filtration in order to concentrate the MAP. The new extraction protocol combined with Adiavet® Paratb PCR detection (based on IS900) give consistent detection sensitivities down to 40 mycobacterias/gram of feces.

An inter-laboratory ring trial for the detection and isolation of Mycobacterium avium subsp. paratuberculosis from raw milk artificially contaminated with naturally infected faeces. Donaghy JA, Rowe MT, Rademaker JL, Hammer P, Herman L, De Jonghe V, Blanchard B, Duhem K, Vindel E. Food Microbiol. 2008 Feb;25(1):128-35. Epub 2007 Jul 14