Plasmid-based controls to detect rpoB mutations in Mycobacterium tuberculosis by quantitative polymerase chain reaction-high-resolution melting

Quantitative polymerase chain reaction-high-resolution melting (qPCR-HRM) analysis was used to screen for mutations related to drug resistance in Mycobacterium tuberculosis. We detected the C526T and C531T mutations in the rifampicin resistance-determining region (RRDR) of the rpoB gene with qPCR-HRM using plasmid-based controls. A segment of the RRDR region from M. tuberculosis H37Rv and from strains carrying C531T or C526T mutations in the rpoB were cloned into pGEM-T vector and these vectors were used as controls in the qPCR-HRM analysis of 54 M. tuberculosis strains. The results were confirmed by DNA sequencing and showed that recombinant plasmids can replace genomic DNA as controls in the qPCR-HRM assay. Plasmids can be handled outside of biosafety level 3 facilities, reducing the risk of contamination and the cost of the assay. Plasmids have a high stability, are normally maintained in Escherichia coli and can be extracted in large amounts.

Comparison of resazurin microtiter assay performance and BACTEC MGIT 960 in the susceptibility testing of Brazilian clinical isolates of Mycobacterium tuberculosis to four first-line drugs

We assessed the performance of REMA in comparison with BACTEC MGIT 960 in the susceptibility testing of 80 Mycobacterium tuberculosis clinical isolates from Clemente Ferreira Institute against four drugs. REMA proved to be a rapid and accurate method, providing excellent correlation with BACTEC MGIT 960, with the exception of results for the ethambutol drug.

Assessment of the quality of dna extracted by two techniques from Mycobacterium tuberculosis for fast molecular identification and genotyping

We report a comparative study of two DNA extraction techniques, thermolysis and chemical lysis (CTAB), for molecular identification and genotyping of M. tuberculosis. Forty DNA samples were subjected to PCR and the results demonstrated that with thermolysis it is possible to obtain useful data that enables fast identification and genotyping.

Rhodococcus equi isolation from sputum of patients with suspected tuberculosis

Rhodococcus equi has emerged as an opportunistic pathogen associated with pulmonary, invasive or systemic infections in immunocompromised patients. We report the identification of 51 R. equi isolates found in sputum samples of 546 individuals suspected to have pulmonary tuberculosis in two Public Health Hospital Units in Brazil. The epidemiology of R. equi infection as well as the phenotypic identification and drug susceptibility profile of isolates are described in this paper.

Quantitative trait loci (QTL) mapping for growth traits on bovine chromosome 14

Quantitative trait loci (QTL) mapping in livestock allows the identification of genes that determine the genetic variation affecting traits of economic interest. We analyzed the birth weight and weight at 60 days QTL segregating on bovine chromosome BTA14 in a F2 resource population using genotypes produced from seven microsatellite markers. Phenotypes were derived from 346 F2 progeny produced from crossing Bos indicus Gyr x Holstein Bos taurus F1 parents. Interval analysis to detect QTL for birth weight revealed the presence of a QTL (p < 0.05) at 1 centimorgan (cM) from the centromere with an additive effect of 1.210 ± 0.438 kg. Interval analysis for weight at 60 days revealed the presence of a QTL (p < 0.05) at 0 cM from the centromere with an additive effect of 2.122 ± 0.735 kg. The region to which the QTL were assigned is described in the literature as responsible for some growth traits, milk yield, milk composition, fat deposition and has also been related to reproductive traits such as daughter pregnancy rate and ovulation rate. The effects of the QTL described on other traits were not investigated.

Quantitative trait locus affecting birth weight on bovine chromosome 5 in a F2 Gyr x Holstein population

Segregation between a genetic marker and a locus influencing a quantitative trait in a well delineated population is the basis for success in mapping quantitative trait loci (QTL). To detect bovine chromosome 5 (BTA5) birth weight QTL we genotyped 294 F2 Gyr (Bos indicus) x Holstein (Bos taurus) crossbreed cattle for five microsatellite markers. A linkage map was constructed for the markers and an interval analysis for the presence of QTL was performed. The linkage map indicated differences in the order of two markers relative to the reference map (http://www.marc.usda.gov). Interval analysis detected a QTL controlling birth weight (p < 0.01) at 69 centimorgans (cM) from the most centromeric marker with an effect of 0.32 phenotypic standard-error. These results support other studies with crossbred Bos taurus x Bos indicus populations