scholarly journals Two alternative DNA extraction methods to improve the detection of Mycobacterium-tuberculosis-complex members in cattle and red deer tissue samples

2016 ◽  
Vol 16 (1) ◽  
Author(s):  
Shari Fell ◽  
Stephanie Bröckl ◽  
Mathias Büttner ◽  
Anna Rettinger ◽  
Pia Zimmermann ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Dna Extraction ◽  
Red Deer ◽  
Mycobacterium Tuberculosis Complex ◽  
Extraction Methods ◽  
Tuberculosis Complex ◽  
Tissue Samples ◽  
Dna Extraction Methods

scholarly journals Evaluation of six different DNA extraction methods for detection of Mycobacterium tuberculosis by means of PCR-IS6110: preliminary study

2013 ◽  
Vol 6 (1) ◽  
pp. 561 ◽  
Author(s):  
Isabela de Almeida ◽  
Wânia da Silva Carvalho ◽  
Maria Rossetti ◽  
Elis Regina Costa ◽  
Silvana de Miranda
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Dna Extraction ◽  
Extraction Methods ◽  
Preliminary Study ◽  
Dna Extraction Methods

scholarly journals Comparison of Roche Cobas Amplicor Mycobacterium tuberculosis Assay with In-House PCR and Culture for Detection of M. tuberculosis

1998 ◽  
Vol 36 (7) ◽  
pp. 2023-2029 ◽  
Author(s):  
Bodo R. Eing ◽  
Andrea Becker ◽  
Arthur Sohns ◽  
Ronald Ringelmann
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Dna Extraction ◽  
Extraction Methods ◽  
Predictive Values ◽  
Three Samples ◽  
Smear Negative ◽  
Negative Sputum ◽  
Dna Extraction Methods ◽  
Sensitivity Specificity ◽  
Roche Cobas

The new Roche Cobas Amplicor Mycobacterium tuberculosisassay, which is a semiautomated version of the manually performed Roche Amplicor M. tuberculosis test, was compared to culture and an IS6110-based in-house PCR protocol. A total of 1,681 specimens from 833 patients, including specimen types other than sputum, were tested in parallel by both the in-house PCR and the Cobas Amplicor M. tuberculosis assay. After we resolved discrepant PCR results, the sensitivity, specificity, and positive and negative predictive values for the Cobas Amplicor M. tuberculosis assay were 66.33, 99.71, 94.36, and 97.66%, respectively. The corresponding values for the in-house PCR were 91.08, 99.85, 97.87, and 99.37%, respectively. For culture- and smear-positive specimens, the sensitivity of the Cobas AmplicorM. tuberculosis test was 96.42% (in-house PCR, 100%). If only smear-negative sputum specimens were considered, the Cobas Amplicor M. tuberculosis assay exhibited a sensitivity of 45.45% (in-house PCR, 63.63%) relative to that of culture. With a modified protocol for DNA extraction (washing of samples plus ultrasonication), both PCR methods performed better with gastric aspirates than with sputum samples (sensitivity of the Cobas AmplicorM. tuberculosis assay with smear-negative gastric aspirates, 70.00%; sensitivity of in-house PCR, 90.00%). With dithiothreitol being used for liquefaction of specimens in this study, the Cobas Amplicor M. tuberculosis assay exhibited an inhibition rate of 9.16%. In our view, the new Cobas Amplicor M. tuberculosis test (i) is well suited for typing of smear-positive specimens, (ii) may also be applied to gastric aspirates and other types of specimens if DNA extraction methods are modified appropriately, and (iii) exhibits a sensitivity with smear-negative sputum specimens which makes it recommendable that a minimum of three samples from the same patient be tested.

scholarly journals Mycobacterium tuberculosis complex detected by modified fluorescent in situ hybridization in lymph nodes of clinical samples

2011 ◽  
Vol 6 (01) ◽  
pp. 58-66 ◽  
Author(s):  
Juan Rodriguez-Nuñez ◽  
Francisco J Avelar ◽  
Francisco Marquez ◽  
Bruno Rivas-Santiago ◽  
Cesar Quiñones ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Lymph Node ◽  
Mycobacterium Tuberculosis Complex ◽  
Extrapulmonary Tuberculosis ◽  
Clinical Samples ◽  
Tuberculosis Complex ◽  
Tissue Samples ◽  
Negative Results ◽  
Modified Dna ◽  
Pna Fish

Introduction:  Lymph node tuberculosis (TB) is the leading cause of extrapulmonary tuberculosis and is the most frequently identified type in Aguascalientes, Mexico. Conventional diagnosis has serious limitations for rapid detection of extrapulmonary tuberculosis in clinical samples. Here PCR and modified FISH have been tested as complementary diagnosis methods for extrapulmonary tuberculosis. Methodology: The specific insertion sequence IS6110 for Mycobacterium tuberculosis complex was used to perform PCR and build DNA and PNA FISH probes (20bp). PCR and modified DNA and PNA FISH assays were performed to evaluate 41 lymph node paraffin-embedded tissue samples, in comparison with the histopathology diagnosis, which was considered the gold standard (22 positive and 19 negative). Results: In comparison with histopathology diagnosis PCR showed 62.5 % sensitivity and 77.8 % specificity (χ2 = 4.583 p < 0.05). Modified DNA FISH showed 71.4% sensitivity and 84.6% specificity (χ2 = 11.21 p < 0.05). PNA FISH showed 66.7% sensitivity and 60.0% specificity (χ2 = 2.93 p > 0.05). Ziehl Neelsen stain was positive in only four cases of 22 lymph node samples positive to histopathology.  In contrast, PCR and modified DNA FISH were positive in 20 cases of the same group. The negative cases were coincident in all tests.Conclusions: PCR and DNA FISH showed a significant increase in the number of cases detected and also showed higher sensitivity and specificity compared with data reported by traditional methodology. In developing countries, these techniques could help to complement the early diagnosis and timely treatment of extrapulmonary tuberculosis.

scholarly journals Comparison of different DNA extraction methods from hair root follicles to genotype Finnish Landrace boars with the Illumina PorcineSNP60 BeadChip

2008 ◽  
Vol 20 (2) ◽  
pp. 143 ◽  
Author(s):  
A. SIRONEN ◽  
P. UIMARI ◽  
J. VILKKI
Keyword(s):  
Dna Extraction ◽  
High Throughput ◽  
Extraction Methods ◽  
Sequence Information ◽  
Hair Root ◽  
Tissue Samples ◽  
Preparation Methods ◽  
Large Populations ◽  
Recent Developments ◽  
Dna Extraction Methods

Recent developments in sequencing methods have enabled whole genome sequencing of several species and the available sequence information has allowed the development of high throughput genotyping chips. However, these genotyping methods require high quality DNA. The possibility to genotype samples based on DNA from non-invasive sources would permit retrospective genotyping of previously collected samples and also facilitate the analysis of large populations e.g. for genomic selection. In this study we have developed and evaluated different DNA preparation methods from porcine hair root follicles for high throughput genotyping with the PorcineSNP60 Genotyping BeadChip (Illumina). We describe a method for DNA extraction from porcine hair root samples, which produces results from high throughput genotyping with the same high degree of accuracy as previously reported for DNA extracted from sperm, blood or tissue samples. This method was used for the genotyping of 273 hair follicle samples. When the DNA concentration was > 30 ng/ìl all samples had the same high call rate ( > 99%) as sperm samples confirming the robustness of this DNA extraction method for high throughput genotyping. Our data also establishes the suitability of the PorcineSNP60 BeadChip for genotyping the Finnish Landrace population.;

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