Detection of Mycobacterium in clinical samples by multiprimer polymerase chain reaction

Barouni, A. S.; Saridakis, Halha Ostrensky; Vidotto, Marilda Carlos

doi:10.1590/s1517-83822004000100004

Cited by 5 publications

(5 citation statements)

References 11 publications

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“…The PCR result exhibited 96.3% sensitivity and 96.7% specificity, with three false negative and two false positive samples. The presence of falsenegative results can be due to insufficient number of samples or the presence of inhibitors Barouni, AS (2004). Although the MTB-F and MTB-R primers did not show any DNA amplification for some Gram negative and positive bacteria, they produced two false positive results.…”

Section: Discussionmentioning

confidence: 97%

Research Article Comparison Between Real-Time Polymerase Chain Reaction and DNA-Microarray in Detection and Identification of Mycobacterium Species

Gaber¹,

Hamed²,

Elsawy³

2017

Genet. Mol. Res.

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Low-cost and low-density (LCD) DNA arrays offer an easy way to detect resistance as minimal laboratory instrumentation is needed. Nucleic acid-based amplification tests allow the rapid detection of Mycobacterium tuberculosis. Recently, a real-time PCR assay for M. tuberculosis complex was introduced. Real-Time PCR and DNAmicroarray techniques were compared with the classical methods of Ziehl-Neelsen (ZN) staining and culturing. Regarding to the standard culture method, 80 positive individuals were identified out of 140 urine samples. RT-PCR showed 96.3% sensitivity and 96.7% specificity with Mycobacterial tuberculosis complex (MTB) (n=10) and nontuberculous mycobacteria (NTM) (n=70). The DNA-microarray analysis exhibited 100% sensitivity and specificity. One species belonging to (MTB) was identified as M. tuberculosis and positively represented by 12.5% (n=10). Five species belonging to nontuberculous mycobacteria (NTM) were identified and represented as M. kansasii 37.5% (n=30), M. celatum 21.25% (n=17), M. gordonae 11.25% (n=9), M. chelonae 10% (n=8), and M. phlei 7.5% (n=6). The results recommend the use of our simple and rapid PCR technique for early diagnosis of mycobacteria's. Gaber A, et al 2 Genetics and Molecular Research 16 (4): gmr16039837 Also, the fast LCD-microarray protocol is very useful for species identification and differentiation between MTB and NTM.

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Section: Discussionmentioning

confidence: 97%

Research Article Comparison Between Real-Time Polymerase Chain Reaction and DNA-Microarray in Detection and Identification of Mycobacterium Species

Gaber¹,

Hamed²,

Elsawy³

2017

Genet. Mol. Res.

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“…Primer selection of present study was done based on local characteristics of strains and their importance in the diagnosis of the disease (tabulated in Table 4). The nucleotide sequences of primers used for the detection of Mycobacterium in this study were described and validated as diagnostic markers in the past [17,18,29,[37][38][39][40][41][42][43][44][45][46]. This antigen would provide a target, which is universally present.…”

Section: Resultsmentioning

confidence: 99%

“…All samples collected were examined by H & E staining [30,31,32], Z-N staining for AFB [32,33,34], cultured on L-J egg media [34,35,36] and MTB specific multi-gene PCR method [17,18,29,[37][38][39][40][41][42][43][44][45][46] by which FGTB was confirmed and correlated with laparoscopic findings. The diagnosis was done based on morphological [19] and molecular investigations.…”

Section: Case Groupmentioning

confidence: 99%

Detection of Mycobacterium Tuberculosis among Infertile Patients Suspected with Female Genital Tuberculosis

Bhanothu¹,

Theophilus²,

Rozati³

2014

AJIDM

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Female genital tuberculosis (FGTB) is a symptomless disease that evidences itself only when it is investigated for infertility. Demonstration of the etiologic agent by H & E staining or Z-N staining for acid fast bacilli, smear microscopy, culture of menstrual blood, urine and sputum were often unsuccessful. We therefore, proposed to use the endo-ovarian tissue biopsies and pelvic aspirated fluids for the detection of FGTB among infertile women by conventional versus genotypic methods. A prospective case-control study was undertaken. A total of 302 specimens were collected from 202 infertile women highly suspected of having FGTB on laparoscopic examination and from 100 control women of reproductive age. Out of these 302 specimens, 150 (49.67%) were premenstrual endometrial tissue biopsies (ETBs), 95 (31.46%) were ovarian tissue biopsies (OTBs) and 57 (18.87%) were pelvic aspirated fluids (PAFs). All specimens tested by conventional/ phenotypic methods were later compared with multi-gene/ multi-primer PCR (multi-gene PCR) method using four sets of primers for the detection of Mycobacterium tuberculosis (MTB) DNA in a single tube-single step reaction and correlated with laparoscopic findings. The presence of MTB DNA was observed in 49.5% of ETBs, 33.17% of OTBs and 5.44% of PAF specimens collected from highly suspected FGTB patients. All control women were confirmed as negative for tuberculosis. The conventional methods showed 99% to 100% specificity with a low sensitivity, ranging from 21.78% to 42.08% while H & E staining showed a sensitivity of 51.48%. Multi-gene PCR method was found to have a much higher sensitivity of 70.29% with MTB64 gene, 86.63% with 19kDa antigen gene at species and TRC4 element at regional MTB complex level and 88.12% with 32kDa protein gene at genus level (Pearson χ2 =214.612, 1df, McNemar's test value <0.0001). The specificity of multi-gene PCR was 100%. We suggest site specific sampling, irrespective of sample type and amplification of the 19kDa antigen gene in combination with TRC4 element as a successful multi-gene PCR method for the diagnosis of FGTB and differentiation of mycobacterial infection among endo-ovarian tissue biopsies and PAFs taken from infertile women.

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“…On the other hand, the sensitivity of PCR methods may be influenced by the presence of PCR inhibitors, such as DNA-degrading enzymes in a template or in reaction reagents, and by the insufficient quality of a DNA template. The presence of inhibitors interfering with nucleic acid extraction and/or amplification has been observed in different clinical samples, especially in tissue specimens [ 3 , 8 ]. The presence of large amounts of eukaryotic DNA in a template has also been shown to have inhibitory properties [ 8 ].…”

Section: Discussionmentioning

confidence: 99%

Development and evaluation of the internal-controlled real-time PCR assay for <i>Rhodococcus equi</i> detection in various clinical specimens

Stefańska

Witkowski

Rzewuska

et al. 2016

The Journal of Veterinary Medical Science

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Rhodococcus equi is the causative agent of rhodococcosis in horses, resulting in significant morbidity and mortality in foals. This bacterium has also been isolated from a variety of animals and is being increasingly reported as a cause of infection in humans, mainly in immunosuppressed individuals. Laboratory diagnostics of R. equi infections based only on conventional microbiological methods shows low accuracy and can lead to misidentification. The objective of the study was to develop and evaluate a real-time PCR assay for direct detection of R. equi in various clinical specimens, including tissue samples. The species-specific region of the gene encoding R. equi cholesterol oxidase, choE, was used as a qPCR-target. The diagnostic applicability of the assay was confirmed by testing various tissue specimens obtained from horses with clinical signs of rhodoccocal infection and swine submaxillary lymph nodes. The rate of R. equi detection in clinical specimens by the developed assay was higher in comparison to the culture method (90% vs. 60.0% of positive samples) and conventional PCR (90.0% vs. 20.0% of positive samples). In case of 13 samples that were negative in the culture-based method, R. equi was detected by the developed assay. Only in one case, it gave negative result for culture-positive sample. The assay may provide a simple and rapid tool to complement the classical methods of R. equi detection based on culture and phenotypic identification of isolates, as the performed evaluation indicated a high specificity and accuracy of the results.

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Detection of Mycobacterium in clinical samples by multiprimer polymerase chain reaction

Cited by 5 publications

References 11 publications

Research Article Comparison Between Real-Time Polymerase Chain Reaction and DNA-Microarray in Detection and Identification of Mycobacterium Species

Research Article Comparison Between Real-Time Polymerase Chain Reaction and DNA-Microarray in Detection and Identification of Mycobacterium Species

Detection of Mycobacterium Tuberculosis among Infertile Patients Suspected with Female Genital Tuberculosis

Development and evaluation of the internal-controlled real-time PCR assay for <i>Rhodococcus equi</i> detection in various clinical specimens

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