Detection of 123 bp fragment of insertion element IS6110 Mycobacterium tuberculosis for diagnosis of extrapulmonary tuberculosis

Maurya, Ashok Kumar; Kant, Surya; Nag, Vijayalakshmi; Kushwaha, Rashmi; Tn, Dhole

doi:10.4103/0255-0857.96688

Cited by 19 publications

(10 citation statements)

References 17 publications

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“…For PCR assay, choosing one or more appropriate target (s), can be a great way to achieve a high sensitivity. PCR targeting rpoB , IS 6110 genes and nested PCR have been generally used to detect MTBC in EP samples ( Yun et al, 2005 ; Huang et al, 2009 ; Maurya et al, 2012 ).…”

Section: Discussionmentioning

confidence: 99%

Detection of Mycobacterium tuberculosis in extrapulmonary biopsy samples using PCR targeting IS6110, rpoB, and nested-rpoB PCR Cloning

et al. 2015

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Present study was aimed to examine the diagnostic utility of polymerase chain reaction (PCR) and nested PCR techniques for the detection of Mycobacterium tuberculosis (MTB) DNA in samples from patients with extra pulmonary tuberculosis (EPTB). In total 80 formalin-fixed, paraffin-embedded (FFPE) samples comprising 70 samples with definite diagnosis of EPTB and 10 samples from known non- EPTB on the basis of histopathology examination, were included in the study. PCR amplification targeting IS6110, rpoB gene and nested PCR targeting the rpoB gene were performed on the extracted DNAs from 80 FFPE samples. The strong positive samples were directly sequenced. For negative samples and those with weak band in nested-rpoB PCR, TA cloning was performed by cloning the products into the plasmid vector with subsequent sequencing. The 95% confidence intervals (CI) for the estimates of sensitivity and specificity were calculated for each method. Fourteen (20%), 34 (48.6%), and 60 (85.7%) of the 70 positive samples confirmed by histopathology, were positive by rpoB-PCR, IS6110-PCR, and nested-rpoB PCR, respectively. By performing TA cloning on samples that yielded weak (n = 8) or negative results (n = 10) in the PCR methods, we were able to improve their quality for later sequencing. All samples with weak band and 7 out of 10 negative samples, showed strong positive results after cloning. So nested-rpoB PCR cloning revealed positivity in 67 out of 70 confirmed samples (95.7%). The sensitivity of these combination methods was calculated as 95.7% in comparison with histopathology examination. The CI for sensitivity of the PCR methods were calculated as 11.39–31.27% for rpoB-PCR, 36.44–60.83% for IS6110- PCR, 75.29–92.93% for nested-rpoB PCR, and 87.98–99.11% for nested-rpoB PCR cloning. The 10 true EPTB negative samples by histopathology, were negative by all tested methods including cloning and were used to calculate the specificity of the applied methods. The CI for 100% specificity of each PCR method were calculated as 69.15–100%. Our results indicated that nested-rpoB PCR combined with TA cloning and sequencing is a preferred method for the detection of MTB DNA in EPTB samples with high sensitivity and specificity which confirm the histopathology results.

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Section: Discussionmentioning

confidence: 99%

Detection of Mycobacterium tuberculosis in extrapulmonary biopsy samples using PCR targeting IS6110, rpoB, and nested-rpoB PCR Cloning

et al. 2015

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“…In addition, PCR was also positive in 21 cases which were smearnegative and 3 cases which were culturenegative. In smear-negative cases, PCR is reported to have a lower sensitivity as compared with liquid culture [23][24][25].…”

Section: Discussionmentioning

confidence: 99%

Diagnosis of pediatric pulmonary tuberculosis with special reference to polymerase chain reaction based nucleic acid amplification test

Tiwari¹,

Nataraj²,

Kanade³

et al. 2015

International Journal of Mycobacteriology

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“…complex family. [27][28][29] In this work, a small fragment of the IS6110 sequence was used as the target biomarker and detected directly via hybridization to a specific probe sequence. There are many techniques that are used to detect DNA hybridization with the aid of a label molecule, [30][31][32][33] however, these methods typically have low sensitivity, require significant method development, and most importantly the target affinity of the DNA probes may be reduced due to labelling with fluorescent or electroactive species.…”

Section: Introductionmentioning

confidence: 99%

Development of an electrochemical surface-enhanced Raman spectroscopy (EC-SERS) aptasensor for direct detection of DNA hybridization

Karaballi

Nel

Krishnan

et al. 2015

Phys. Chem. Chem. Phys.

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Rapid detection of disease biomarkers at the patient point-of-care is essential to timely and effective treatment. The research described herein focuses on the development of an electrochemical surface-enhanced Raman spectroscopy (EC-SERS) DNA aptasensor capable of direct detection of tuberculosis (TB) DNA. Specifically, a plausible DNA biomarker present in TB patient urine was chosen as the model target for detection. Cost-effective screen printed electrodes (SPEs) modified with silver nanoparticles (AgNP) were used as the aptasensor platform, onto which the aptamer specific for the target DNA was immobilized. Direct detection of the target DNA was demonstrated through the appearance of SERS peaks characteristic for adenine, present only in the target strand. Modulation of the applied potential allowed for a sizeable increase in the observed SERS response and the use of thiol back-filling prevented non-specific adsorption of non-target DNA. To our knowledge, this work represents the first EC-SERS study of an aptasensor for the direct, label-free detection of DNA hybridization. Such a technology paves the way for rapid detection of disease biomarkers at the patient point-of-care.

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Detection of 123 bp fragment of insertion element IS6110 Mycobacterium tuberculosis for diagnosis of extrapulmonary tuberculosis

Cited by 19 publications

References 17 publications

Detection of Mycobacterium tuberculosis in extrapulmonary biopsy samples using PCR targeting IS6110, rpoB, and nested-rpoB PCR Cloning

Detection of Mycobacterium tuberculosis in extrapulmonary biopsy samples using PCR targeting IS6110, rpoB, and nested-rpoB PCR Cloning

Diagnosis of pediatric pulmonary tuberculosis with special reference to polymerase chain reaction based nucleic acid amplification test

Development of an electrochemical surface-enhanced Raman spectroscopy (EC-SERS) aptasensor for direct detection of DNA hybridization

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