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

Meghdadi H, Khosravi AD, Ghadiri AA, Sina AH, Alami A - Front Microbiol (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences , Ahvaz, Iran.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

The area and the sensitivity and specificity of ROC curves of the methods applied in present study.
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Figure 3: The area and the sensitivity and specificity of ROC curves of the methods applied in present study.

Mentions: The rpoB-PCR alone showed low sensitivity in this study, but when this target gene was used in nested PCR, we had a high sensitivity of 85.7%. This was in concordance with similar study of Huang et al. (2009) which a rate of 86.1% was reported. By using TA cloning and subsequent sequencing of the cloned plasmid, we gained an overall 95.7% sensitivity compared to histopathology examination leading to construct a ROC curve analysis (Figure 3), which the PCR methods results variable related to curve are presented in Table 4. The nucleic acid sequence analysis revealed a 98–100% homology in comparison with MTB H37Rv reference strain (Genbank). TA cloning showed much higher sensitivity compared to other applied techniques. By cloning, we were able to first, detect seven additional positive samples among the initial 10 negative samples in the PCR methods including nested rpoB-PCR and second, improve the quality of eight samples with weak positive results by nested rpoB- PCR, allowing its sequencing.


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

Meghdadi H, Khosravi AD, Ghadiri AA, Sina AH, Alami A - Front Microbiol (2015)

The area and the sensitivity and specificity of ROC curves of the methods applied in present study.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4490800&req=5

Figure 3: The area and the sensitivity and specificity of ROC curves of the methods applied in present study.
Mentions: The rpoB-PCR alone showed low sensitivity in this study, but when this target gene was used in nested PCR, we had a high sensitivity of 85.7%. This was in concordance with similar study of Huang et al. (2009) which a rate of 86.1% was reported. By using TA cloning and subsequent sequencing of the cloned plasmid, we gained an overall 95.7% sensitivity compared to histopathology examination leading to construct a ROC curve analysis (Figure 3), which the PCR methods results variable related to curve are presented in Table 4. The nucleic acid sequence analysis revealed a 98–100% homology in comparison with MTB H37Rv reference strain (Genbank). TA cloning showed much higher sensitivity compared to other applied techniques. By cloning, we were able to first, detect seven additional positive samples among the initial 10 negative samples in the PCR methods including nested rpoB-PCR and second, improve the quality of eight samples with weak positive results by nested rpoB- PCR, allowing its sequencing.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences , Ahvaz, Iran.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus