Fluorescent quantitative PCR detection ofMycobacterium tuberculosisin tissue sections from granulomatous lesions retrieved using EDTA.
OBJECTIVE
This study aimed to use EDTA to retrieve paraffin-embedded tissue sections of inflammatory granulomatous lesions and increase the detection rate of tuberculosis (TB)/non-tuberculous mycobacteria. Due to the influence of chemical reagents during the fixation process, the amplification of fluorescent quantitative PCR was blocked after DNA extraction, and the results were not ideal.


METHODS
Special staining technologies (acid-fast and Auramine O) and fluorescent quantitative PCR were used to detect TB/non-tuberculous mycobacteria in 125 cases of inflammatory granulomatous lesions in paraffin-embedded tissue sections with and without EDTA retrieval.


RESULTS
In 125 cases of inflammatory granulomatous lesions, 75 cases (60%) were positive for mycobacteria using fluorescent quantitative PCR without EDTA retrieval, of which 74 cases (59.2%) were detected with TB mycobacteria and 1 case (0.8%) with non-tuberculous mycobacteria. The average cycle threshold value of positive specimens ranged from 29 to 32 (30.5). However, 88 cases (70.4%) were positive for mycobacteria using fluorescent quantitative PCR with EDTA retrieval, of which 83 cases (66.4%) were detected with TB mycobacteria and 5 cases (4%) with non-tuberculous mycobacteria. The average Ct value of positive specimens ranged from 27 to 30 (28.0). Statistical differences were found between the two groups (p<0.05; p<0.01).


CONCLUSIONS
This study showed that compared with special staining technologies (acid-fast and Auramine O) and molecular pathology detection, fluorescent quantitative PCR with EDTA retrieval could greatly improve the detection rate of TB/non-tuberculous mycobacteria and increase the sensitivity of the fluorescent quantitative PCR.
Department of Pathology, Dongfang Hospital, Fujian Medical University, Fuzhou, China.
Xuzhou
X
Wang
Department of Pathology, Dongfang Hospital, Fujian Medical University, Fuzhou, China.
Feilai
F
Xie
Department of Pathology, Dongfang Hospital, Fujian Medical University, Fuzhou, China.
Qiaoling
Q
Zheng
Department of Pathology, Dongfang Hospital, Fujian Medical University, Fuzhou, China.
Xingfeng
X
Qi
Department of Pathology, Dongfang Hospital, Fujian Medical University, Fuzhou, China.
Min
M
Li
Department of Pathology, Dongfang Hospital, Fujian Medical University, Fuzhou, China.
Xiaoling
X
Zhou
Department of Pathology, Dongfang Hospital, Fujian Medical University, Fuzhou, China.
Zhiyong
Z
Zheng
D000328:Adult; D000368:Aged; D001706:Biopsy / Q000379:methods; D004269:DNA, Bacterial / Q000302:isolation & purification; D004492:Edetic Acid; D005260:Female; D005453:Fluorescence; D006099:Granuloma / Q000175:diagnosis / Q000382:microbiology* / Q000473:pathology; D006801:Humans; D008168:Lung / Q000382:microbiology / Q000473:pathology; D008297:Male; D008875:Middle Aged; D009165:Mycobacterium Infections, Nontuberculous / Q000175:diagnosis / Q000382:microbiology* / Q000473:pathology; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000302:isolation & purification*; D009170:Nontuberculous Mycobacteria / Q000235:genetics / Q000302:isolation & purification*; D016612:Paraffin Embedding; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D014376:Tuberculosis / Q000175:diagnosis / Q000382:microbiology* / Q000473:pathology
D004269:DNA, Bacterial; D004492:Edetic Acid
DIAGNOSTICS; MOLECULAR BIOLOGY; PCR