Fluorescent quantitative PCR detection of in tissue sections from granulomatous lesions retrieved using EDTA.

Aims: 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.