Abstract:

Objective　To increase the detection rate of Mycobacterium tuberculosis(MTB) in real-time fluorescence quantitation polymerase chain reaction (PCR) by increasing sputum volume and modifying ultrasonic extraction of MTB DNA. Methods　Sputum samples from 206 tuberculosis patients and 103 non-tuberculosis patients as controls were analyzed. Routine sputum MTB smear and liquid culture of MTB by American BD MAGIT960 system were performed. At least 5mL sputum was collected from each patient,and 2-3-fold volume of 4% NaOH solution was added to the sputum. After liquefaction,1 mL solution was used for the routine DNA extraction procedure,and the remaining part was used for the modified ultrasonic extraction procedure. Both of the extracted DNA were quantitated by real-time fluorescence quantitation PCR. Results　By the modified ultrasonic extraction procedure,the MTB DNA positive detection rate increased from 87.5%(confidence interval 81.4%-93.6%)to 95.5% (confidence interval 91.7%-99.4%)(P<0.05)in the smear positive and culture positive tuberculosis patients,and the positive detection rate increased from 57.4%(confidence interval 47.5%-67.4%) to 83%(confidence interval 75.4%-90.6%) in the smear negative and culture positive tuberculosis patients (P<0.01). The quantity results of increasing sputum volume and modifying ultrasonic extraction procedure increased 14 folds in average by the modified ultrasonic extraction procedure. Conclusions　The increasing sputum volume and modifying ultrasonic extraction procedure increase the positive detection rate of real-time fluorescence quantitation PCR for tuberculosis,which can be recommended for routine laboratory use.

Key words: Mycobacterium tuberculosis, Fluorescence quantitative polymerase chain reaction, Ultrasound, Sputum