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J Thorac Dis. 2013 Jun;5(3):251-7. doi: 10.3978/j.issn.2072-1439.2013.05.08.

Comparison of four DNA extraction methods for detecting Mycobacterium tuberculosis by real-time PCR and its clinical application in pulmonary tuberculosis.

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1
Department of Laboratory Medicine, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China; ; National Key Clinical Department of Laboratory Medicine, Nanjing 210029, China;

Abstract

OBJECTIVES:

China is one of the countries with a high burden of Mycobacterium tuberculosis (MTB) infection. One challenge for the earlier diagnosis of tuberculosis is the DNA extraction of MTB. This study was to compare four MTB DNA extraction methods, and use the best one in the diagnosis of pulmonary tuberculosis.

METHODS:

A total of 43 serum and 94 plasma samples were collected from 124 clinical diagnosed pulmonary tuberculosis patients. Four different MTB DNA extraction methods, including phenol-chloroform method, Qiagen kit, Omega kit and magnetic bead method, were compared to determine which method displayed the highest sensitivity. A quantitative fluorescent PCR assay was also designed for the detection of MTB DNA.

RESULTS:

The highest DNA extraction efficiency (52.8%) and the best reproducibility (coefficient of variance =26.7%) were observed using the magnetic bead method. For 39 of the 124 (31.5%) pulmonary tuberculosis patients, MTB DNA was detected in their plasma or serum samples. Interestingly, 35.3% (12/34) of smear-negative cases were MTB DNA positive.

CONCLUSIONS:

In conclusion, magnetic bead method is the best one for the DNA extraction of MTB. The detection of MTB DNA may provide valuable information for the diagnosis of acid-fast bacilli (AFB) negative pulmonary tuberculosis patients.

KEYWORDS:

Mycobacterium tuberculosis (MTB); plasma DNA; pulmonary tuberculosis; quantitative fluorescent PCR

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