Development of tools for detection of DNA and mRNA from mycobacteria in clinical samples
Abstract number: p1110
Tuberculosis is one of the leading causes of mortality among infectious diseases. The conventional methods for detection of Mycobacteria in clinical samples are based on the demonstration of the acid-fast organisms following cultivation. In the last years polymerase chain reaction (PCR) has been used for detection of M.tuberculosis in different tests and has been shown to be at least as sensitive as the classical methods.
In this study development of Real-Time PCR-based tests for identification of DNA and mRNA from M.tuberculosis is described. Prokaryotic mRNA has a short half-life and can be found only in viable organisms.
Clinical samples. A total of 70 samples of sputum were obtained from patients treated for a period of severel months. DNA and RNA isolation was conducted with Trizol reagent (Life Technologies) according to the manufacturer's specification. Primers. The specific mRNA target is transcribed from Mtp-40 gene. One of the primers (37 nucleotides) was used for reverse transcription. First 20 nucleotides of this primer were complementary to mRNA. Other 17 nucleotides were chosen randomly and were identical to one of primers for PCR. Two other primers (20 and 17 nucleotides) were used for Real-Time PCR.
Reverse Transcription was performed at 42°C. Annealing temperature for PCR amplification of cDNA was 70°C. Annealing temperature for PCR amplification of DNA was 60°C.
Different methods for detection of M.tuberculosis in sputum of 70 patients were compared. These were: the developed Real-Time PCR with previous reverse transcription and classical methods (bacteriology and microscopy). The research showed high correlation between the data sets obtained by the PCR and RT-PCR and classical methods.
The developed tools for identification of DNA and mRNA from M.tuberculosis could be applied for differentiation of viable and nonviable M.tuberculosis. It is useful for rapid diagnostics and monitoring of the efficacy of treatment of tuberculosis.
|Session name:||XXIst ISTH Congress|
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