MutaPLATE® MYCOBACTERIUM TUBERCULOSIS REAL TIME PCR (TAQMAN) KIT
INTENDED USE
The MutaPLATE® Mycobacterium tuberculosis real time PCR (TaqMan) kit is a qualitative screening assay for specific detection of Mycobacterium tuberculosis (complex) in clinical specimens (gut biopsies, stool samples) using open real time PCR systems (e. g. Applied Biosystems, Stratagene).
INTRODUCTION
Tuberculosis is – along with AIDS and Malaria – the most widely spread infectious disease worldwide. The World Healh Organization (WHO) estimated that a third of the world`s population is infected and tuberculosisi accounts for three million deaths annually. One-fifth of all deaths in adults in developing countries relate to TB. Two-thirds of the worlds tuberculsis-infected people reside in Asia and this will have a significant impact on the control of TB in other countries as a result of increased immigration.
TB is caused by the tubercle bacillus Mycobacterium tuberculosis and rarely by M. bovis or M. africanum. The initial pulmonary infection usually goes unnoticed with lesions healing, sometimes leaving traces of calcified scar tissue. The infection may however progress to pulmonary tuberculosis, or through blood or lymphatic spread produce military, meningeal or other extrapulmonary involvement.
The key to interrupting the chain of transmission is the early diagnosis and treatment of pulmonary TB. For many years, diagnosis was based on staining smears for acid-fast bacilli (AFB) and culturing for mycobacteria. More recently, a number of nucleic acid amplification methods have been developed and made commercially available for rapid detection and identification of Mycobacterium tuberculosis complex (MTB) in clinical specimens.
PRINCIPLE OF THE TEST
MutaPLATE® Mycobacterium tuberculosis real time PCR (TaqMan) kit
The MutaPLATE® Mycobacterium tuberculosis real time PCR (TaqMan) kit contains specific primers, hybridisation probes and additional material for the detection of all bacteria from Mycobacterium tuberculosis complex. Starting material for DNA extraction are clinical samples from the respiratory or bronchial tract (e. g. sputum, lung biopsy).
The amplification of a gene fragment specific for Mycobacterium tuberculosis during PCR (polymerase chain reaction) process is done by the use of thermostable DNA polymerase. In the same step the specifity of the generated amplicon is proofed by hybridisation with probes (with fluorophor and quencher labelled oligonucleotides) specific for Mycobacterium tuberculosis (real time PCR).
In case of Mycobacterium tuberculosis specific amplification, the emitted fluorescence signal is detected and quantified by the real time PCR microplate system`s optical unit (ABI: PRISM SDS-, Stratagene: MxPRO-Software).
Mycobacterium tuberculosis specific amplification is measured by FAM fluorescence (470 nm excision/ 510 nm detection).
To exclude a possible PCR inhibition, the amplification mix contains an internal control. The amplification of this internal control does not affect the Mycobacterium tuberculosis detection and is measured by a probe`s VIC / HEX fluorescence (530 nm excision/ 555 nm detection).
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