Mycobacterium tuberculosis – Double Check (Ready to use RealTime PCR-Kit)
13A Mycobacterium tuberculosis: double check (Ready to use real time PCR kit) [Technical details]
Mycobacterium tuberculosis is the causative agent of tuberculosis (TB) in the world. It is a chronic disease, which spreads through coughing, sneezing, speaking and singing as the bacteria spread in air. There are a number of persons with latent infections of the TB around the around the world, where latently infected patient rarely experience the symptoms of the disease, but act us the source for other infections. In the active form of TB, the symptoms are loss of weight, coughing, sputum with blood, fever, chills, pain in chest, cough lasting for more than 3 weeks. TB can be a compounding factor in immunosuppressive conditions like HIV. The TB can be detected with help of tuberculin test (skin test) or interferon test and culturing the sputum or blood samples. Latent TB can not be detected properly as there is problem of cross reaction in tuberculin and interferon tests in many diseases e.g. allergies, where these tests can lead to wrong results. These test may fail to differentiate between vaccinated and non-vaccinated cases. In the literature, there are many publications, which are pointing out the drawbacks of these tests, but still these tests are being used in diagnostic regularly. Similarly weeks are needed to get the results with culturing methods. Application of serological tests should be done carefully as there may be cross reactions leading to false results. Therefore there is a need of reliable tests, which can give accurate, highly specific and sensitive results within a short period. This can be done with PCR method. Real time PCR method can give the results within a short period and it can indicate the load of bacterial in form Ct values in the samples, which can be compared between the samples in order to find the success of the therapy. PCR method is also in position to detect the latent TB. But this can be improved combining the other parameters e.g. detecting the mRNA expression of different cytokines.
TB can be cured with different kinds of therapies. The drugs available are rifampin, isoniazid, ethambutol and pyrazinamide. The course of treatment can be over the months. During the treatment, there is chance of development of drug resistance strains. The prevention of drug strains of Mycobacterium is one of biggest challenge in medicine.
The vaccination is available, therefore people should be vaccinated to reduce the TB in the world. This vaccine is based on BCG strain. The recommendation of application of this vaccine varies country to country.
Tuberculosis (TB) is one of major public health problem throughout the world. In order to overcome this disease, one has to develop rapid, sensitive and specific identifcation methods. Traditional laboratory methods such direct microscopy and culture methods have drawbacks in view of identification of Mycobacterium tuberculosis as they are not so sensitive or they need skilled manpower (who give again different results of the same probe) or need a long time. The detection of Mycobacterium tuberculosis DNA sequence might be representing a more sensitive and fast daignostic target. One needs efficient method to detect the Mycobacterium tuberculosis in sputum and moreover to monitor the drug therapy, there is need of quantitative PCR assay. At present, the laboratories are using the PCR assay, which detects only one target gene and there are a certain strains of Mycobacterium tuberculosis, which does not contain this strain, therefore they can escape this test, therefore Genekam has brough a ready to use PCR kit, which can detect one most common gene target (which is widely accepted as standard throughout the world in all laboratories carrying the identification of Mycobacterium tuberculosis) and an extra gene, which is available in certain strains without the most common gene target.
However Genekam offers the assay in two forms:
1. ready to use kit for one target gene
2. ready to use kit for detectiong 2 different target genes
target gene: one IS gene (multicopy gene) in simplex version and two highly conversed gene targets (one is IS: it is multicopy gene and an other gene) in duplex version
Types of the samples: Sputum, strains from the culture, Respiratory fluid, lung, tissue, blood etc. DNA can be isolated through commecial available kits or home made good methods. This kit need good quality of DNA.
Contents of Kit: primern, fluorogenic primers (filter FAM to used in one step PCR and filters FAM and VIC are to be used in 2 step PCR), nucleotides, buffers, molecular water, negative control, positive control and manual. Dyes used are 6-carboxy-fluorscein, YYE and 6-carboxytetramethylrhodamine.
Validations: The assay has been validated for cross reactions with related like Mycobacterium avium sp paratuberculosis, Mycobacterium avium sp avium etc and non related micro organisms like Salmonella, Legionella, Staphyloccus etc. along with the different viruses of human and animal origin. The assy is 98% sensitive in most common used IS gene against the microscopic method.
detectionlimit: 10-50 genome equivalent per reaction
Storage: -20 degree
- only for in vitro use
- only for research use
- to be used through technical person
