Spoligotyping Kit with Primers and Controls
Why is genotyping so important when working on Mycobacterium tuberculosis?
On the one hand, it is important to differentiate the species from the Mycobacterium tuberculosis complex (MTBC) which include Mycobacterium tuberculosis, M. africanum, M. bovis, M. canettii, M. microti, M. caprae, and M. pinnipedii, in order to distinguish between strict human and zoonotic TB and to initiate an appropriate therapy. In particular, it is very important to distinguish between M. tuberculosis and M. bovis, as the second species has a natural resistance to the anti-tuberculous drug pyrazinamide. On the other hand, genotyping of M. tuberculosis isolates is useful as a means of addressing evolutionary questions but also as a means of surveying the transmission dynamics of this pathogen and identifying new outbreaks.
What is the Spoligotyping Kit?
Spoligotyping is a PCR-Based Method to Simultaneously Detect and Type M. tuberculosis strain H37RV and M. bovis strain BCGP3. The typing method is based on DNA polymorphism present at one particular chromosomal locus, the “Direct Repeat” (DR) region, which is uniquely present in H37Rv and BCGP3. The presence or absence of the strains can be detected by hybridization of CR amplified sample to a set of immobilized oligonucleotides on the membranes. Spoligotyping is similar to Southern blotting when rapid results are required. By Spoligotyping one can detect the presence or absence of the spacers by the known sequence.
Why is the Spoligotyping kit superior to the existing commonly used methods?
Most molecular methods for MTBC species differentiation are based on the analysis of genomic deletions by PCR, followed by agarose gel electrophoresis, or are based on the detection of single nucleotide polymorphisms (SNPs) by PCR-restriction fragment length polymorphism (PCR-RFLP) analysis, which consists of digestion of the PCR product with a restriction enzyme, followed by agarose gel electrophoresis.
The Spoligotyping kit provides a sensitive and precise way to differentiate the strains H37RV (M. tuberculosis) BCGP3 (M. bovis) in just two major steps – 1) PCR amplification of the DNA sample and 2) hybridization of the biotin-labeled PCR products to the immobilized on a membrane spacer-oligos that represent spacers of known sequence. This way the Spoligotyping kit is not only more accurate but also time and cost efficient. Another cost saving feature is that the membrane can be regenerated and used for at least 10 consecutive hybridizations.
How can I get a price quotation and order the kit?
We strongly encourage all of our customers to contact us via any means that are most convenient to them. Give us a call or write us an e-mail, contact us through skype or our live chat to get a quotation on our Soligotyping kit.
About Mycobacterioum tuberculosis: Mycobacterium tuberculosis is a bacteria which is an obligate pathogen due to its inability to live outside the body of the host. It belongs to the family Mycobacteriaceae. M. tuberculosis causes most cases of of tuberculosis. M. tuberculosis has a quite unusual and waxy coating which covers the cell’s surface. This coating exists mainly because of the presence of mycolic acid (a fatty acid with long chains). This waxy coating makes the bacteria impervious to staining by Gram. However, due to various reasons, the M. tuberculosis can appear both Gram-negative and Gram-positive in clinical settings. Because of the unrealiable results using Gram staining for M. tuberculosis, another method is used – the Ziehl-Neelsen stain, a.k.a “acid-fast stain” is used. This method utilizes carbol fuchsin, acid alcohol, and methylene blue. Acid-fast bacilli will be coloured in bright red after staining. Due to its highly aerobic physiology, M. tuberculosis requires high levels of oxygen, therefore it is mostly a pathogen living in the respiratory system of mammals infecting the lungs. The most frequently used diagnostic methods for tuberculosis are the tuberculin skin test, acid-fast stain, and chest radiographs. The M. tuberculosis genome was sequenced in 1998, opening the door to creating such highly sensitive and accurate tests like the Spoligotyping.
Description of the method: Spoligotyping is a method based on the Polymerase Chain Reaction (PCR). The spoligotyping can both detect and type at the same time a complex of Mycobacterium tuberculosis in a big amount of clinical samples, including any suspected nosocomial infections. Spoligotyping offers an alternative for typing of Mycobacterium tuberculosis via Southern blotting especially in the case when obtaining results rapidly is needed. The Spoligotyping method has a lower level of differentianion compared to the IS6110 fingerprinting when the strains have 5 or more copies of the IS6110, but is considerably higher when IS6110 has less than 5 copies. This is the reason why the Spoligotyping is the preferred method for performing typing of Mycobacterium bovis strains, as they usually contain only 1 or 2 copies of the IS6110. Tip for regonizzing M. bovis: by the absence of reactivity with spacers 39-43.
About the product: IM9701 is the full Spoligotyping Kit. It contains four vials: 2 vials of primers – Primer Dra ( biotinylated ) and Primer Drb and and 2 vials of controls: Positive control 1 ( M. tuberculosis strain H37Rv ) and Positive control 2 ( M. bovis BCG P3). In the IM9701 Spoligotyping kit also is included the Spoligotyping Membrane (cat. number IM9702) which is bound with spacer oligonucleotides. The Spoligo-membrane (cat. numebr IM9702) is needed for the Southern blot analysis of the samples, after the PCR is performed. The IM9702 Spoligotyping Membrane is also available for purchasing as a separate item. The Spoligo-membrane is shipped in 20 mM EDTA.