Molecular Genetics

Molecular methods to detect drug resistance

In order to control TB, is necessary to diagnose and treat the patient fast and properly. For this purpose, molecular methods can be used to rapidly detect the possible resistance of an isolate. Below is a summary of the molecular methods used in Hospital Germans Trias i Pujol.

Line probe assay

Line probe assays are reverse hybridization tests. They are based in the amplification of Mycobacterium tuberculosis DNA regions associated with resistance to first- and second-line drugs, and the subsequent hybridization of  these products to nitrocellulose strips where specific probes are bound. When the hybridization takes place, it triggers a colorimetric reaction that shows a band.

Pyrosequencing

Pyrosequencing is a semi-automated test based in the amplification of Mycobacterium tuberculosis DNA regions associated with resistance to first- and second-line drugs, and the following pyrosequencing reaction. This is a real-time reaction that detects the pyrophosphate released when a nucleotide is bound to the DNA growing chain. This pyrophosphate enters in an enzyme cascade reaction that ends in a light peak transformed into a graphic.

Molecular epidemiology

Molecular epidemiology studies are carried out in order to establish chains of transmission and detect outbreaks, to differentiate between reinfection and relapse, and to detect laboratory cross-contamination. Below is a summary of the current molecular methods that can be used.

Restriction-fragment length polymorphism

Restriction fragment length polymorphism (RFLP) is based in the insertion sequence 6110 (IS6110) present only in Mycobacterium tuberculosis complex. The protocol consists in the digestion of the DNA with a restriction enzyme and hybridization with a IS6110 probe. The different number of copies of the IS6110 and their localization in the chromosome yields different patterns for each strain tested. This method has a high discriminative power, but it is laborious and time-consuming.

Spoligotyping

Spoligotyping (spacer oligonucleotide typing) is based in the presence of 43 different spacers between the directed repeat (DR) sequences in the DR locus of the Mycobacterium tuberculosis chromosome. In this method, the DR locus is amplified and hybridized with oligos of the inter-DR spaces. The presence or absence of the different spacers allows a specific pattern for each strain. The protocol is shorter than in RFLP but it presents less discriminative power.

Mycobacterial interspersed repetitive units - Variable number tandem repeats (MIRU-VNTR)

This method consists in the amplification 15-20 loci of MIRU located in different regions of the Mycobacterium tuberculosis chromosome and the determination of the number of repetitive units according to the size in electroforesis gel. This method is rapid, simple and automatic and with discriminative power similar to RFLP.

 
Tuesday the 25th.