**2. Structural organization and function of the IS***6110*

IS*6110* was initially named IS*986*. It is a genomic insertion element of 1361 bp long and shows 28 bp imperfect IRs, and duplications of 3 or 4 bp next to the insertion site. It has two overlapping ORFs (*orf*A and *orf*B) coding for a transposase, showing similarities with elements of the IS*3* family of prokaryotes (Accesion No.: X17348, M29899; Fig. 1).

The IS*6110* was found to be specific of mycobacteria belonging to the MTBC (Thierry et al., 1990a) and it was considered as the main target of the first reference genotyping tool, due to the high degree of polymorphism observed comparing strains of the MTBC (see part 3.2; Otal et al., 1991), turning into an important factor involved in the evolution of the *M. tuberculosis* genome. The sequences of IS*6110* and IS*986/*IS*987* identified in MTBC were practically identical and considered the same IS (Thierry et al., 1990b; McAdam et al., 1990).

IS*6110* the Double-Edged Passenger 63

Because of the increased accessibility and convenience of PCR-based detection techniques, these are suitable to replace conventional culture methods. Since bacterial growth is not required, PCR can give results rapidly in as short a period as 1 day. Further PCR modifications, as nested–PCR or multiplex-PCR, can be used to improve results. Over the years, a significant improvement of PCR technologies has been achieved with the development of real-time PCR for the detection of target genes of *M. tuberculosis* in clinical specimens. The main advantages of real-time PCR are a shortened turnaround time, automation of the amplification and product detection and a decreased in the risk of cross-

To obtain species-specific pathogen identification and detection in clinical samples, specific primers have been designed and tested using PCR-based methods, targeting different genomic sequences of *M. tuberculosis*. These have included IS*6110*, *hsp65*, TRC4 and *mpt40* (Bannalikar et al., 2006; Narayanan et al, 2001; Savekoul et al., 2006; Tumwasorn et al., 1996; Wei et al., 1999). Among these, the most widely investigated has been the IS*6110* being reported as a specific sequence of MTBC (Brisson-Noel et al. 1991; Eisenach, 1994; Sankar et al., 2011a). IS*6110* is an ideal target for PCR. IS*6110* is usually a multi-copy element and randomly distributed throughout the genome. The presence of multiple copies improves the

Different oligonucleotides derived from that sequence have been successfully used to detect *M. tuberculosis* in all type of clinical specimens. Table 2 summarizes a list of the primers more frequently used in the literature. A problem found was that authors give different names to the same primers. The primers IS1 and IS2 (Eisenach et al., 1990) are the most frequently used, these oligonucleotides amplified a final product of 123 bp from 759 to 881

A search in the databases PubMed since 1991 using "IS*6110*" and "diagnostic" as keywords, allowed the identification of 138 papers that showed how IS*6110* could be a useful tool in diagnostic of TB. In 105 of these papers the diagnostic is based on PCR. Up to 5 of the 11 works published during the seven first months of 2011 applied the real time PCR technique

In most of the cases the authors applied in-house PCR methods and compared results to other methods. Some authors concluded that IS*6110*-based PCR could be used routinely in clinical laboratories for rapid detection of *M. tuberculosis*, in sputum samples allowing early diagnosis and treatment (Ereqat et al., 2011). Evaluation of in-house PCR showed that

The usefulness of IS*6110* in the detection and identification of MTBC in clinical samples has been demonstrated in many studies, either detecting IS*6110* as single target (Sankar et al., 2010a; Gupta et al., 2010; Inoue et al., 2011) or together to other specific targets (Sankar et al., 2010b; Leung et al., 2011). Multiplex PCR assay can be used for the simultaneous detection

Additionally, in some cases, the location of IS*6110* specific to one strain can be used. PCR with primers targeting IS*6110* and the flanking region allowed identify and differentiate that

sensitivity of the PCR amplification (Mathema et al., 2006; Sankar et al., 2011a).

contamination (Espy et al., 2006).

nucleotide position of IS*6110* (Table 2).

in tuberculosis diagnostic using IS*6110* as target sequence.

variability in sensitivity and specificity is high (Cho et al., 2007).

of other coinfections in clinical samples (Boondireke et al., 2010).

**3.1.1 Advantages of IS***6110* **as target of the MTBC** 

Recently, Sankar and cols (2011b) have been suggested variations into the sequence of IS*6110* from different strains of *M. tuberculosis*, which could have implications in its usefulness as target of PCR detection.

Fig. 1. Structural organization of IS*6110*

IS*6110* does not have a known target or consensus sequence, it has been found within ORFs and intergenic regions (see part 4.1). It may be present up to 25 copies per genome in *M. tuberculosis* (Brosch et al., 2000), only a few number of strains have no copies of this IS (see part 3.1.2).

Many functions have been shown by the IS*6110*: (i) activation of genes during infection (Safi et al., 2004) (ii) participation in the evolution as an epidemiological marker (van Embden et al., 1993) (iii) activation of downstream genes with an activity promoter orientationdependent (Soto et al., 2004). Finally, it has been suggested that the presence of IS*6110* in *M. bovis* could participate in the adaptation of this bacteria to a particular host, animal or human (Otal et al., 2008). Several of these features are being reviewed herein.
