A Novel Method of Mycobacterium Tuberculosis Complex Strain Differentiation Using Polymorphic Gc-rich Gene Sequences

Tuberculosis is one of the leading infectious diseases. In this work, a new genotyping method of Mycobacterium tuberculosis (Mtb) complex strain is presented. 27 Mtb genomes were analyzed for the presence of length poly-morphism within polymorphic GC-rich gene sequences. Four genes, Rv3345c, Rv3507, Rv0747 and Rv3511, showing variation in length depending on the Mtb strain were selected for designing primer sequences flanking variable regions for the PCR method. Identification of 16 genotypes among 27 analyzed genomes demonstrated usefulness of our genotyping method in differentiation of Mtb genomes based on sequence polymorphism in the four PGRS genes.


INTRODUCTION
The IS6110-RFLP strain identification method (van Embden et al., 1993) has been used for many years as a golden standard in studying diversity of the Mycobacterium tuberculosis complex strains, mainly because of high discriminatory power.Currently, the most popular and internationally recognized genotyping methods for studying molecular epidemiology of Mycobacterium tuberculosis complex strain outbreak are Spoligotyping (Kamerbeek et al., 1997) and MIRU-VNTR (Supply et al., 2001).The main advantages of these two methods are simplicity of performance and reproducibility of results between different laboratories, as well as access to international databases that contain genotyping results from different countries.This is of a particular importance in the analysis of the route of transmission of drug-resistant strains that are crossing borders between countries and continents.Since Spoligotyping is limited to the differentiation of Beijing type strains, MIRU-VNTR has a higher discriminatory power.However, in spite of many papers describing overall technical robustness, resolution power and clonal stability of the individual MIRU-VNTR loci, there is still a need for new genotyping methods that are better in reflecting variation in the genes encoding virulence features and antigenic properties of the Mtb strains.For this reason, a new genotyping method targeting the PGRS genes is presented in this study (Ramakrishnan et al., 2000;Brennan et al., 2001;Delgou et al., 2001;Singh et al., 2001;Banu et al., 2002;Brennan & Delgou, 2002;Lamichhane et al., 2003;Delogu et al., 2004;Chaitra et al., 2005;Talarico et al., 2005).We hypothesized that identification of specific genotypes will be possible based on the DNA sequence variation within the PGRS genes that are targeted in our new genotyping method.Analysis of defined lengths of amplicons will allow differentiation of strains using combination of agarose and polyacrylamide gel electrophoresis.

METHODS
In our study, 27 M. tuberculosis complex genomes were analyzed.For collection of genome sequences, GenBank databases of the National Center for Biotechnology Information, DNA Data Bank of Japan, Wellcome Trust Sanger Institute and Broad Institute were used (Benson et al., 2010).Blast, ClustalX (Larkin et al., 2007) and Clone Manager 7 computer programs were applied for DNA comparison and designing of primers.Methodology presented in this work relayed on application of six novel pairs of primers designed for the PCR method specific to the PGRS regions (Table 1).

RESULTS
Three different genotyping methods were evaluated in this work, a new genotyping method (Table 2), Spoligotyping (Table 3) and MIRU-VNTR (Table 4).For the new genotyping method, six sets of primers were selected based on theoretical analysis of 27 genomic sequences of the M. tuberculosis complex strains (Table 1, Fig. 1).The annealing temperature calculated for the designed primer sequences is about 68°C.Initial size-polymorphism results between the analyzed strains, presented in Table 2, showed that the designed pairs of primers flanking variable regions allowed for differentiation of 16 out of 27 genotypes (Fig. 2) For MIRU-VNTR and Spoligotyping methods the numbers of genotypes identified were 22 (Fig. 3) and 18 (Fig. 4), respectively.HGDI indexes for all genotyping methods are presented in table V.In addition, genotyping analysis showed that for M. tuberculosis Beijing type strains CCDC5079 and CCDC5180, two different banding patterns were obtained using our new method and MIRU-VNTR technique in contrast to Spoligotyping.The first strain was sensitive to all four first line drugs while the second one was resistant (Zhang et al., 2011).Also M. africanum with the specific size of one

DISCUSSION
Molecular biology methods used for tracking the evolution of an Mtb strain during outbreak, when passed from person to person, are very helpful in the early detection and mapping of the transmission of strains and allow for determination of whether the recurring tuberculosis is due to relapse or recurrence.In this work, a novel genotyping method of Mtb strain differentiation is presented.The method relies on a single-locus amplification reaction using PCR, and the analysis of results is based on determination of the length of amplicons.Reproducible sizes of the PCR products among 27 analyzed genomes depending on strains and relatively big differences in the length of amplicons within I, II, III and V pairs of primers, allow for precise determination of the size of PCR products based on DNA ladder standards.However, for the IV and VI pairs of primers, the polyacrylamide gel separation or sequencing techniques are recommended because of similarity of PCR band sizes.
The method proposed, like other single-locus amplification PCR techniques, will allow for detection of similar intensity of PCR products in contrast to the PCR-fingerprinting techniques that yield an unexpected number and size of bands as a result, varying from dozens to a few thousands of nucleotide base pairs (Friedman et al., 1995;Kotlowski et al., 2004).Ligation of oligonucleotide adapters to the enzymatically digested fragments of the genomic DNA allows to improve discriminatory power of the fingerprinting techniques and reproducibility of results (Goulding et al., 2000), however, there are still significant differences between the intensities of PCR products in each sample which makes it difficult to identify the right number of amplicons for correct and reproducible banding pattern analysis.
The first genotyping method using high GC-rich sequences concerning PCR amplification of variable regions between IS6110 insertion sequences and PGRS regions was DRE-PCR (Friedman et al., in 1995).A com-parative study using 90 M. tuberculosis strains has shown that the DRE-PCR method has a slightly higher discriminatory power than Spoligotyping, however, the main disadvantage of this method was reproducibility of results which equals 58% (Kremer et al., 1999).Interestingly, for another PGRS-RFLP(AluI) hybridization method using PGRS probe against AluI digested chromosomes ( Kremer et al., 1999), highly reproducible results were obtained, however, in this case sample preparation procedure is very laborious and more difficult in contrast to the single-locus amplification methods like MIRU-VNTR.

A B C
The method presented in this study is the first single-locus PCR amplification technique utilizing the PGRS regions.Although the discriminatory power of the method presented is still relatively low in comparison to the MIRU-VNTR technique, the number of variable GC-rich amplicons can possibly be extended, improving the discriminatory power, after obtaining more data from sequencing results of a greater number of Mycobacterium tuberculosis complex genomes.
NC_020245_BCG str.Korea 1168PHGDI index for all strains = 0.9573 amplicon for sets of primers IV was differentiated from other 26 strains.

Table 3 .
Figure 1.Histogram presenting the distribution of high-GC content Mtb2 sequence: 5' CGG-CGG-CAA-CGG-CGG-C in the genome of M. tuberculosis.Location of amplicons for each set of primers is indicated by Roman numbers.