INT J TUBERC LUNG DIS 18(3):302–309 © 2014 The Union http://dx.doi.org/10.5588/ijtld.13.0538

Development of a new ligation-mediated PCR method for the differentiation of Mycobacterium tuberculosis strains A. Z˙aczek,* A. Brzostek,† A. Kuron´,† A. Wojtasik,† A. Sajduda,‡ J. Dziadek*† * Department of Biochemistry and Cell Biology, University of Rzeszów, Rzeszów, † Institute of Medical Biology, Polish Academy of Science, Łódz´, ‡ Department of Microbial Genetics, Faculty of Biology and Environmental Protection, University of Łódz´, Łódz´, Poland SUMMARY BACKGROUND:

There is a need for rapid, inexpensive methods for analysing a limited number of Mycobacterium tuberculosis strains. The ligation-mediated polymerase chain reaction (LM-PCR) method appears to be sufficiently discriminative and reproducible to be considered as a molecular tool for the initial evaluation of hospital outbreaks, laboratory cross-contamination, and family or small community transmission. O B J E C T I V E : To develop a new LM-PCR method based on PCR amplification of the 5′-flanking region of insertion sequence (IS) 6110 consisting of Sal I/PvuII digestion of chromosomal DNA, ligation of a Sal I linker and differentiation of IS6110-carrying restriction fragments by suppression subtractive hybridisation. D E S I G N : The fast ligation amplification polymorphism (FLAP) method was applied in the analysis of 62 M. tuberculosis clinical isolates and compared with IS6110restriction fragment length polymorphism (RFLP) and

mycobacterial interspersed repetitive units–variable number of tandem repeat (MIRU-VNTR) analyses of the same strains. R E S U LT S : The sensitivity of FLAP was estimated at 0.25 ng/μl. FLAP yielded 32 patterns among the 62 M. tuberculosis strains compared to respectively 28 and 36 patterns obtained using MIRU-VNTR and IS6110RFLP. Its Hunter-Gaston discriminatory index value (0.973) is similar to that of MIRU-VNTR (0.966) and IS6110-RFLP (0.971). The specificity of the FLAP patterns was also confirmed. C O N C L U S I O N : FLAP proved highly discriminating, sensitive and specific and could be a valuable molecular tool, especially for analysing a limited number of M. tuberculosis strains. K E Y W O R D S : Mycobacterium tuberculosis epidemiology; molecular typing; ligation-mediated PCR; FLAP

MYCOBACTERIUM TUBERCULOSIS, the causative agent of tuberculosis (TB) identified by Koch in 1882, is still one of the most dangerous human pathogens. Molecular methods developed since the 1990s have substantially improved the epidemiological typing of M. tuberculosis clinical isolates and enabled molecular-guided control of the disease.1,2 The most widely applied and generally accepted reference method—the international gold standard—is insertion sequence (IS) 6110 restriction fragment length polymorphism (IS6110-RFLP) typing, which is based on the detection of variability in the number of copies and chromosomal locations of IS6110 insertion sequences.3,4 However, this method is not without its drawbacks: it is labour-intensive and does not lend itself to the construction of inter-laboratory databases. Alternative methods to IS6110-RFLP have therefore been developed. One such method is spoligotyping (spacer oligonucleotide typing), a polymerase chain

reaction (PCR) based method that analyses a single locus in chromosomal DNA carrying a variable number of direct repeats. Given its low discriminatory power, this method may be most appropriate for initial screening of a large collection of M. tuberculosis complex strains or in cases of strains with a low copy number of IS6110.1,5 For epidemiological analyses, isolates classified into one spoligotype should be analysed further using other, more discriminatory methods.6,7 A PCR-based method with discriminatory power similar to that of IS6110-RFLP is mycobacterial interspersed repetitive unit–variable number of tandem repeats (MIRU-VNTR).8,9 From among the 41 identified loci that contain MIRUVNTR repeats, 12 loci were originally used in genetic typing; currently, 15 or 24 of the most hyper-variable loci are used.10 MIRU sequences are repeated between 2 and 20 times in a single locus.11 The number of MIRU-VNTR sequence repeats in each locus can

Correspondence to: Jaroslaw Dziadek, Department of Genetics and Physiology, Institute for Medical Biology, Polish Academy of Sciences, Lodowa 106, Łódz´ 93-232, Poland. Tel: (+48) 42 272 36 10. Fax: (+48) 42 272 36 30. e-mail: jdziadek@ cbm.pan.pl Article submitted 25 July 2013. Final version accepted 25 October 2013.

LM-PCR differentiation of M. tuberculosis

be expressed as a numeric code, which can be used to easily compare strains isolated at different times and in different laboratories. On the other hand, current configurations of the MIRU-VNTR analysis require 15 (or 24) individual amplification reactions for each strain and identification of PCR product sizes. Although standardisation on automated sequencers has made the procedure less labour-intensive, it still requires expensive equipment and fluorescein-labelled oligonucleotides.11 Interesting alternatives to the above methods are those based on ligation-mediated PCR (LM-PCR), which has proven useful in the epidemiological analysis of a number of bacterial species.12–14 The direct application of these methods to M. tuberculosis isolates is usually not successful due to the low genetic variability among M. tuberculosis strains.15,16 However, LM-PCR methods can be adapted for use in mycobacteria if they are based on variability in IS6110 flanking regions.17–20 In the present study, we describe a new LM-PCR method, termed fast ligation amplification polymorphism (FLAP), present the results of its application to a published reference set,21,22 and compare its discriminatory power to that of IS6110-RFLP23 and MIRUVNTR methods.

MATERIALS AND METHODS Strains The 62 M. tuberculosis strains used in the study were collected from patients hospitalised at the Centre for Lung Disease Treatment and Rehabilitation in Łód´z, Poland, between 1 January 2006 and 31 December 2007, for which ethics approval was obtained from the Medical University of Łódz. ´ During the 2-year study, two patients returned to the hospital and additional samples were collected from them.21,23 All strains were tested for susceptibility to isoniazid, rifampicin, pyrazinamide, streptomycin and ethambutol using the BACTEC™ 460TB System (BD, Sparks, MD, USA), as described previously.21 All strains were previously subjected to epidemiological analysis using spoligotyping, 15-locus MIRU-VNTR and RFLP-IS6110.21 Sample preparation Genomic DNA was extracted from M. tuberculosis strains using a previously described protocol.24 DNA in samples was quantified using an ND-1000 Spectrophotometer (NanoDrop Technologies Inc, Wilmington, DE, USA). Genomic DNA (20 ng) was restriction digested by incubating a mixture containing 10 U of SalI (1.0 μl), 10 U of PvuII (1.0 μl; Fast Digest; Fermentas, Vilnius, Lithuania) and 2.0 μl of reaction buffer in a total volume of 20 μl at 37ºC for 30 min.

303

Adaptor synthesis For adaptor synthesis, equimolar amounts of two oligonucleotides (Eurogentec, Seraing, Belgium), SSDRpomlongSalI (5′-TCG ACG TGG GTC GCC TGA CCG ACC AGC AGG TTG CGT GCG C-3′) and SSDRliglongSalI (5′-GCG CAC GCA ACC TGC TGG TCG GTC AGG CGA CCC ACG-3′) were dissolved in 100 μl water at a final concentration of 10 μM. The adapter was synthesised by precisely annealing complementary sequences using a DNA thermal cycler (Applied Biosystems, Foster City, CA, USA), with an initial denaturation step of 94ºC for 15 min, followed by cycling the temperature three times between 58ºC for 10 min and 70ºC for 5 min. After synthesis, the adaptor was cooled to 4ºC and stored at −20ºC. Ligation of oligonucleotide adaptor and polymerase chain reaction The first step of FLAP was ligation of the DNA restriction fragments to the adaptors (1 μM) using 0.05 U of T4 ligase and 2.5 μl of 1× ligation buffer (Fermentas) in a total volume of 25 μl for 2 h at 25ºC. A total of 2.5 μl of this mixture was amplified by PCR (Veriti Thermocycler; Applied Biosystems) in a reaction mixture consisting of 20 pmol of each primer (SSDRSalshort-prim, 5′-CCT GCT GGT CGG TCA GGC GAC-3′; SSDR6110rev, 5′-GAC CAT CCG CAC GCG CCG-3′; Eurogentec), 1× Taq buffer, 1.5 mM magnesium chloride, 0.8 mM nucleoside triphosphates, and 1 U Taq polymerase (Fermentas) in a total volume of 25 μl. The following thermocycling conditions were used: initial denaturation for 2 min at 94ºC, followed by 30 cycles of denaturation for 1 min at 94ºC, annealing at 68ºC for 1 min and elongation at 72ºC for 1 min, with a final elongation at 72ºC for 5 min. Electrophoresis and analysis Each PCR product (8 μl) was run on 6% polyacrylamide gel (AppliChem, Darmstadt, Germany), and amplification patterns were determined by examination of ultraviolet (UV) light-illuminated (Intas Science Imaging, Göttingen, Germany), ethidium bromide (0.7%) stained gels. Amplicon sizes were determined by comparing bands with a 100 base pair (bp) DNA mass ladder (Fermentas). All bands between 100 and 1000 bp were used in dendrogram analyses. Five independent FLAP reactions were conducted using DNA samples from the control strain M. tuberculosis H37Rv to confirm the reproducibility of the FLAP method. Epidemiological data were analysed using the BioNumerics package 5.0 (Applied Maths, Sint Martens-Latem, Belgium). All dendrograms were generated with BioNumerics software by applying the Dice coefficient for calculating the similarities between patterns and the unweighted pair group method with arithmetic mean algorithm for clustering. A

304

The International Journal of Tuberculosis and Lung Disease

cluster was defined as all isolates sharing the same FLAP pattern. The Hunter-Gaston discriminatory index (HGDI) was calculated as described,25 and used to evaluate the discriminatory power of the typing methods. Hybridisation The FLAP fragments of selected strains were blotted onto a membrane and hybridised with a 642-bp probe corresponding to the PCR-amplified DNA fragment of IS6110. The probe was non-radioactively labelled with an enhanced chemiluminescence kit (ECL Direct Nucleic Acid Labeling and Detection System; Amersham Biosciences, Piscataway, NJ, USA). The hybridised FLAP fragments detected IS6110, demonstrating the specificity of amplification.

RESULTS It was previously reported that LM-PCR analysis could be applied for M. tuberculosis typing if based on IS6110 polymorphism.22 Here, we developed a new method that allowed us to distinguish M. tuberculosis clinical strains with an HGDI value similar to that of MIRU-VNTR and the IS6110-RFLP reference method. Using our LM-PCR method, the genomic DNA of M. tuberculosis strains was digested with PvuII and SalI restriction enzymes. The PvuII endonuclease recognises a single nucleotide sequence within IS6110 and generates blunt ends. After the digestion step, oligonucleotide adaptors (36 and 40 nucleotides in length) were ligated to SalI cohesive ends. The resulting restriction fragments could be flanked with 1) two PvuII blunt ends, 2) a PvuII blunt end and an adaptor sequence ligated to a SalI restriction site, or 3) adaptors ligated to SalI restriction sites at both ends. All restriction fragments were used as templates for PCR amplification, with one primer complementary to the adaptor sequence and the second complementary to the inner fragment of IS6110. The amplification of fragments carrying adaptors at both sites (SalI-SalI fragments) is inhibited by suppression subtractive hybridisation (SSH). The amplification of fragments carrying a single adaptor sequence (SalIPvuII) requires the presence of a 5′ end of IS6110 as a template for the second primer. The fragment containing two PvuII blunt ends can be amplified only when two closely located IS6110 are in a head-tohead orientation. The PCR products were separated on polyacrylamide gel and visualised by UV light illumination to generate the FLAP patterns (Figure 1). To determine the sensitivity of the FLAP method, we performed stepwise dilutions of M. tuberculosis H37Rv genomic DNA. The lowest DNA concentration that allowed us to obtain a FLAP pattern was estimated at 0.25 ng/μl (data not shown). The FLAP

Figure 1 Flow chart of the general strategy for FLAP. The discrimination between specific (Sal I-Pvu II-carrying IS6110) and non-specific restriction fragments is based on Sal I adaptors, and the hybridisation of one primer to the adaptor sequence and a second primer to IS6110. Ligation of adapters at both sides of restriction fragments (Sal I-Sal I) inhibits amplification through suppression subtractive hybridisation. FLAP = fast ligation amplification polymorphism; IS = insertion sequence.

method was then applied to a reference set of 62 DNA samples isolated from M. tuberculosis clinical strains. The number of bands in the FLAP patterns obtained for our collection of M. tuberculosis strains ranged from 4 to 12, with the majority of strains (n = 51) yielding 7 to 9 bands. The same set of 62 M. tuberculosis strains was previously evaluated using spoligotyping, 15-locus MIRU-VNTR and IS6110-RFLP analysis and grouped into 21, 28 and 36 patterns, respectively.21,23 FLAP typing yielded 32 patterns among the 62 M. tuberculosis strains, 14 (22.6%) of which were unique (Figure 2). The remaining 18 patterns contained 48 (77.4%) strains. These strains could be grouped into two clusters of five strains each with identical FLAP patterns, three clusters consisting of four strains each, and 13 clusters consisting of two strains each (two of which were repeat isolates from the same patients). The discriminatory power of the

LM-PCR differentiation of M. tuberculosis

305

Figure 2 Differentiation of a collection of 62 M. tuberculosis strains using the FLAP method. Genetic relationships among 62 M. tuberculosis strains based on FLAP patterns. The strain numbers (key) are indicated to the right of the band pattern. The similarity among strains is indicated above the dendrograms as percentages. The tree was generated using the Pearson correlation and unweighted pair group method, as described in Materials and Methods. FLAP = fast ligation amplification polymorphism.

306

The International Journal of Tuberculosis and Lung Disease

FLAP method for the 62 M. tuberculosis strains, calculated as HGDI, was 0.973 compared to respectively 0.966 and 0.971 for the MIRU-VNTR and IS6110RFLP methods. The specificity of the resulting FLAP patterns was verified for selected strains by Southern blot hybridisation, which showed that all PCRamplified DNA fragments from tested strains hybridised with the IS6110 insertion sequence (Figure 3). Both FLAP and IS6110-RFLP methods differentiated four MIRU-VNTR clusters (M16, M18, M23 and M27). Two MIRU-VNTR clusters (M19 and M28) were differentiated exclusively by the FLAP method. On the other hand, four MIRU-VNTR clusters (M1, M6, M21 and M22) were differentiated using IS6110-RFLP, but not by FLAP typing. Detailed information about the congruence and divergence of results generated by the three methods is presented in the Table.

Figure 3 The specificity of FLAP patterns verified using Southern blot hybridisation. A. FLAP-PCR fragments separated on 1% agarose gel. B. Southern blot hybridisation of separated FLAP-PCR fragments in 3A with an IS6110 inner fragment used as a probe. Lane 1 = strain 319/7; lane 2 = strain 386/7; lane 3 = strain 391/7; lane 4 = strain 412/7; lane 5 = strain 490/7; lane 6 = strain 41/7; lane 7 = strain 34/7; lane 8 = strain 704; lane 9 = strain 9/7; lane 10 = strain 696; lane 11 = strain 253; lane 12 = strain 671; lane 13 = strain 50/8; lane 14 = strain 108/8; lane 15 = strain 176/7; lane 16 = strain 146/7. IS = insertion sequence; FLAP = fast ligation amplification polymorphism; PCR = polymerase chain reaction.

Table A comparison of the differentiation of 62 M. tuberculosis strains by 15 loci-based MIRU-VNTR typing, FLAP and IS6110-RFLP Pattern assigned by No.

Strain

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 22 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62

50/8 674/7 149/8 146/7* 319/7 19/7 118/7 126/7 147/8 54/8 102/7 176/7 171/8† 412/7 129/7 41/7 216/8 307/7 165 218/8 230 9/7 84 513 152/7 34/7 549/7 550 690/7 567/7 696 232/8 571/7 565 80/7 490/7 704 222 459 237/8 253 1/7 10/7 601 120/7 723 91/8 321 179/8 386/7 391/7 65/7 632 611 564 306/7 725 724 108/8 671 305/7 37/7

MIRU-VNTR typing

FLAP

M.1

F.1

M.2 M.3 M.4

F.2 F.3 F.4

M.5

IS6110-RFLP R.1 R.2 R.3 R.4 R.5 R.6

M.6

F.5

M.7

F.6

R.7 R.8 R.9

M.8

F.7

R.10

M.9

F.8

R.11

M.10

F.9

R.12

M.11 M.12 M.13

F.10 F.11 F.12

R.13 R.14 R.15

M.14 M.15 M.16

F.13 F.14 F.15

R.16 R.17 R.18

F.16

R.19 R.18

M.17

F.17

R.20

M.18

F.18 F.19 F.20 F.21

R.21 R.22 R.23

M.19 M.20 M.21

F.22 F.23

R.24 R.25 R.26

M.22

F.24

R.27

M.23

F.25

M.24

F.26 F.27

R.28 R.29 R.30 R.31

F.28 M.25 F.27 M.26 M.27

M.28

F.29

F.30 F.31 F.32

R.32 R.33 R.34 R.35 R.36

* Isoniazid-resistant. † Isoniazid-, rifampicin-, ethambutol- and streptomycin-resistant. MIRU-VNTR = mycobacterial interspersed repetitive units–variable number of tandem repeats; FLAP = fast ligation amplification polymorphism; IS = insertion sequence; RFLP = restriction fragment length polymorphism.

LM-PCR differentiation of M. tuberculosis

DISCUSSION It seems apparent that appropriate epidemiological analyses of M. tuberculosis clinical strains should be based on more than one molecular method. An epidemiological database with a composition that allows the comparison of strains isolated in different places

307

and at different times requires the use of reproducible methods with high discriminatory power, such as IS6110-RFLP and MIRU-VNTR. On the other hand, routine laboratory methods should also be fast and reliable to allow the differentiation of a relatively limited number of isolates. These methods could initially identify laboratory contamination, small hospital

Figure 4 LM-PCR method variations used for the molecular analysis of M. tuberculosis strains; A) Palittapongarnpim et al.17 B) Prod’hom et al.18 C) Haas et al.19 D) Reisig et al.20 LM-PCR = ligation-mediated polymerase chain reaction; UDG = uracil DNA glycosylase.

308

The International Journal of Tuberculosis and Lung Disease

outbreaks or patient links within the same family or in isolated groups (e.g., prisoners, the homeless, residents of homes for the elderly). Such a method could also be used for further verification of molecular clusters identified by gold standard methods. One-step PCR methods based on LM-PCR were previously described as being useful for the discrimination of a number of bacterial species.12–14 In the case of M. tuberculosis clinical isolates with low genetic variability, LM-PCR methods should also take into account variability in IS6110 flanking regions.17–20 The ligation-mediated PCR amplification of both sides of IS6110 was proposed by Palittapongarnpim et al.17 In this method, non-phosphorylated BamHI-compatible linker and primers that bind to the 5′ and 3′ ends of IS6110 were used, generating from 5 to 12 bands ranging from about 100 bp to 2 kb in length. Prod’hom et al. used a LM-PCR procedure to amplify the flanking sequence located on the 5′ side of IS6110.18 This method used one primer specific for IS6110 and a second specific for a linker ligated to a SalI-restriction site; the amplification of SalI-SalI restriction fragments unrelated to IS6110 was avoided by using non-phosphorylated linkers. In the latter study, LM-PCR applied to the analysis of 98 M. tuberculosis complex strains generated up to 11 bands (median 6) compared to 20 (median 9) generated by IS6110-RFLP. When LM-PCR was used to examine 40 strains grouped into 13 clusters using RFLP, the strains within each cluster appeared to be identical between the two methods. These authors further observed that the number of bands generated using LM-PCR was equal to or less than the IS6110 copy number. The variation in LM-PCR was used by Haas et al. to differentiate M. tuberculosis strains.19 In this study, the authors applied one primer specific for IS6110 and a second complementary to a linker ligated to the HhaI restriction site. In one strain, the linker contained uracil in place of thymidine, which made it possible to avoid amplification of IS6110-unrelated restriction fragments after treatment with uracil Nglycosylase. Using this method to analyse 24 M. tuberculosis isolates, the authors found full agreement between LM-PCR and RFLP fingerprinting methods. This method was further modified by Reisig et al. in the form of FLiP (fast ligation-mediated PCR).20 A blinded analysis of 90 M. tuberculosis complex strains using FLiP identified 81 patterns, compared to 84 differentiated using IS6110-RFLP. A comparison of the abovementioned LM-PCR-based methods is shown in Figure 4. The FLAP method described here can also be classified as LM-PCR. FLAP is based on double digestion of chromosomal DNA with SalI and PvuII endonucleases, ligation of a SalI-compatible linker and amplification of a 5′ flanking region of IS6110 using a specific linker and IS6110 primers (Figure 1).

The linkers were designed to self-hybridise, thereby inhibiting the amplification of SalI-SalI restriction fragments through SSH. We applied FLAP to analyse 62 strains isolated in a single city in Poland. The calculated discriminatory power of FLAP was very similar to that of IS6110-RFLP and greater than that of MIRU-VNTR (15 loci) for the same collection of strains. Consistent with previous LM-PCR studies, the number of bands identified using FLAP was usually lower (65%) than that obtained using IS6110RFLP. For nine strains, FLAP yielded more bands than IS6110-RFLP, an outcome that could reflect some non-specificity or overlap of IS6110-RFLP bands. On the other hand, all FLAP bands tested hybridised with the IS6110 probe, confirming the specificity of the amplification procedure. Our data and those of others demonstrate the usefulness of different LM-PCR IS6110-related methods, particularly for the analysis of a limited collection of M. tuberculosis strains. These methods are highly discriminating, inexpensive and rapid, and could be valuable molecular epidemiology tools for analysing collections with a limited number of strains. Acknowledgements This work was supported by a grant from the National Science Centre, Krakow, Poland (project no. N N302 111338) and by a grant from the European Regional Development Fund (POIG. 01.01.02-10-107/09) under the Operational Programme Innovative Economy. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Conflict of interest: none declared.

References 1 Allix-Beguec C, Fauville-Dufaux M, Supply P. Three-years population-based evaluation of standardized mycobaterial interspersed repetitive-unit-variable-number tandem-repeat. J Clin Microbiol 2008; 46: 1398–1406. 2 Bidovec-Stojkovic U, Zolnir-Dovc M, Supply P. One year nationwide evaluation of 24-locus MIRU-VNTR genotyping on Slovenian Mycobacterium tuberculosis isolates. Respir Med 2011; 105 (Suppl 1): S67–S73. 3 Thierry D, Brisson-Noël A, Vincent-Lévy-Frébault V, et al. Characterization of a Mycobacterium tuberculosis insertion sequence, IS6110, and its application in diagnosis. J Clin Microbiol 1990; 28: 2668–2673. 4 Thierry D, Cave M D, Eisenach K D, et al. IS6110, an IS-like element of Mycobacterium tuberculosis complex. Nucleic Acids Res 1990; 18: 188. 5 Cowan L S, Mosher L, Diem L, Massey J P, Crawford J T. Variable-number tandem repeat typing of Mycobacterium tuberculosis isolates with low copy numbers of IS6110 by using mycobacterial interspersed repetitive units. J Clin Microbiol 2002; 40: 1592–1602. 6 Gutacker M M, Smoot J C, Migliaccio C A, et al. Genomewide analysis of synonymous single nucleotide polymorphisms in Mycobacterium tuberculosis complex organisms: resolution of genetic relationships among closely related microbial strains. Genetics 2002; 162: 1533–1543. 7 Rozo-Anaya J C, Ribón W. Molecular tools for Mycobacterium tuberculosis genotyping. Rev Salud Publica (Bogota) 2010; 12: 510–521.

LM-PCR differentiation of M. tuberculosis

8 Supply P, Mazars E, Lesjean S, Vincent V, Gicqeul B, Locht C. Variable human minisatellite-like regions in the Mycobacterium tuberculosis genome. Mol Microbiol 2000; 36: 762–771. 9 Allix-Beguec C, Harmsen D, Weniger T, Supply P, Niemann S. Evaluation and strategy for use of MIRU-VNTRplus, a multifunctional database for online analysis of genotyping data and phylogenetic identification of Mycobacterium tuberculosis complex isolates. J Clin Microbiol 2008; 46: 2692–2699. 10 Supply P, Lesjean S, Savine E, Kremer K, van Soolingen D, Locht C. Automated high-throughput genotyping for study of global epidemiology of M. tuberculosis based on mycobacterial interspersed repetitive units. J Clin Microbiol 2001; 39: 3563–3571. 11 Supply P, Allix C, Lejean S, et al. Proposal for standardization of optimized mycobacterial interspersed repetitive unitvariable-number tandem repeat typing of M. tuberculosis. J Clin Microbiol 2006; 44: 4498–4510. 12 Masny A, Płucienniczak A. Ligation-mediated PCR performed at low denaturation temperatures-PCR melting profiles. Nucl Acids Res 2003; 31: e114. 13 Krawczyk B, Samet A, Leibner J, Sledzinska A, Kur J. Evaluation of a PCR melting profile technique for bacterial strain differentiation. J Clin Microbiol 2006; 44: 2327–2332. 14 Krawczyk B, Leibner-Ciszak J, Stojowska K, Kur J. The new LM-PCR/shifter method for the genotyping of microorganisms based on the use of a class IIS restriction enzyme and ligation mediated PCR. J Microbiol Biotechnol 2011; 21: 1336– 1344. 15 de Wit D, Steyn L, Shoemaker S, Sogin M. Direct detection of Mycobacterium tuberculosis in clinical specimens by DNA amplification. J Clin Microbiol 1990; 28: 2437–2444. 16 Frothingham R H, Hills G, Wilson H. Extensive DNA sequence conservation throughout the Mycobacterium tuberculosis complex. J Clin Microbiol 1994; 32: 1639–1643.

309

17 Palittapongarnpim P, Chomyc S, Fanning A, Kunimoto D. DNA fingerprinting of Mycobacterium tuberculosis isolates by ligationmediated polymerase chain reaction. Nucleic Acids Res 1993; 21: 761–762. 18 Prod’hom G, Guilhot C, Gutierrez M C, Varnerot A, Gicquel B, Vincent V. Rapid discrimination of Mycobacterium tuberculosis complex strains by ligation-mediated PCR fingerprint analysis. J Clin Microbiol 1997; 35: 3331–3334. 19 Haas W H, Butler W R, Woodley C L, Crawford J T. Mixedlinker polymerase chain reaction: a new method for rapid fingerprinting of isolates of the Mycobacterium tuberculosis complex. J Clin Microbiol 1993; 31: 1293–1298. 20 Reisig F, Kremer K, Amthor B, et al. Fast ligation-mediated PCR, a fast and reliable method for IS6110-based typing of Mycobacterium tuberculosis complex. J Clin Microbiol 2005; 43: 5622–5627. 21 Krawczyk M, Brzostek A, Gorna A, et al. Epidemiological analysis of Mycobacterium tuberculosis strains isolated in Lodz, Poland. Int J Tuberc Lung Dis 2011; 15: 1252–1258. 22 Zaczek A, Brzostek A, Gorna A, Sajduda A, Dziadek J. Application of FLiP method for differentiation of Mycobacterium tuberculosis strains in comparison to commonly used methods, spoligotyping and MIRU-VNTR typing. Pol J Microbiol 2013; 62: 73–76. 23 Z˙aczek A, Ziółkiewicz M, Wojtasik A, Dziadek J, Sajduda A. IS6110-based differentiation of Mycobacterium tuberculosis strains. Pol J Microbiol 2013; 62: 201–204. 24 van Embden J D A, Cave M D, Crawford J T, Dale J W, Eisenach K D, Gicquel B. Strain identification of M. tuberculosis by DNA fingerprinting: recommendations for a standardized methodology. J Clin Microbiol 1993; 31: 406–409. 25 Hunter P R, Gaston M A. Numerical index of the discriminatory ability of typing systems: an application of Simpson’s index in diversity. J Clin Microbiol 1988; 26: 2465–2466.

LM-PCR differentiation of M. tuberculosis

i

RÉSUMÉ C O N T E X T E : Il y a un besoin de méthodes rapides et peu coûteuses d’analyse d’un nombre limité de souches de Mycobacterium tuberculosis. La réaction polymérase en chaine par ligation (LM-PCR) semble suffisamment discriminative et reproductible pour être considérée comme une technique moléculaire pour l’évaluation initiale des épidémies hospitalières, des contaminations croisées en laboratoire ou de la transmission dans les familles ou les petites communautés. O B J E C T I F : Elaborer une nouvelle LM-PCR basée sur l’amplification par PCR de la séquence flanquante 5′ de l’IS6110 qui consiste en digestion SalI/PvuII de l’ADN chromosomique en Sa/I/P, ligation du SalI et différenciation des fragments de restriction porteurs du IS6110 par l’hybridation suppressive soustractive. S C H É M A : La méthode FLAP a été appliquée à l’analyse de 62 isolats cliniques de M. tuberculosis et comparée

aux analyses IS6110-polymorphisme de la taille des fragments de restriction (RFLP) et analyse par la méthode des unités répétitives dispersées sur le génome mycobactérien– nombre variable de répétitions en tandem (MIRU-VNTR) des mêmes souches. R É S U LTAT S : La sensibilité de FLAP a été estimée à 0,25 ng/μl. Le FLAP a produit 32 profils parmi les 62 souches comparée à 28 et 36 profils obtenus précédemment avec MIRU-VNTR et IS6110-RFLP, respectivement. La valeur HGDI (0,973) était similaire à celle de MIRU-VNTR (0,966) et d’IS6110-RFLP (0,971). La spécificité des profils FLAP a également été confirmée. C O N C L U S I O N : FLAP s’est avéré une méthode très discriminante, sensible et spécifique et pourrait être un outil moléculaire précieux surtout pour analyser un nombre limité de souches de M. tuberculosis.

RESUMEN M A R C O D E R E F E R E N C I A : Se reconoce la necesidad creciente de métodos rápidos y de bajo costo de análisis de un número limitado de cepas de Mycobacterium tuberculosis. El método de reacción en cadena de la polimerasa mediada por ligación (LM-PCR) ofrece una capacidad discriminatoria y reproducibilidad suficientes para constituir una herramienta molecular en la evaluación inicial de los brotes epidémicos hospitalarios, las contaminaciones cruzadas en el laboratorio y los casos de transmisión comunitaria en pequeña escala. O B J E T I V O : Desarrollar un método de MD-PCR basada en la amplificación de la región adyacente a la secuencia de inserción IS6110 hacia 5′; el método consistió en digerir el ADN cromosómico con SalI y PvuII, realizar una ligación con un conector de ADN Sal I y practicar la diferenciación de las secuencias que contenían IS6110 que comportaban fragmentos de restricción mediante hibridación sustractiva. M É T O D O S : Se aplicó el método rápido de ligación y amplificación de los fragmentos de restricción (FLAP) al

análisis de 62 cepas clínicas de M. tuberculosis y se compararon con el análisis de la longitud de los fragmentos de restricción (RFLP) con el IS6110 y el análisis por genotipado con marcadores para locus múltiples de las secuencias repetitivas en tándem (MIRU-VNTR) de las mismas cepas. R E S U LTA D O S : La sensibilidad del método FLAP se calculó en 0,25 ng/μl. El FLAP aportó 32 perfiles en las 62 cepas de M. tuberculosis, en comparación con los 28 perfiles obtenidos previamente con el MIRU-VNTR y 36 con el RFLP-IS6110. El FLAP exhibió un índice de discriminación de Hunter-Gaston (0,973) equivalente al del MIRU-VNTR (0,966) y del RFLP-IS6110 (0,971). También se confirmó la especificidad de los perfiles del FLAP. C O N C L U S I Ó N : El método FLAP ofreció un alto poder discriminatorio y fue un método sensible y específico que puede constituir una valiosa herramienta molecular en el análisis de un número limitado de cepas de M. tuberculosis.

Development of a new ligation-mediated PCR method for the differentiation of Mycobacterium tuberculosis strains.

There is a need for rapid, inexpensive methods for analysing a limited number of Mycobacterium tuberculosis strains. The ligation-mediated polymerase ...
982KB Sizes 0 Downloads 4 Views