Heminested inverse PCR for IS6110 fingerprinting of Mycobacterium tuberculosis strains

0095-1137/96/$04.0010

Copyrightq1996, American Society for Microbiology

Heminested Inverse PCR for IS6110 Fingerprinting of

Mycobacterium tuberculosis Strains

SUSHIL PATEL,1_{SUSAN WALL,}2

ANDNICHOLAS A. SAUNDERS1*

Molecular Biology Unit, Virus Reference Division, Central Public Health Laboratory, London NW9 5HT,1

and Molecular Microbiology Group, School of Biological Sciences, University of Surrey,

Guildford, Surrey GU2 5XH,2_{United Kingdom}

Received 16 January 1996/Returned for modification 20 March 1996/Accepted 10 April 1996

A heminested inverse PCR (HIP) for the amplification of sequences flanking theMycobacterium tuberculosis

insertion sequence IS6110has been developed. The method depends upon primers that anneal to IS6110at sites between its 5*end and the closestBsrFI site. The accuracy of HIP was demonstrated by the amplification of sequences within plasmid constructs carrying one or two copies of the insertion sequence IS986in different orientations. The identities of the amplicons produced from strains carrying a single copy of IS6110were verified by nucleotide sequencing. Analyses of 204M. tuberculosisstrains including those involved in outbreaks showed that IS6110HIP is highly discriminatory and reproducible. HIP fingerprinting of these 204 strains generated 136 distinct types, and its discriminatory power was equivalent to that of standard restriction fragment length polymorphism analysis. The method is therefore of value for the rapid fingerprinting ofM. tuberculosisstrains for epidemiological purposes.

The resurgence of tuberculosis has stimulated the develop-ment of improved methods for the diagnosis and epidemiolog-ical tracing of Mycobacterium tuberculosis infections. These investigations are an essential aid to the understanding and control of the disease. They have benefited greatly from the discovery of repetitive DNA elements which enable strain-specific polymorphisms to be identified. The insertion se-quence (IS) IS6110 (17, 18), which is virtually identical to the more recently described IS986 (6, 10, 23), is the strain-specific marker of choice for the epidemiological typing of M.

tubercu-losis isolates (19). Southern blot analysis of PvuII restriction

fragment length polymorphisms (RFLPs) is used for analysis of the copy number and distribution of this IS. This typing method (19), which depends upon IS6110 having a high inter-strain variation in copy number and chromosomal location coupled to an appropriate rate of transposition (21), is highly effective and widely used.

The standard RFLP typing method is, however, laborious and requires the preparation of several micrograms of chro-mosomal DNA, a procedure which may take several weeks because of the slow growth rate of M. tuberculosis organisms. To avoid the necessity for prolonged culture, alternative PCR-based typing protocols have been developed (3, 4, 9, 12, 13, 15, 16). To date, three general approaches have been used. The first is to amplify the DNA separating adjacent copies of IS6110 or between IS6110 and another M. tuberculosis repeti-tive DNA sequence (3, 15, 16). The main drawback of this approach is that production of the PCR amplicons is depen-dent on the priming sites provided by the repeated elements being close enough for efficient PCR. In practice, few ampli-cons are produced, and this limits the level of discrimination achieved. The second approach is to use ligation-mediated PCR, in which a specific synthetic oligonucleotide priming site is ligated to a restriction site flanking IS6110 to allow PCR of

the sequence between the IS and the restriction site (4, 12). These relatively complex methods require careful optimiza-tion. Although the level of discrimination provided by ligation-mediated PCR strategies is satisfactory, it seems that to im-prove their reproducibilities, additional steps are required, and these extra manipulations increase the complexity of the pro-cedure still further. The third method, random amplified poly-morphic DNA analysis, also referred to as arbitrary primer PCR (9, 13), does not use repetitive elements or require se-quence data. Random amplified polymorphic DNA analysis simply depends on PCR amplification from random primer-binding sites under conditions of low stringency, but there are significant problems regarding its reproducibility and discrim-inatory ability (9, 13).

We report here a method for the analysis of IS6110 integra-tion sites based on a heminested inverse PCR (IS6110 HIP) (11). This technique results in the amplification of part of the IS6110 sequence together with its upstream flanking sequence. The size and number of PCR amplicons produced depend on the number of copies and sites of integration of IS6110. The method is technically simple and has a discriminatory power which is comparable to that of standard RFLP analysis.

MATERIALS AND METHODS

Bacterial strains and plasmids.Two hundred four isolates of M. tuberculosis submitted to the Central Public Health Laboratory for standard IS6110 typing (19) were tested. Strains were identified by standard biochemical methods in the source laboratories. The plasmid constructs pUS251, pUS252, pUS255, and pUS256 are based on a pGEM-3Zf1vector carrying one to two copies of IS986 as well as a kanamycin resistance cassette each cloned into the polylinker in the orientations shown in Fig. 1. Construction of a similar set of plasmids in pUC18 has been described previously (2).

DNA preparation.Total DNA was extracted from clinical M. tuberculosis strains as follows. A loopful of cells, grown on Lo¨wenstein-Jensen slopes, was harvested and heat killed at 808C for 20 min in 0.5 ml of sterile distilled water. Following lysozyme (5 mg/ml in TE buffer containing 50 mM Tris-HCl and 5 mM EDTA [pH 7.0]) digestion at 378C for 30 min, the cells were lysed by treatment with TE buffer containing 100mg of proteinase K per ml and 1% sodium dodecyl sulfate (SDS) at 658C for 1 h. The SDS concentration was raised to 4%, the temperature was increased to 758C, and the lysis mixture was left for a further 30 min. The cell lysate was purified by phenol-chloroform and ethanol precipitation and was then resuspended in 10 mM Tris-HCl–1 mM EDTA (pH 7.0) buffer.

Preparation of PCR template.Aliquots of genomic or plasmid DNA (0.1mg) were digested with 2 U of BsrFI (New England Biolabs, Hitchin, United

King-* Corresponding author. Mailing address: Molecular Biology Unit, Virus Reference Division, Central Public Health Laboratory, London NW9 5HT, United Kingdom. Phone: 0181 200 4400, extension 3072. Fax: 0181 200 1569. Electronic mail address: [email protected] .ac.uk.

1686

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dom) for 2 h at 378C. The restriction endonuclease was inactivated by treatment with diethyl pyrocarbonate (0.1% [vol/vol]) at 378C for 20 min, and the digest was then neutralized by the addition of 0.06 volume of 1 M Tris-HCl (pH 8.0). The digested DNA was incubated at 508C for 5 min to dissociate spontaneously reannealed restriction fragments, chilled on ice, and circularized at a concentra-tion of 0.5mg/ml in 20ml of ligation buffer (50 mM Tris-HCl [pH 7.6], 10 mM MgCl2, 1 mM dithiothreitol, 5% [wt/vol] polyethylene glycol 8000). T4 DNA

ligase (20 U/ml; New England Biolabs) was added, and the mixture was incu-bated at 168C for 16 h.

Standard heminested PCR amplification.The first round of amplification was performed in a volume of 50ml, overlaid with mineral oil, by using a thermocy-cler (OmniGene; Hybaid, Teddington, United Kingdom). The PCR mixture contained 1ml (0.5 ng) of the circularized DNA, 10 mM Tris-HCl (pH 8.8; at 258C), 50 mM KCl, 1.5 mM MgCl2, 0.1% Triton X-100, 200mM (each)

de-oxynucleoside triphosphates (Boehringer Mannheim, Mannheim, Germany), 0.15mM (each) primers PA1 (59-TGAACCACCTGACATGACCC-39) and PB (59-GTCTACTTGGTGTTGGCTGC-39), and 1 U of Taq DNA polymerase (Gibco BRL Life Technologies Inc., Gaithersburg, Md.). A hot-start protocol was used; this was followed by 1 cycle at 948C for 3 min and then 15 cycles at 948C for 1 min, 628C for 1 min, and 728C for 2 min, with a final extension cycle at 728C for 3 min. Second-round heminested amplification was carried out as described for the first round, except that 1ml of the first-round PCR product and 0.6mM (each) primers PA2 (59-GACATGCCGGGGCGGTTC-39) and PB were used. The PCR mixture was subjected to an initial denaturation cycle at 948C for 3 min and then 25 cycles at 948C for 1 min, 608C for 1 min, and 728C for 2 min, with a final extension cycle at 728C for 3 min.

Rapid heminested capillary PCR amplification.The time taken for hemin-ested PCR was considerably reduced by the use of a capillary PCR machine (Rapidcycler; Idaho Technology, Idaho Falls, Idaho). Amplification was carried

out in a volume of 10ml containing 0.1 ng of ligated DNA, 50 mM Tris-HCl (pH 8.3), 3 mM MgCl2, 500mg of bovine serum albumin per ml, 200mM (each)

deoxynucleoside triphosphates, 0.15mM (each) primers PA1 and PB, and 0.4 U of Taq DNA polymerase. First-round amplification comprised 1 cycle at 948C for 45 s and then 25 cycles at 948C for 0 s, 628C for 0 s, and 728C for 30 s, with a final extension step at 728C for 2 min. Denaturation and annealing times of “0” s of hold are sufficient for rapid-cycle DNA amplification (22). The heminested amplification used conditions identical to those used in the first round of ampli-fication, except that 0.5ml of the first-round product was used as the template, 0.6mM (each) primers PA2 and PB was added, and the annealing temperature was reduced to 608C. The PCR products were visualized by ethidium bromide staining after electrophoresis in a 2% Metaphor agarose gel (FMC BioProducts, Rockland, Maine).

The nucleotide bases of IS6110 were numbered according to the published sequence (GenBank accession number M29899) (18). The relative positions of the inverse PCR primers in IS6110 are depicted in Fig. 2. For the nested primer set PA1 and PA2, the sequence given corresponds to the complement of the published sequence.

RESULTS

The strategy for HIP analysis requires DNA to be digested to completion with BsrFI and the restriction fragments pro-duced to be ligated at low concentrations, which favors in-tramolecular bonding. Of the resulting circularized restriction fragments, only those containing the 59 end of IS6110 should act as a substrate for priming during inverse PCR.

A number of primer sets-restriction endonuclease combina-tions were evaluated in initial experiments which suggested that primer set location and the choice of restriction enzyme were important for successful fingerprinting. For example, a primer set designed for amplification of sequence downstream (39) from IS6110 distinguished epidemiologically related from unrelated strains, but several of the bands produced were not attributable to IS6110-flanking sequences, and these nonspe-cific bands were not reproducible. However, a primer set that was based on the left (59) end of IS6110 and that gave ampli-fication upstream from the IS, coupled to digestion with the restriction enzyme BsrFI, produced reproducible and discrim-inatory fingerprints in which nonspecific products were of min-imal intensity. BsrFI was found to be particularly suitable be-cause it produces fragments in the ideal size range for PCR with relatively stable cohesive ends that facilitate ligation.

Heminested PCR amplification with a single nested primer (i.e., primer PA2) in the second round of amplification pro-duced patterns similar to those propro-duced in a single-step 35-cycle PCR. However, the patterns obtained by HIP were gen-erally clearer, with a lower yield of high-molecular-weight material together with an enhanced quantity of the desired amplicons (Fig. 3). The use of a Rapidcycler machine (Idaho Technology) considerably reduced the time required to carry out heminested PCR, allowing completion of two sets of am-plification in approximately 1 h. The use of such a machine is not essential, however, and indistinguishable fingerprint

pro-FIG. 1. Maps of plasmid constructs pUS251, pUS252, pUS255, and pUS256. The sizes of the expected PCR products are indicated. Within IS6110, only relevant BsrFI sites are marked.

FIG. 2. Schematic representation of IS6110 showing the positions of the primers PA1, PA2, and PB used in HIP. The arrows indicate the direction of priming on the sequence. The location of the BsrFI restriction enzyme site proximate to the 59end of IS6110 is also indicated.

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files were obtained with a standard thermocycler (data not shown).

In order to demonstrate that the PCR products represented 59 IS6110-flanking sequences, plasmids of known sequence were analyzed. The plasmids pUS251, pUS252, pUS255, and pUS256 carry either one or two copies of IS986 (Fig. 1). The numbers and sizes of the products generated from these tem-plates by IS6110 HIP (Fig. 4) were exactly as predicted (Fig. 1). To determine the ability of the technique to reliably amplify DNA flanking chromosomal copies of IS6110, M. tuberculosis strains known to carry a single copy of this IS by the standard RFLP method (19) were analyzed. Within such strains IS6110 is often located in a specific chromosomal location that is an integration hot spot (5). This region is characterized by mul-tiple 36-bp direct repeats (DRs) interspersed with unique spac-ers of 35 to 41 bp in length (5). The location of the single IS6110 within the DR region in these strains was verified by Southern blotting with an oligo-DR probe (20). Sequence analysis of the single HIP amplicons produced by these strains also re-vealed the presence of 36-bp DRs in each case (data not shown). The ability of HIP to distinguish clinical isolates of M.

tu-berculosis was demonstrated by analysis of a series of 204

strains. Of the 204 isolates which had been analyzed by stan-dard IS6110-RFLP and shown to consist of 137 distinct multi-band types (types defined by only one or two multi-bands are gen-erally treated separately in IS6110 typing studies, since the discriminatory power of the method is known to be lower for these strains), HIP fingerprinting discriminated between 136 types. In the remaining instance two apparently unrelated

iso-lates gave indistinguishable HIP patterns. Figure 5 shows the HIP fingerprints of 15 M. tuberculosis strains that had previ-ously been typed by standard RFLP analysis. The 15 strains analyzed represented 14 distinct IS6110-RFLP types (unpub-lished data), but 15 distinct HIP fingerprints were generated. Two strains, in lanes 15 and 16, produced a single band of the same molecular size by IS6110-RFLP analysis. HIP also pro-duced a single band for each of these strains, but the bands were of a different molecular size. RFLP analysis generated patterns with 1 to 16 bands for these strains, whereas HIP produced between 1 and 11 bands. The strains in lanes 6, 8, 9, 10, and 17 produced apparently related fingerprints that in-cluded a doublet of approximately 550 bp and several other conserved bands, and IS6110 RFLP analysis of these strains (which were all from patients in the same hospital) also showed an unusually high proportion of common bands, particularly those with a larger molecular size. The significance of this finding remains unclear, but could have indicated the predom-inance of several closely related strains in the sample area.

The HIP technique was also used to examine 13 isolates from known case clusters (clusters A to D) (Fig. 6). Standard RFLP analysis of these strains had shown indistinguishable banding patterns for strains within cluster A (lanes 2 and 3) and cluster B (lanes 4, 5, 6, 7, and 9). Within clusters C (strains represented in lanes 10 to 12) and D (strains in lanes 13 to 15), two different patterns were discernible by the standard IS6110 typing method. One strain in each of these clusters (for cluster C, lane 11; for cluster D, lane 15) differed from the others by the presence or absence of a single band. HIP fingerprinting produced indistinguishable patterns for strains within clusters

FIG. 3. Comparison of standard and heminested inverse capillary PCR. Lanes 1 to 5, HIP fingerprints generated with primers PA1 and PB in the first round of amplification and then primers PA2 and PB in the second round; the fingerprints of four clinical isolates of M. tuberculosis are shown; lanes 3 and 4, separate preparations from the same isolate; lane 6, molecular size standards (fX174 replicative-form DNA-HaeIII fragments; Gibco BRL Life Technologies Inc.); lanes 7 to 11, standard inverse PCR with primers PA1 and PB in a single round of amplification. The fingerprints of the same four M. tuberculosis isolates including the repeat isolate (lanes 9 and 10) are shown.

FIG. 4. HIP products generated following BsrFI digestion and ligation of plasmid DNA. Lane 1,lPstI molecular size markers; lanes 2 to 5, products from

plasmid constructs pUS251, pUS252, pUS255, and pUS256, respectively.

FIG. 5. HIP analysis of 15 isolates of M. tuberculosis. Lanes 2 to 6, 8 to 12, and 14 to 18, clinical isolates; lanes 1, 7, and 13, DNA molecular size standards (fX174 replicative-form DNA-HaeIII fragments).

FIG. 6. HIP analysis of 13 isolates from known case clusters. Lanes 1 and 8, molecular size markers (fX174 replicative-form DNA-HaeIII fragments); lanes 2 and 3, strains isolated from an outbreak in a family (cluster A); lanes 4 to 7 and 9, strains isolated during an outbreak in a renal ward (cluster B); lanes 10 to 12, strains isolated from an outbreak in a family (cluster C); lanes 13 to 15, strains isolated from a suspected outbreak in a hospital (cluster D).

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A, B, and C. For the divergent isolate in cluster D (lane 15), HIP also produced an extra band compared with the numbers of bands in the other isolates in the cluster.

The reproducibility and stability of the HIP fingerprints over time were demonstrated by analysis of a set of three isolates collected from the same patient over a period of 18 months. These gave indistinguishable fingerprints (Fig. 6, lanes 4, 7, and 9).

The sensitivity of the HIP method was assessed by analysis of a serial dilution of template DNA (Fig. 7). HIP was able to generate a fingerprint pattern from as little as 100 fg of chro-mosomal DNA. At DNA concentrations of less than 100 fg, nonspecific bands were observed, with the concomitant dimi-nution of the IS-flanking amplicons.

Although the HIP fingerprints obtained were reproducible and discriminatory, a progressive reduction in the clarity of the banding patterns with increasing IS6110 copy number was ob-served (Fig. 5; cf. lanes 14 and 15). Altering the concentration of DNA, the Taq polymerase used, and the thermocycler con-ditions did not significantly reduce this problem. The smearing observed was suspected to be due to single-stranded DNA, and this was verified by post-PCR digestion with S1 nuclease (Fig. 8). S1 nuclease treatment reduced the smearing considerably without affecting the PCR amplicons, but it produced an ad-ditional small-molecular-size band equivalent to the section of

IS6110 amplified during HIP. The use of the Stoffel fragment (Perkin-Elmer) (a modified Taq DNA polymerase lacking 59-39 exonuclease activity) for HIP resulted in significantly cleaner fingerprints, but it only produced fragments with a molecular size of,500 bp (data not shown).

DISCUSSION

The results of the present study demonstrate that sequences flanking IS6110 (IS986) in plasmid constructs can be amplified reliably. Similarly, sequences flanking chromosomally located copies of this insertion element are also amplifiable. This rapid, novel fingerprinting method was highly discriminatory for a variety of M. tuberculosis strains. The results for epide-miologically related strains, which gave indistinguishable pat-terns for strains shown to be indistinguishable by standard IS6110-RFLP typing, illustrate the potential of the method for identifying and monitoring outbreaks. Single band changes exhibited by a strain during an outbreak are not uncommon by standard IS6110 analysis, and HIP can also detect such changes, which are attributable to single replicative transposi-tion or deletransposi-tion events (1, 20). The rapid identificatransposi-tion of clusters of infection is important because this allows effective disease control measures to be implemented. Furthermore, during an outbreak in which the drug resistance profile of a strain has been determined, it may be possible to infer the resistance of isolates shown to be related to it by HIP finger-printing.

Although in theory an HIP amplicon can be generated from each copy of IS6110, in practice the number of bands produced by HIP is usually less than the number of copies of IS6110. There are two reasons for this. First, a proportion of the flanking BsrFI restriction enzyme sites may be located too far from IS6110 for efficient PCR, although this factor was mini-mized by choosing an enzyme that generally produces frag-ments of a size suitable for amplification. Second, the bands generated from different copies of IS6110 may be of similar molecular size and thus comigrate. Although fewer bands are produced by HIP compared with the number produced by the standard RFLP method, analysis of a large number of strains has shown the discriminatory ability of the PCR method to be similar to that of standard RFLP analysis. For strains carrying a single copy of IS6110, HIP may be more discriminatory than standard RFLP analysis; for example, two strains with single copies of IS6110 that were indistinguishable by RFLP analysis could be discriminated by HIP (Fig. 5, lanes 15 and 16).

S1 nuclease treatment of the crude PCR product eliminated most of the diffusely staining (ethidium bromide) background material on the gels which was observed for strains with high copy numbers of IS6110 (data not shown). This material may therefore be presumed to mainly comprise single-stranded DNA. S1 nuclease treatment also resulted in the appearance of a band of approximately 60 bp. This double-stranded DNA residue is of the size expected for the ends of the inverse PCR amplicons corresponding to IS6110. It can be deduced from these findings that the single-stranded material likely com-prises mismatched amplicons held together by complementary IS6110-derived ends. Such heteroduplexes are likely to be formed by simple hybridization during the final annealing step of the PCR, which is driven by the high amplicon concentra-tion. In addition, small quantities of hairpin molecules are formed during previous annealing steps. These intermediate molecules are paired at one end but are mismatched in the middle (being products of different flanking sequences), and they are prevented from hybridizing at the distal end because of the annealing of the primer. When extension from the

FIG. 7. Sensitivity of HIP. Lane 1, molecular size markers (fX174 replica-tive-form DNA-HaeIII fragments); dilutions of purified M. tuberculosis DNA were subjected to PCR; lanes 2 to 9, PCR products from serial (10-fold) dilutions of 0.1 ng to 0.1 fg of purified M. tuberculosis DNA.

FIG. 8. S1 nuclease treatment of HIP product. Lane 1, untreated HIP prod-uct from an M. tuberculosis isolate; lane 2, HIP prodprod-uct of isolate (lane 1) treated with 20 U of S1 nuclease (Boehringer Mannheim) for 15 min; lane 3, HIP product of isolate (lane 1) treated with 20 U of S1 nuclease for 30 min; lane 4, HIP product of isolate (lane 1) treated with 5 U of S1 nuclease for 30 min. The arrow indicates the position of an extra band (of approximately 60 bp) that can be seen following S1 nuclease treatment of the HIP products (lanes 2 to 4).

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primer reaches the hairpin fork, the mismatched strand is cleaved because of the 59-39 exonuclease activity of the Taq polymerase (7). In this way single-stranded amplicons lacking a priming site may accumulate in the reaction mixture. Evidence that this process was involved in the production of the single-stranded debris was obtained by using the Stoffel fragment (Perkin-Elmer), of Taq polymerase which lacks 59-39 exonucle-ase activity. In reactions with the Stoffel fragment, the propor-tion of single-stranded material was greatly reduced (data not shown). However, PCRs with the Stoffel fragment only pro-duced amplicons of up to 500 bp because of the lower proces-sivity of this enzyme (8). Consequently, Stoffel fragment-de-rived fingerprints were less discriminatory than those generated with conventional Taq polymerase.

The HIP method is technically simple and reproducible and requires considerably less DNA than the standard RFLP typ-ing method. It is most comparable to ligation-mediated PCR (4, 12), which also uses PCR to amplify sequences flanking IS6110. The main advantage of HIP over ligation-mediated PCR is that an intermolecular ligation is not involved. Small-scale ligations designed to yield predictable intermolecular as-sociations require careful optimization. In contrast, intramo-lecular ligation requires only that the input DNA is sufficiently dilute. Additional advantages of inverse PCR over ligation-mediated PCR are that the specificity of amplification is con-ferred by both primers and that unused linkers cannot interfere in the reaction (4) since they are not required.

The sensitivity of HIP avoids the requirement for prolonged mycobacterial growth, since as little as 100 fg of DNA (equiv-alent to 30 genomes of strain H37Rv [2.53109_{Da]) is}

suffi-cient to produce a fingerprint. Furthermore, the ligation con-ditions used require only very small quantities of DNA. Thus, HIP may be applicable to fingerprinting directly from clinical material such as sputa (if enough organisms are present), avoiding the need for culture. A heminested amplification pro-cedure was used because nested amplifications tend to have greater specificity and are usually more sensitive (14), an im-portant consideration if the method were ultimately to be applied to clinical material. Since the banding patterns pro-duced become unreproducible at very low DNA concentra-tions, it would be necessary to perform replicate analyses on samples derived directly from clinical specimens. Samples yielding nonmatching banding patterns from replicates could then be discounted.

Currently, the rate-limiting step in the procedure is the ex-traction and purification of M. tuberculosis DNA. In theory, because the majority of the HIP amplicons produced are smaller than 1 kb, the method should tolerate more vigorous extraction procedures than those used with the standard RFLP method, which requires a large quantity of high-molecular-weight DNA. However, the requirement of HIP for double-stranded DNA suitable for digestion limits the choice of ex-traction methods available.

By its nature, the IS6110 HIP results in amplification of IS6110-flanking sequences. Hence, direct sequencing of differ-ent HIP amplicons should provide a rapid alternative to clon-ing in studies of IS6110 transposition. Certain hot spots of IS6110 integration have already been described (5), and HIP appears to be an ideal strategy for determining whether other such sites exist.

ACKNOWLEDGMENT

We thank J. Dale for critical reading of the manuscript.

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