![[Feedback]](/icons/banner/feedbackACT.gif)
![[For Subscribers]](/icons/banner/subscriberACT.gif)
![[Archive]](/icons/banner/archiveACT.gif)
![[Search]](/icons/banner/searchACT.gif)
![[Contents]](/icons/banner/tocACT.gif)
Previous Article | Next Article 
Journal of Clinical Microbiology, April 1999, p. 1224-1226, Vol. 37, No. 4
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Specificity of IS6110-Based DNA
Fingerprinting and Diagnostic Techniques for Mycobacterium
tuberculosis Complex
Willie A.
Githui,1,2,3,*
Stuart M.
Wilson,1 and
Francis A.
Drobniewski1
PHLS Mycobacterium Reference Unit, Dulwich
Public Health Laboratory and Department of Microbiology, Kings
College School of Medicine and Dentistry, Dulwich
Hospital,1 and Department of
Infectious and Tropical Diseases, London School of Hygiene and
Tropical Medicine,2 London, United Kingdom;
and Centre for Respiratory Diseases Research, Kenya Medical
Research Institute, Nairobi, Kenya3
Received 4 September 1998/Returned for modification 3 November
1998/Accepted 1 January 1999
 |
ABSTRACT |
Restriction fragment length polymorphism and hybridization of DNA
extracted from Mycobacterium tuberculosis, nontuberculous mycobacteria, and nonmycobacterial species with a probe derived from
IS6110 confirmed that IS6110 was specific to
M. tuberculosis complex. In addition, DNA amplification
with IS6110-specific primers yielded a 181-bp fragment only
in DNA from M. tuberculosis complex isolates.
| |
TEXT |
The discovery of polymorphic DNA
sequences in Mycobacterium tuberculosis and the subsequent
use of methods based on the insertion sequence IS6110 both
for amplification and for typing of isolates of M. tuberculosis complex have facilitated early diagnosis of tuberculosis and differentiation of strains, respectively.
However, the specificity of these methods for M. tuberculosis has recently been questioned.
Kent et al. (13) reported that strains of
nontuberculous mycobacteria (NTM) were positive by PCR
amplification of a sequence homologous to a central 181-bp fragment of
IS6110. In a subsequent study, Gillespie and colleagues
(7) emphasized that the observed homology was demonstrated
by Southern blotting. They also described the presence in PCR products
of nonmycobacterial species of discrete bands that hybridized with a
probe derived from the 181-bp fragment. They suggested that the
central sequence found to be homologous in the NTM isolates overlaps
that of the internationally recognized probe, IS6110
(19), for the standard restriction fragment length polymorphism (RFLP) method. In a subsequent study by Hellyer et al. (9) no DNA amplification of any of the 27 NTM isolates analyzed, 26 of which had been used in the study by Kent et al. (13), was observed. Similar results were obtained by Mulcahy et al. (15).
McHugh et al. (14) described the presence of
IS6110 elements in multiple copies in 14 strains of NTM that
hybridized with a probe derived from the 181-bp fragment of
IS6110 at 50°C. They did not, however, clarify the exact
conditions under which hybridization was performed, including details
of the hybridizing solution as well as the time taken for both
hybridization and posthybridization washes. For instance, protocols
incorporating formamide use hybridization temperatures of 42°C
(8), while those that do not use higher hybridization
temperatures of 60 to 68°C (1, 12). In both techniques,
appropriate posthybridization stringency washes at temperatures ranging
between 65 and 68°C are usually used. In this study, we investigated
the specificity of IS6110-based assays for identification
and typing of M. tuberculosis complex.
The M. tuberculosis reference strain Mt. 14323, clinical isolates of NTM (M. avium complex,
M. xenopi, M. kansasii, M. gordonae, M. fortuitum, M. chelonae, M. malmoense, and M. marinum), and nonmycobacterial species
(Nocardia sp. and Escherichia coli NCTC 10418) were analyzed. Genomic DNA was extracted by using procedures described previously (20). The concentration of the
extracted DNA was determined by measuring absorbance at 260 nm. DNA
from a Mycobacterium bovis BCG strain (Glaxo, Greenford,
United Kingdom) was included to serve as a positive control for PCR
amplification. Twenty-five nanograms of each DNA sample was amplified
in a two-tube nested protocol with IS6110 specific primers;
the outer primers were Tb 294 (5'-GGACAACGCCGAATTGCGAAGGGC-3') and Tb 850 (5'-TAGGCGTCGGTGACAAAGGCCACG-3'), and the inner primers were
Tb 505 (5'-ACGACCACATCAACC-3') and Tb 670 (5'-AGTTTGGTCATCAGCC-3') (13, 14, 21). All
primers were used at concentration of 0.5 pmol/µl. Using the outer
primers, reactions were amplified for 30 cycles (denaturation at 93°C
for 20 s, annealing at 65°C for 30 s, and extension at
72°C for 1 min). One microliter of the product of the reaction with
the outer primers was amplified by the inner primers for 30 cycles
(denaturation at 93°C for 20 s, annealing at 48°C for 30 s, and extension at 72°C for 30 s). PCR products were analyzed
by agarose gel electrophoresis.
One microgram of DNA from each isolate was digested in duplicate with
PvuII. One duplicate was hybridized with the
INS1/INS2-derived IS6110 probe (19). The other
duplicate was hybridized with a digoxigenin-labeled 312-bp 16S RNA
fragment of M. tuberculosis H37Rv (nucleotides
248 to 560) (2) which is reported to be conserved in
all prokaryotes (5, 18). Hybridization was carried out at
42°C in a solution containing 50% formamide, 6× SSC (1× SSC is
0.15 M NaCl plus 0.015 M sodium citrate), 0.1% (wt/vol) N-lauroylsarcosine, 0.02% (wt/vol) sodium dodecyl sulfate,
and 5% (wt/vol) skimmed milk powder, followed by two posthybridization washes of 2× SSC, 0.1% (wt/vol) sodium dodecyl sulfate, and 0.02% (wt/vol) N-lauroylsarcosine at 65% for 15 min each. The
digoxigenin-labeled DNA probe was detected with alkaline
phosphatase-conjugated anti-digoxigenin antibody (Boehringer, Mannheim, Germany).
Hybridization with the IS6110 probe was seen with DNA from
M. tuberculosis but not with DNA from NTM or
nonmycobacterial species (Fig. 1). Even
loading of DNA in all lanes was confirmed visually by ethidium bromide
staining and from the comparable hybridization signals from all lanes
when hybridization was performed on the duplicate membrane with the
conserved 16S rRNA gene fragment (data not shown). This indicated that
appropriate amounts of DNA were used for IS6110
hybridization analysis.
View larger version (80K):
[in this window]
[in a new window]
|
FIG. 1.
Isolates of mycobacterial and nonmycobacterial species
hybridized with the international INS1/INS2 IS6110 probe.
Lane(s): M, Lambda HindIII molecular weight (size)
marker; 1 and 12, M. tuberculosis Mt. 14323; 2, M. avium complex; 3, M. xenopi; 4, M. kansasii; 5, M. gordonae; 6, M. fortuitum; 7, M. chelonae; 8, M. malmoense; 9, Nocardia sp.; 10, M. marinum; and 11, E. coli NCTC 10418.
|
|
Amplified PCR products of the expected size (181 bp) were seen in DNA
extracted from M. tuberculosis complex isolates but not
in DNA from NTM or from nonmycobacterial isolates (Fig.
2). Some amplified products were seen in
M. avium complex, M. xenopi and
E. coli isolates, but these were of the wrong size. Low
levels of nonspecific priming can often cause the generation of
nonspecific products in a sensitive nested PCR format such as this
(10, 21). All seven negative controls were negative for
amplified products.
View larger version (59K):
[in this window]
[in a new window]
|
FIG. 2.
Amplified products of various mycobacterial and
nonmycobacterial species obtained by using IS6110 two-tube
nested PCR. Lane(s): M, 100-bp ladder (Promega, Madison, Wis.); 1, M. avium complex; 2, M. xenopi; 3, M. kansasii; 4, M. gordonae; 5, M. fortuitum; 6, M. chelonae; 7, M. malmoense; 8, Nocardia sp.; 9, M. marinum; 10, E. coli NCTC 10418; 12 and
13, M. tuberculosis Mt. 14323; 15 through 21, negative
controls; 23 and 24, BCG strain (Glaxo) positive controls
(amplification of 0.05 and 0.5 pg of BCG DNA, respectively); 11, 14 and
22, empty. Arrows indicate the position of a 181-bp IS6110
fragment.
|
|
The results obtained in this study support the recent observations by
Hellyer et al. (11). Although some homology between members
of the IS3 family of insertion sequences exists, this should
not cause a problem in PCR or RFLP assays performed under appropriate
conditions. RFLP requires carefully controlled hybridization conditions
and high-stringency washes to minimize low-homology binding. In the
study by McHugh et al. (14) bacteriophage lambda molecular
weight markers had undefined bands on their Southern blots,
demonstrating binding which presumably was due to nonspecific hybridization. Concurrently analyzed M. tuberculosis
controls which might help to differentiate nonspecific binding are not shown.
Although Gillespie et al. (7) dismissed the likelihood of
PCR contamination, it remains possible that their product became contaminated during the DNA extraction procedure. The apparent `reduced specificity' shown in some studies (3, 6, 16, 17)
could be explained by contamination and carryover of PCR products in
samples. Moreover, in a French study (4), cited by McHugh et
al. (14), in which the false PCR-positive rate was
7%, the authors did not attribute this to DNA from
NTM. Cross contamination with M. tuberculosis DNA or amplicons was an equally likely explanation.
Results obtained in this study confirm that the IS6110-based
assays used both for DNA fingerprinting of M. tuberculosis complex and for diagnosis of tuberculosis disease are
specific. We highlight the importance of maintaining appropriate
stringency during hybridization and washing when performing RFLP assays
and of excluding contamination in PCR analysis.
| |
ACKNOWLEDGMENTS |
This work was supported by a grant from The Commonwealth
Scholarship Commission in the United Kingdom, ACU, and in part by KEMRI, LSHTM, PHLS & Wellcome Trust.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: PHLS
Mycobacterium Reference Unit, Dulwich Public Health Laboratory and
Department of Microbiology, Kings College School of Medicine and
Dentistry, Kings College Hospital (Dulwich), East Dulwich Grove, London
SE22 8QF, United Kingdom. Phone: 44 (0) 181-693-2830. Fax: 44 (0)
171-346-6477. E-mail: w.githui{at}lshtm.ac.uk.
| |
REFERENCES |
| 1.
|
Chaves, F.,
Z. Yang,
H. El-Haji,
M. Alonso,
W. J. Burman,
K. D. Eisenach,
F. Droda,
J. H. Bates, and M. D. Cave.
1996.
Usefulness of the secondary probe pTBN12 in DNA fingerprinting of Mycobacterium tuberculosis.
J. Clin. Microbiol.
34:1118-1123[Abstract].
|
| 2.
|
Cole, S. T.,
R. Brosch,
J. Parkhill,
T. Garnier,
C. Churcher,
D. Harris,
S. V. Gordon,
K. Eiglmeier,
S. Gas,
C. E. Barry III,
F. Tekeia,
K. Badcock,
D. Bashma,
D. Brown,
T. Chillingworth,
R. Connor,
R. Davies,
K. Devlin,
T. Feltwell,
S. Gentles,
N. Hamlin,
S. Holroyd,
T. Hornsby,
K. Jagels,
A. Krogh,
J. McLean,
S. Moule,
L. Murphy,
S. Oliver,
J. Osborne,
M. A. Quail,
M. A. Rajandream,
J. Rogers,
S. Rutter,
K. Seeger,
S. Skeleton,
S. Squares,
R. Squares,
J. E. Sulston,
K. Taylor,
S. Whitehead, and B. G. Barrell.
1998.
Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence.
Nature
393:537-544[Medline].
|
| 3.
|
Dalovisia, J. R.,
S. Montenegro-James,
S. A. Kemmerly,
C. F. Genre,
R. Chambers,
D. Greer,
G. A. Pankey,
D. M. Failla,
K. G. Haydel,
L. Hutchinson,
M. F. Lindley,
B. M. Nunez,
A. Praba,
K. D. Eisenach, and E. S. Cooper.
1996.
Comparison of the amplified Mycobacterium tuberculosis (MTB) Direct Test, Amplicor MTB PCR, and IS6110-PCR for detection of MTB in respiratory specimens.
Clin. Infect. Dis.
23:1099-1106[Medline].
|
| 4.
|
Doucet-Populaire, F.,
V. Lalande,
E. Carpentier,
A. Bourgoin,
M. Dailloux,
C. Bollet,
A. Vachee,
D. Moinard,
J. Texier-Maugein,
B. Carbonelle, and J. Grosset.
1996.
A blind study of the polymerase chain reaction for the detection of Mycobacterium tuberculosis DNA.
Tubercle Lung Dis.
77:358-362[Medline].
|
| 5.
|
Edwards, U.,
T. Rogall,
H. Blocker,
M. Emde, and E. C. Bottger.
1989.
Isolation and direct complete nucleotide determination of entire genes. Characterization of a gene coding for 16S ribosomal RNA.
Nucleic Acids Res.
17:7843-7853[Abstract/Free Full Text].
|
| 6.
|
Eisenach, K. D.,
M. D. Sifford,
M. D. Cave,
J. H. Bates, and J. T. Crawford.
1991.
Detection of Mycobacterium tuberculosis in sputum samples using a polymerase chain reaction.
Am. Rev. Respir. Dis.
144:1160-1163[Medline].
|
| 7.
|
Gillespie, S. H.,
T. M. McHugh, and L. E. Newport.
1997.
Specificity of IS6110-based amplification assays for Mycobacterium tuberculosis complex.
J. Clin. Microbiol.
35:799-801[Medline].
|
| 8.
|
Hang, Z. H.,
B. C. Ross, and B. Dwyer.
1991.
Identification of Mycobacterium kansasii by DNA hybridization.
J. Clin. Microbiol.
29:2125-2129[Abstract/Free Full Text].
|
| 9.
|
Hellyer, T. J.,
L. E. DesJardin,
M. K. Assaf,
J. H. Bates,
M. D. Cave, and K. D. Eisenach.
1996.
Specificity of IS6110-based amplification assays for Mycobacterium tuberculosis complex.
J. Clin. Microbiol.
34:2843-2846[Abstract].
|
| 10.
|
Hellyer, T. J.,
L. E. DesJardin,
M. K. Assaf, and K. D. Eisenach.
1997.
Authors' reply.
J. Clin. Microbiol.
35:800-801.
|
| 11.
|
Hellyer, T. J.,
L. E. DesJardin,
M. L. Beggs,
Z. Yang,
K. D. Eisenach,
M. D. Cave,
J. H. Bates,
M. K. Assaf, and J. T. Crawford.
1998.
IS6110 homologs are present in multiple copies in mycobacteria other than tuberculosis-causing mycobacteria.
J. Clin. Microbiol.
36:853-854[Free Full Text]. (Letter to the editor.)
|
| 12.
|
Hermans, P. W. M.,
D. van Soolingen,
J. W. Dale,
A. R. J. Schuitema,
R. A. McAdam,
D. Catty, and J. D. A. van Embden.
1990.
Insertion element IS986 from Mycobacterium tuberculosis: useful tool for diagnosis and epidemiology.
J. Clin. Microbiol.
28:2051-2058[Abstract/Free Full Text].
|
| 13.
|
Kent, L.,
T. M. McHugh,
O. Billington,
J. W. Dale, and S. H. Gillespie.
1995.
Demonstration of homology between IS6110 of Mycobacterium tuberculosis and DNAs of other Mycobacterium spp.
J. Clin. Microbiol.
33:2290-2293[Abstract].
|
| 14.
|
McHugh, T. D.,
L. E. Newport, and S. H. Gillespie.
1997.
IS6110 homologs are present in multiple copies in mycobacteria other than tuberculosis-causing mycobacteria.
J. Clin. Microbiol.
35:1769-1771[Abstract].
|
| 15.
|
Mulcahy, G. M.,
Z. C. Kaminski,
E. A. Albanese,
R. Sood, and M. Pierce.
1996.
IS6110-based PCR methods for detection of Mycobacterium tuberculosis.
J. Clin. Microbiol.
34:1348-1349[Medline].
|
| 16.
|
Noordhoek, G. T.,
A. J. Kolk,
G. Bjune,
D. Catty,
J. W. Dale,
P. E. M. Fine,
P. Godfrey-Faussett,
S. N. Cho,
T. M. Shinnick,
S. B. Svenson,
S. Wilson, and J. D. A. van Embden.
1994.
Sensitivity and specificity of PCR for detection of Mycobacterium tuberculosis: a blind comparison study among seven laboratories.
J. Clin. Microbiol.
32:277-284[Abstract/Free Full Text].
|
| 17.
|
Noordhoek, G. T.,
J. D. A. van Embden, and A. H. Kolk.
1996.
Reliability of nucleic acid amplification for detection of Mycobacterium tuberculosis: an international collaborative quality control study among 30 laboratories.
J. Clin. Microbiol.
34:2522-2525[Abstract].
|
| 18.
|
Rogall, T.,
J. Wolters,
T. Flohr, and E. C. Bottger.
1990.
Towards a phylogeny and definition of species at the molecular level within the genus Mycobacterium.
Int. J. Syst. Bacteriol.
40:323-330[Abstract/Free Full Text].
|
| 19.
|
van Embden, J. D. A.,
M. D. Cave,
J. T. Crawford,
J. W. Dale,
K. D. Eisenach,
B. Gicquel,
P. Hermans,
C. Martin,
R. McAdam,
T. M. Shinnick, and P. M. Small.
1993.
Strain identification of Mycobacterium tuberculosis by DNA fingerprinting: recommendations for a standardized methodology.
J. Clin. Microbiol.
31:406-409[Abstract/Free Full Text].
|
| 20.
|
van Soolingen, D.,
P. W. M. Hermans,
P. E. W. de Haas,
D. R. Soll, and J. D. A. van Embden.
1991.
Occurrence and stability of insertion sequence-dependent DNA polymorphism as a tool in the epidemiology of tuberculosis.
J. Clin. Microbiol.
29:2578-2586[Abstract/Free Full Text].
|
| 21.
|
Wilson, S. M.,
R. McNerney,
P. M. Nye,
P. D. Godfrey-Faussett,
N. G. Stoker, and A. Voller.
1993.
Progress towards a simplified polymerase chain reaction and its application to diagnosis of tuberculosis.
J. Clin. Microbiol.
31:776-782[Abstract/Free Full Text].
|
Journal of Clinical Microbiology, April 1999, p. 1224-1226, Vol. 37, No. 4
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Saves, I., Lewis, L.-A., Westrelin, F., Warren, R., Daffe, M., Masson, J.-M.
(2002). Specificities and Functions of the recA and pps1 Intein Genes of Mycobacterium tuberculosis and Application for Diagnosis of Tuberculosis. J. Clin. Microbiol.
40: 943-950
[Abstract]
[Full Text]
Top
Abstract
Text
