0095-1137/90/081751-09$02.00/0
CopyrightC 1990, American Society for Microbiology
Detection and
Identification of Mycobacteria
by Amplification of rRNA
BORIS BODDINGHAUS,1 TILL ROGALL,' THOMAS FLOHR,' HELMUT BLOCKER,2 ANDERIKC. BOTTGERî*
Institutefur Medizinische Mikrobiologie, Medizinische HochschuleHannover, Konstanty-Gutschow-Strasse 8, D-3000
Hannover61,1 and Gesellschaft fur Biotechnologische Forschung, D-3300 Braunschweig,2 Federal Republic of Germany
Received 21 February 1990/Accepted 23 May 1990
Oligonucleotides specific at a genus, group, orspecies level were defined by a systematic comparison of
small-subunit rRNA sequencesfrom Mycobacteriumtuberculosis,M.bovis,M. africanum,M. bovisBCG,M.
avium,M.kansasii,M.marinum,M.gastri,M.chelonae,M.smegmatis,M.terrae,M.nonchromogenicum,M.
xenopi,M.malmoense,M.szulgai,M.scrofulaceum,M.fortuitum, M.gordonae,M. intracelulare,M.simiae,
M.flavescens,M.paratuberculosis,M. sphagni,M. cookii,M.komossense,M.phlei,and M.farcinica. On the
basis of the defined oligonucleotides, the polymerase chain reaction technique was explored to develop a
sensitivetaxon-specific detectionsystemfor mycobacteria. By usingM. tuberculosis asamodelsystem, fewer
than 10 bacteria could bereliably detected by this kind ofassay. Theseresults suggestthatamplification of rRNAsequencesby the polymerase chain reactionmayprovideahighly sensitive and specific toolfor the direct
detection of microorganisms withouttheneedfor prior cultivation. ThegenusMycobacterium consists ofa diverse groupof
acid-fast bacilli which includes a number of human and
animal pathogens (23). In addition to such overt human
pathogens as Mycobacterium tuberculosis and M. leprae,
there are a number of opportunistic pathogens (e.g., M.
avium, M. intracellulare, M. kansasii, M. simiae, and M.
szulgai) which cancause severe lung disease whenever the
normal cellular defenses have been temporarily depressed
(6). Furthermore, occasional clinical reports describe most
of the mycobacterial species defined so far as potential
pathogens (e.g., references 19 and 25). Despite progress in
biochemical, biological, and immunological techniques, the
reliable identification of mycobacteria remains aproblem.
Commonly, two approaches are used in clinical practice
for the diagnosis of mycobacterial infections. The direct
identification of acid-fast organisms by microscopy is rapid
but does not permit the identification of the mycobacterial
species observed and lacks sensitivity because large
num-bers oforganisms (>104/ml) mustbe present tobe reliably
detected(2). Culture, when positive, permits the subsequent
identification of the mycobacterial species investigated, but
the slowgrowth of mycobacteria in vitro entailsadelay of 3
to6 weeks.
Nucleic acid technology provides a radically different
approach and maybegin a new erain clinical microbiology
provided that itcanbe established in clinicallaboratories in
additiontothetraditionalserological, cultural, and
biochem-ical methods. Recently, amethod which permits the
ampli-fication ofspecific DNAsequencesand whichcanbe usedto
multiplyeven a single copyofagiven DNA sequence bya
factor of 1012 has been developed (21). This technique has
already been applied as an effective tool for bacteriological
diagnosis (13, 14, 18).
Severalreasonsmake rRNAsappealingtargetsfor
detec-tionassays based on nucleic acid structure. (i) rRNA is an
essential constituent of bacterial ribosomes (24). (ii)
Com-parative analysis of rRNAsequencesrevealssomestretches
ofhighly conserved sequences and others with a
consider-ableamountofvariability (24). (iii) rRNA ispresentinlarge
*Corresponding author.
copy numbers, i.e., 103 to 104 molecules per cell, thus
facilitating the development of sensitive detection assays
(11, 17, 20).(iv) The nucleotide sequenceof16S rRNAcan
rapidly be determined withoutanycloning procedures (3, 10,
16). The rRNA sequence is characteristic fornearly every
organism and has been used for establishing phylogenetic relationships (22, 24) as well as for identification of
micro-organisms (11, 12, 20).
Inthepresentstudy, the feasibility of using rRNA-derived
DNA probes to differentiate between highly related taxa
within the genus Mycobacterium was investigated. Our
results demonstrate that despite the high sequence
conser-vation of rRNA cistrons, such probes can be used to
differentiate between very closely related mycobacterial
species. We have developed asensitive andrapid technique
for the detection of mycobacteria which is based on the
amplification of nucleic acidsequencesfrom16S rRNA. The
subsequent identification of the amplified DNA fragment is
carried outby hybridization to oligonucleotides, the
speci-ficity of whichis definedatagenus, group, orspecieslevel.
MATERIALSANDMETHODS
Bacterialstrains.Table 1 lists the strains whose16SrRNA
sequences were determined for this study. The complete
sequences willbepublished elsewhere.
Extraction of nucleic acids. Culturesgrown on
Lowenstein-Jensen slants wereharvested and washed in 10mM Tris-i
mM EDTA(TE). The pelletwasdissolved in 10 mMTris-10
mM EDTA-0.1% Tween 80-2mg oflysozyme (Boehringer
GmbH, Mannheim, Federal Republic of Germany) ml-',
incubated for 2 hat37°C with shaking, and centrifuged. The
bacterial pelletwas lysed by redissolvingin TE containing
100 Ftg of proteinase K (Boehringer) ml-'-1% (wt/vol)
sodium dodecyl sulfate and incubated for 1 h at 37°C.
Nucleic acidswereextractedby adding anequal volumeof
TE-saturated phenol-chloroform-isoamyl alcohol (25:24:1,
vol/vol/vol). The aqueousphase wastransferred toanother
tube and 0.1 volumes of cold 3 M sodium acetate (pH 5.2)
were added. Afterbeing mixed by inversion, samples were
placedonice for 10 min beforecentrifugationina
microcen-trifuge for 10min. The supernatant fluidwastransferredto
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TABLE 1. Bacterialstrains used in this study
Species andstrains Source
M. tuberculosis
M. bovis... Institute for Medical
Microbi-ology, Hannover, Federal Republicof Germany M. bovis BCG
M. tuberculosis H37
M. aviumChester DSM 43216...DSM
M.gastriATCC 15754T...ATCC M. kansasii DSM 43224...DSM
M. marinum... K. Schroder,
Forschungsinsti-tutBorstel,Borstel,Federal Republic ofGermany M. africanum... K. Schroder
M. chelonae ATCC 14472...DSM
M. smegmatisATCC 14468...DSM
M. terrae ATCC15755T...DSM
M.gordonae ATCC14470T...ATCC
M.scrofulaceumATCC 19981T...ATCC
M.szulgaiATCC 2573T...ATCC
M. intracellulare ATCC 15985...DSM
M. nonchromogenicum
ATCC 19530T... ATCC
M.xenopiATCC 19250T...ATCC
M. malmoense ATCC29571T...ATCC
M. simiaeATCC25275T...ATCC
M.flavescens ATCC 14474T...ATCC
M.fortuitumATCC 6841T...ATCC
M.paratuberculosisATCC 19698...B.
J0rgensen,
NationalVeter-inaryLaboratory, Copen-hagen, Denmark
M.sphagniATCC33027T...J. Kazda, Forschungsinstitut Borstel
M. cookii... J. Kazda
M. komossense ATCC33013T...J. Kazda
M.farcinicaDSM 43294...DSM M.phleiATCC 35784...J.Kazda
aATCC, American Type Culture Collection,Rockville, Md.; DSM,
Deut-scheStammsammlungfurMikroorganismen, Braunschweig, Federal Repub-lic ofGermany.Tindicatestypestrain.
another tube, andthenucleic acids wereprecipitated bythe
addition of20pg ofacrylamide
ml-',
0.05volumes of 3 Msodiumacetate,and 2.5 volumes ofethanol,washed, dried, redissolved inH20orTE, and storedat-20°C. Concentra-tions of nucleic acids were determined spectrophotometri-cally at 260 nm and verified by judging the intensities of bands afterelectrophoresis on0.8%
(wt/vol)
agarose gels.To produce specimens containing a known number of
mycobacteria, a culture of M. tuberculosis H37 was
ob-tained, and serial 10-fold dilutions were prepared from this culture.Samples ofthedilutionsweregrownon
Lowenstein-Jensenslantstodeterminethe number oforganismspresent.
Reverse transcription and amplification reaction. Nucleic
acidextractswereheatedfor 5 minat65°Cbefore rRNAwas
transcribed into cDNAin 20 ,ul of 50mMTris (pH 8.3)-30
mM KCI-8 mM MgCl2 containing 100 pmol of synthetic oligonucleotide primer, 10 Uof AMV reversetranscriptase
(Seikaguku,
Rockville, Md.),and 16 Uof RNasin(Promega Corp., Madison, Wis.) by incubationfor 1hat 37°C.The amplification ofDNA sequences was performed as
described elsewhere (10). Briefly, the polymerase chain
reaction (PCR) was performed in a total volume of 100 pi
with 2.5 U of Taq polymerase (Perkin-Elmer Cetus,
Nor-walk, Conn., or GIBCO BRL, Gaithersburg,
Md.)-0.05%
detergent
W-1 (GIBCOBRL)-50
mM KCI-10 mM Trishydrochloride(pH 8.3)-1.5 mM
MgCl2-0.01% (wt/vol)
gela-tin-100 pmol ofeach ofthetwo primers and 200 ,uM each
deoxynucleoside triphosphate (dATP,
dCTP, dGTP,
anddTTP).
The90-pul
mixture was coveredby
100pul
oflight
mineral oil
(Perkin-Elmer Cetus).
DNA wasalways
added last ina10-puI
quantity whilethereaction mixturewaskeptat70°C. BeforeDNAwas
added,
itwasincubatedfor 2 minat95°Candfor15minatroomtemperature. Similarly, 10pulof
the reverse transcriptase reaction mixture was used in the
PCR. The thermalprofile involved36cyclesofdenaturation
at
93°C
for 1min, primer annealing at60or63°C for2min,
andextension at 72°Cfor 6 min.
Oligonucleotides
weresynthesized
on aGene AssemblerPlus (Pharmacia) and purified with a shadow-casting
poly-acrylamide gel
(1).
Hybridization and analysis ofamplified samples.
Aliquots
of
amplified samples (10 pul)
wereelectrophoresed
through
0.8% agarose gels, and the DNA was visualized by UV
fluorescence after itwas stained with ethidium bromide.
Forslot blotanalysis,
20-pul
aliquots of amplifiedsamples
ornucleic acids from each
organism
weredenatured bytheaddition of100pul of0.5 M NaOH-25 mM EDTA,incubated for30 minatroomtemperature, andneutralized with120
pul
of2 M ammonium acetate. The samples were loaded into
wells of a manifold (Minifold II; Schleicher & Schuell,
Dassel, Federal Republic of Germany) fitted witha
nitrocel-lulose membrane (BA 85; Schleicher & Schuell)
previously
wetted in 10x SSC (lx SSC is 0.15 MNaCl plus 0.015 M
sodium
citrate,
pH7.0). Each wellwasrinsed with 0.4 ml of10x SSC, and the membranes were heatedfor 2 h at80°C.
Hybridization was performed by standard techniques (1).
The membraneswererinsedin 6x SSCandprehybridizedin
6x SSC-5x Denhardt solution (lx Denhardt solution is
0.02% Ficoll-0.02% polyvinylpyrrolidone-0.02% bovine se-rumalbumin)-0.1% sodiumdodecyl sulfate-100 ,ug of tRNA
(Boehringer) ml1 for 1 to 2 h at 50°C. Hybridization was
performedinthesamesolutioncontaining2 x 106to1 x
107
cpm of 5'-end-labeled oligonucleotide ml1 overnight. The
blots were washed three times for 20 min each at room
temperaturein 6x SSCandthree timesfor20min each in6x
SSC-0.1% sodium dodecyl sulfate at the appropriate
tem-peraturegiven by the Tm in degrees Celsius of the
oligonu-cleotide. The blots were exposed to XAR-5 film (Eastman Kodak Co., Rochester, N.Y.) in cassettes containing an
amplifying screen for various lengths of time at -70°C.
The oligonucleotides werelabeledin 15 to 20 pi of 50 mM
Tris (pH
7.6)-10
mM MgCl2-5 mM dithiothreitol-0.1 mMspermidine-0.1 mM EDTA with 8 pmol of synthetic
oligo-nucleotide, 100 ,uCi of [-y-32P]ATP(specific activity, >5,000 Ci/mmol; Amersham, Braunschweig, Federal Republic of
Germany), and 10 U of T4 polynucleotide kinase (New
England BioLabs, Schwalbach, Federal Republic of
Ger-many) by incubation for 1 h at 37°C. The kinase was
inactivated byincubation for 10 min at75°C; the nucleotides
were extracted by phenol, and the labeled oligonucleotide
was purified and separated from residual [-y-32P]ATP with
push columns (Stratagene, Heidelberg, Federal Republic of
Germany). The resulting specific activity of the labeled oligonucleotide was 2 x 108 to5 x 108 cpm/,ug.
RESULTS
Sequence data. Evolutionarily less-conserved regions of
potential use as target sites for genus- or species-specific
oligonucleotideprobes were identified by comparison within
the collection of determined mycobacterial 16S rRNA
se-quences as well as with published sequences (9). On the
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100 bp
266 247 267
"r-l
5,
260264
r-ir-FIG. 1. Physicalmapof the oligonucleotides used withrespect to theirtargetsites in the16SrRNA. Thespecies-specific region in the rRNA molecule is indicated byan arrow.
basis of the extent ofsequence variability, several regions
werechosen;aselectionof these is described here. Figure1
illustrates a physical map of the selected oligonucleotide
targetsites onthe 16S rRNA.
Aregion knowntobe highly variable in eubacteria, i.e.,
positions 200 to 240 according to the IUB numbering of
Escherichia coli, ispartially contained within thepartof 16S
rRNAdeterminedtobeapossible species-specific region for
mycobacteria (Fig. 2). Inspection of the aligned sequences
revealed small butconsistent sequencedifferences between
thespecies investigated. In detail, uniquesequencestretches
can be definedfor the M. tuberculosis group (including M.
bovis and M. africanum), theM. aviumgroup(including M.
paratuberculosis), M. kansasii (including M. gastri, M.
simiae,and M. scrofulaceum),M.chelonae,M.smegmatis,
M. terrae, M. gordonae, M. szulgai, M. malmoense, M.
intracellulare, M. nonchromogenicum, M.xenopi, M.
fortu-itum, M. flavescens, M. marinum, M. sphagni, M. cookii,
M. komossense, M.farcinica,and M.phlei.
A few regions were initially characterized as potentially
genusspecific (Fig. 3). These regions showed mismatches of
atleast two bases when compared to published 16S rRNA
sequences(4, 7-9),includingsequencesfromclosely related
species belongingtothe
Corynebacterium-Nocardia-Actino-myces group.
Oligonucleotide 414 (Fig. 3A) is invariable in the
myco-bacterial species investigated, except for M. tuberculosis
(onemismatch) and M. xenopi (characterized byaninsertion
of threenucleotides). The region of oligonucleotide 247 (Fig.
3B) is characterized by single mismatches ofonebase each
with M.xenopi andM.flavescens aswellas amismatchof
two bases with M. nonchromogenicum. Oligonucleotides
266 and 267(Fig. 3C) targetthe sameregion in 16S rRNA.
Except for M. simiae, the sequence of which is less
con-served within thispartof the small-subunitrRNA,
mycobac-teria show nucleotide stretches identical tooligonucleotide
266or267. Notably,M.flavescens showsasingle mismatch
tooligonucleotide 267. Oligonucleotide 260 (Fig. 3D),aswell asoligonucleotide 264 (Fig. 3E), is well conserved within the
mycobacterial species investigated.
Specificity of oligonucleotides. Selected oligonucleotides
wereinvestigated inmoredetailtoevaluate theirspecificity.
Sufficient differences in 16S rRNA positions 161to215to
defineoligonucleotide probes which should distinguish
my-cobacteria at the species level were present. Thus, as an
example, synthetic 20-mers within this region which were
complementary to nucleotides from the M. tuberculosis
group(including M. bovis andM. africanum),M. avium-M.
paratuberculosis, and M. intracellularewere32Plabeledand
hybridized with nucleic acid extracts. The probes
comple-mentary toM.tuberculosis,M.avium, andM.intracellulare
hybridized onlytothe nucleic acids from thecorresponding
species but not to nucleic acids fromany other
mycobacte-rial species investigated (Fig. 4).
Thecomplement of oligonucleotide 247 (reverse 247)was
hybridized to nucleic acid extracts of various bacteria,
including close relatives of the genus Mycobacterium, and
AAC TGG GTC TAA TAC CGG ATA GG-ACCA
.A ..T.- ..G
.A ..T.-..
..A ... T-C..T
..A ... CACC.TG
... ... ... ... ... ... ... ..-.... .TT .GC
... ... ... ... ... ... ... ..-.... .TT .GC
... ... ... ... ... ..A ... ..-...C ..A .GC
... ... ... ... ... ... ... ...C ..A .GC
*-- ... ... ... ... ... ... ... ... ...
... ... ... ... ... ..A ... T.-.T.. T.C TC.
... ... ... ... ... ... ... ..-..T. ... AC.
... ... ... ... ... ... ... T.-.... T.. AC.
... ... ... ... ... ... ... ..C... .C.
... ... ... ... ... ... ... CACC.TT .T. .T.
CGG GAT GCA TG--TCT-TGT
..C AC.
..C .C.
NT. .TC
.T. .TC
.TT .GC
.AT .C.
... ... ... ... ... ... ... .. T.. ...
... ..--C..-...
... ..--c ..-...
... ..--C..-..G ... ..--C..-..G
T.. ..--...C-...
T.. ..--GGG-...
... ..G-...
T.. C.G-.T.--..
T.. ..G-.G.--.. ... ..G--..-G.G
GGT GGA AAG C
T.C ..G-.G.--.. ... ... ... .
T.. ..G-.G... ... ... ... .
... .GCCTGG.A- ..- ... ... .
... ..GCCTGG.A- ..- ... ... .
... .. .. ... ... ... * .
... ..--GTG-... ... ... ... .
... ..-T...--.. ... ... ... .
*. .. .. .
*...-*. ... .
*. ...--.
*.. ... *...
A. G... ... .
M.tuberculosis
M.chelonae
M.fortuitum
M.flavescens
M.smegmatis
M.simiae
M.nonchromogenicum
M.terrae
M.xenopi M.gordonae M.scrofulaceum M.avium
M.paratuberculosis M.intracellulare M.gastri
M.kansasii M.malmoense
M.szulgai M.marinum M.sphagni
M.cookii
M.komossense
M.farcinica
M.phlei
FIG. 2. Alignment of mycobacterial16SrRNAsequences.The first nucleotide inthefigure correspondstoE.coli 16SrRNAposition161
(IUB nomenclature).The 16SrRNAsequencesofM.bovis,M.bovisBCG,M. tuberculosisH37,and M. africanumareidenticaltothat of M. tuberculosis andthereforearenotshown. M. tuberculosiswasusedasthe referencesequence. Nucleotides differentfrom those of M.
tuberculosisareindicated; dashes indicate deletions. The complementary strand of theunderlined sequenceswasusedas aspecies-specific probe in theexperiment illustrated in Fig. 4.
... ... ... ... ... ... ... .. TTC TGC ... ..--.GG-G.. ... ... ... ... ... ... ..A ... ..-.... .A. ..CA.. ..--..C-... ...
... ... ... ... ... ... ... ..-.... .TT .GC ... ..--C..-... ... ... ... ... ... ... ... ... ..-...T .AA ..C ... ...
... ... ... ... ... ... ... ..-...T AA ..C ... ..-.... C. ... ... ... ... ... ... ... ... ..-...T TTA .GC ... ..--...-.TA ...
... ... ... ... ...
... ... ... ... ...
... ... ... ... ...
... ... ... ... ...
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BODDINGHAUS
A
B
c
ATC ..T .T. ..A TO T...
T... T...
T.. ... ... ... ..
T...
T...
T...
T... T.. ... ... ... ..
T... T... T...
T.. ... ... ... ..
GGTT.. ... ... ... ..
T... T...
TTG TCG CGT TGT TCGTGAAA
... ... ... .. ... ... ... .. ... ... ... .. ... ... ... .. ... ... ... .. ... ... ... .. ... ... ... .. ... ... ... .. ... ... ... ..
T... .. .... .
... ... ... ... .. ...
... ... ... ... ... ... ..
... ... ... ... ... ... .. ... ... ... ... ... ...G. ... . . .. . . C
... ... ... ... ... ... ..
ACGGCT TAA CTG TGA GCG TG
... ... ... ... ... ... .. ... ... ... ... ... .. .... ... .... .... ... ..*. ... ... ... ... ... .. ... ... ... ... ... .. ... ... ... ... ... .. ... ... ... ... ... .. ... ... ... ... ... .. ... ... ... ... ... .. ... ... ... ... ... .. ..A ... ... ... ..G ..A ... ... ... ..G ..A ... ... ... ..G ..A ... ... ... ..G ..A ... ... ... ..G ..A ... ... ... ..G
..A ...C.. ... ..G
GG. CC CG.
TTT... TG.
T.. NA. C
GC. ... M. N
TCT ... TG.
TC... G....N
in. ... CA ... ..
TT. ..CC.A ... TT. ..Cc...
TT. ... CCA ...
NN. ... ... C.GGA- ... ..
.GA..A... C.GAA. .GA ... ... C.GAA.
.N. CTN.
.N. N.... CTG.
.C..CT.
N. C.GGA.
N. C.G AN.
N. CNGNN.
D
GGGTTT GAC ATG CAC AGG AC
... ... ... .. ... ... ... .. ... ... ... .. ... ... ... .. ... ... ... .. ... ... ... .. ... ... ... .. ... ... ... .. ... ... ... .. ... ... ... .. ... ... ... .. ... ... ... .. ... ... ... .. ... ... ... ... .. ... ... ... .. ... ... ... .. ... ... ... .. ... ... ... ... ..
E
TCC CTT GTG GCC-TGT GTG CA
... ... ... .. ... ... ... .. ... ... ... ... .... ... ... ... .. ... ... ... ... ... .... ... ... ... ... .... ... ... ... ... .... ... ... ... .. ... ... ... ... .... ... ... ... .. ... ... ... ... .... ... ... ... .. ... ... ... .. ... ... ... -...;. .. ... ... ... ... .... ... ... ... .. ... ... ... ... .... ... ... ... ... .... H.tuberculosis N.aviuw N.gastri M.kansasii M.arinum N.paratuberculosis N.gordonae N.scrofulaceum N.szulgai N. intracellulare N.malmoense N.fortuitua N.chelonae N.smegmatis N. terrace N.nonchromogenicun N.xenopi N.flavescens N.simiae
G.. TNN T.. T.-G.. N.A G.. T.. T.. ..T-G.. ACA ..
N.T T.N .G. .TN-G.. TCA ..
CT. T.C .GA ..G-CC. TCA ..
N.TN.G .G. ..T-N.. TCA ..
....NG.N .T.-GO.. A.A
C.... T.A .T.-G.. .. ..
C.. ..N T.N.T.-G.. ..A..
CN. TC. T.. ATATG.. ..A ..
Arachnia propionica Propionibacteriuw freudenreichii Propionibacteriumacnes Actinomycesbovis Actinomycesviscosis Corynebacteriumi variabilis Nocardiaalbus Nocardialuteus Pimelobacterjensenii
FIG. 3. Alignment ofmycobacterial 16S rRNAsequenceswithselectedpublished 16S rRNAsequences (4, 7, 8), includingthosefrom
species belonging to the related Corynebacterium-Nocardia-Actinomyces group. N, Undetermined nucleotide. (A) Oligonucleotide 414
region, correspondingto E. coli positions 212 to230; (B) oligonucleotide247 region, corresponding to E. coli positions 590to 609; (C) oligonucleotide 266-267 region, correspondingtoE.coli positions 614to633; (D)oligonucleotide260region,correspondingtoE.coli positions
988to1007;(E) oligonucleotide 264region,correspondingtoE.coli positions 1027to1046.
demonstratedanexclusivespecificity for mycobacteria (Fig.
5). Bacterialspeciesother thanmycobacteriadidnot
gener-atepositivesignals.Asexpectedfrom thesequence
compar-isons, thisprobe didnothybridizeunderstringent conditions
to nucleicacidsextracted fromM. nonchromogenicum, the
sequence ofwhich differs by twobases in thisregion.
Oligonucleotides complementary to oligonucleotide 260,
264, or 266 and 267 were used as primers in the PCR on several differenttemplate DNAs to evaluate the specificity
of these potentially genus-specific regionsforamplification.
Thus, theseprimers shoulddirect thepreferential synthesis
oftheappropriate 16S rRNA genefragmentfrom
mycobac-teriabut notfrom otherspecies. Most importantly, human
DNA, which is present in clinical samples, should not
GC mT .. ... .. ... .. ... .. ... .. ... TT... CT ..A GT ..C CT ... TT .GN AG ... AG ... AG ... AG ...
GG. ... N.. G. ... ...
GG . ... ...
CTN ... N..
CT. ... ...
CG. ... ...
GGT ... ...
GGT ... ...
GGN ... ...
.CC ... ..T ..
..C ..G ..T . .
.CC ... ... ..
... GAG ..T.N
... G.GT.N NN
..C C.G N.... .ACCTN ..C .N
.AACCTN ..C ..
.CTGNT ..T
... ... ...
. .. .NN ...
A.. C...
A.. C.N ...
..A A...
... ... ... ... ... ... ..
..ATG. C....
... TT. T.... ... GTT C.. G.
... TGG GT. TT
..ATG. C.. .T
GG. G0. CC. CA ..A ... C.. .A
G.A ... C.. .A N.A ... C.. .A
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B c
- M. avium-chester
- M.avium-intra.ellulare -M.scrofulaceum -M.kansasii -M.terrae
- M.gastri
M. gordonae
_- - M.tuberculosis
- M.fortuitum
M.flavescens
M.marinum
M.szulgai
M.nonchromogenicum
M.xenopi
M.malmoense
M.simiae
M.chelonae
N.asteroides
A.israelii
P.picketii
FIG. 4. Hybridizationofnucleic acids (1 ,ug)from the indicated bacterialspeciestospecies-specific oligonucleotide probes.P. pick-ettii, Pseudomonas pickettii. (A) Sequence ACC ACA AGACAT GCA TCC CG, specific for M. tuberculosis. Hybridization was
performed at 52°C, and washings were performed at 61°C. (B) Sequence ACC AGA AGA CAT GCG TCT TG, specific for M. avium-M. paratuberculosis. Hybridizationwasperformed at52°C, and washings were performed at60°C. (C) Sequence ACC TAA AGACAT GCG CCTAA, specific forM. intracellulare. Hybrid-ization was performed at 50°C, and washings were performed at 550C.
M. avium -t_
M.intracellulare
M.scrofulaceum
-M.kansasii
M.terrae
M.gastri - _
M.gordonae -M.fortuitum
M.tuberculosis
-M.flavescens _
M.marinum
M.szulgai
M.nonchromogenicum
M. xenopi
-M.malmoense - _
M.simiae
--N.asteroides
- P.acnes
- S.vnds
- C.paurometabourm
- Rhodococcusequi
- S.hirsuta
- N.autotrophica
M. chelonae
M. cookii
M.sphagni ab
M. komossee-s_
M. smegmatis
M.phiei *
FIG. 5. Hybridizationof nucleic acids(1 ,ug)from theindicated
bacterial speciesto anoligonucleotide broadly reactive for
myco-bacteriaatthegenuslevel (reverse 247, with sequence lT- CAC GAA CAA CGC GAC AA;hybridization wasperformed at52°C, andwashingswereperformedat55°C).P.acnes,Propionibacterium
acnes;S.viridis, Saccharomonospora viridis;C.paurometabolum,
Corynebacterium paurometabolum; S.hirsuta,Saccharopolyspora hirsuta;N.autotrophica,Nocardiaautotrophica.
interfere with the amplification of mycobacterial DNA. As
canbeseeninFig.6AtoC(lane 1),thecombination of these primerswitha second primercomplementarytoacommon sequence within bacterial 16S rRNA (i.e., oligonucleotide
246 [10]) resulted in the synthesis ofa prominent band of
amplifiedDNAwiththe appropriate size in thepresence of
mycobacterial DNA. Incontrast, no specificbandof
corre-spondingsizewasvisible whenamplificationwasperformed
with human DNA as a template (Fig. 6A to C, lane 6). In
addition, the simultaneous presence of human DNA and
mycobacterial DNA did not interfere with the specific
am-plification of mycobacterial DNA (Fig. 6Ato C, lane 7). A
faint band ofamplified DNA was observed for the related
species Actinomyces israeliiwhen an oligonucleotide
com-plementary to oligonucleotide 260 was used but not when
reverse264orreverse266-267wasused (Fig. 6AtoC, lane
3). Amplification carried out on total DNA from Nocardia
asteroids, another species closely relatedtothemembers of
the genus Mycobacterium (24), did not result in visible
amplified fragments (Fig. 6A to C, lane 2). Likewise,
per-forming the PCR on DNA from more distantly related
membersof thefamily Enterobacteriaceaedidnotyieldany
band(Fig.6Ato C, lanes 4 and5).
Extractionofnucleicacids, reverse transcription, and
sen-sitivity of the amplification reaction. A simple procedure
basedon chemical and enzymatic methods was established
to yield DNA as well as RNA from mycobacteria. This
method results in extraction of intact, undegraded rRNA
molecules(Fig.7A).Wereasoned that thehighcopynumber of rRNA should facilitate the development of sensitive assaysbasedondetectionof nucleic acids. In ordertomake rRNA accessible to the amplification procedure, reverse
transcriptase is used to preparecDNA before the
conven-tionalPCR. Wegenerated cDNA with aprimerwhichdoes
not interfere with the specificity of the PCR by defining a
short 12-mer oligonucleotide for the reverse transcriptase reaction (Fig. 7B and C). Because of its shortness, this
primerdoes not formastable hybridwithits target DNA at
60°C,the temperature under whichannealingwasperformed
during the amplification cycles; therefore, the use of this
primer in the PCR does not result in an amplified DNA
fragment (Fig. 7D).
Thesensitivity of theamplification procedure was
inves-tigatedwith M. tuberculosis as amodel systemby
amplifi-cation of the probe site in a 1,030-base-pair (bp) fragment
withprimers246 andreverse264. Theeffect ofusingthehigh
copy number of rRNA on the sensitivity of the assay was
investigated by first performing reverse transcription of
rRNAinto cDNA.
Byusing serialdilutions ofpurifiednucleicacids, 10pgof
purifiednucleic acids addedtothereaction mixture could be
amplifiedsufficientlytoyieldadetectable bandinanagarose
gel (Fig. 8). Whenthe presence ofamplified mycobacterial
DNA was identified by hybridization with a 32P-labeled
oligonucleotide by slot blot analysis the sensitivity was
improved approximately 100-fold. Interestingly, when
re-verse transcription of the nucleic acid extracts into cDNA
was performed first, sensitivity was increased 103-fold.
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1756 BODDINGHAUS ET AL.
electrophoresedthrough a 0.8% agarose gel, a discrete band
of approximately 1.0 kilobases (kb) was visible in samples
containing 500 mycobacteria when nucleic acids were used
without prior reversetranscription as a substrate for the Taq
polymerase (Fig. 9). However, reverse transcribing the
rRNA into cDNA before the PCR resulted in a clear-cut
band of theappropriate size in samples containing as few as
approximately 30 mycobacteria. Combining the
amplifica-tion procedure with a slot blot hybridizaamplifica-tion format resulted
ina detection limit of threemycobacteria in a sample (Fig.
9).
Fi
FIG. 6. Specificityof selectedoligonucleotides in the PCR. The PCRwasperformedasdescribedinMaterials andMethods with 100
ngofbacterialDNA and1 ,ug of human DNA. Shown is ananalysis
ofamplifiedDNAby electrophoresison a0.8%agarosegelwith 10
,ul of the amplification reaction mixture. Lanes 1, Mycobacterial DNA; lanes 2,N. asteroides; lanes3, A. israelii; lanes4,
Pseudo-monasspecies; lanes 5,E.coli;lanes 6, humanDNA;lane 7, human
DNA and mycobacterial DNA; lanes 8, noDNA. M. avium was
usedasthesourceof mycobacterialDNA.(A) PCR with primers246
(AGAGTTTGATCC TGG CTC AG) andreverse264(TGC ACA
CAGGCC ACA AGG GA) directing the synthesis ofa 1,030-bp
gene fragment; (B) PCRwith primers 246and reverse 260 (GTC
CTG TGC ATGTCAAACCC) directingthesynthesis ofa990-bp
gene fragment; (C) PCR with primer 246 in combination with a
mixture ofreverse 266(CACGCT CAC AGT TAAGCCGT)and
reverse 267 (CAC GCC CAC AGT TAA GCT GT) directing the
synthesisofa620-bpgenefragment.Themolecularmassmarker is
the 1-kbladder(GIBCO BRL).
Thus, 100 fg of nucleic acids yielded a clearly detectable
band in anagarose gel, and even 0.1 fg of purified nucleic
acids yielded a clear-cut positive signal when the PCR
mixture was hybridized with a 32P-labeled oligonucleotide
(Fig. 8). Given that the size of theM.tuberculosisgenomeis
2.2 x
109
daltons(5), 0.1 fg of nucleic acids corresponds toapproximately 2% of the total nucleic acidcontentofasingle
bacterium.
As a further test to determine the sensitivity of the
amplification procedure, nucleic acids extracted from
se-quential dilutions ofa suspensioncontaining aknown
num-ber of M. tuberculosis cells were added to the reaction
mixtures. When aliquots of the amplification mixture were
DISCUSSION
The purpose of a clinical microbiology laboratory is to
rapidly and accurately provide clinicians with information
about thepresence or absence of amicroorganism that may
be involved in an infectious disease process. Traditionally,
this involves the isolation of a suspected pathogen from a
clinical specimenfollowed by identification of the organism.
Most of the tests necessary for identification require the
isolation of a pure culture of the suspected bacterium.
Consequently, the isolation and identification of slowly
growing pathogens like mycobacteria entails a period of
several weeks.
Inthispaper, wedescribeamethodfortherapid detection
and identification of mycobacteria based on the in vitro
amplificationofmycobacterial nucleic acids. Any detection system based onnucleic acids mustfulfilltwocriteria: (i)it mustresult inreliablerelease ofnucleic acids fromcells, and
(ii) it must be capable of definition of oligonucleotides ofa
characterized specificity.
Thecomplex structure andimpermeability of the cell walls
of mycobacteria makes the lysis of mycobacteria for the
extractionof DNA as well as RNAdifficult. Thus, common
methods forisolatingDNAor RNA usephysicalprocedures
of cell disruption, e.g., the use of French presses (26). However, such methodsare
impractical
inclinicallaborato-ries. We have therefore established asimple and versatile
procedure for isolating DNA and RNAfrom mycobacteria by chemical andenzymatic methods (Fig. 7).
rRNA, with its species-specific variable regions, may
provideageneraltargetfordeveloping taxon-specific oligo-nucleotide probes for virtuallyany species of interest. The
identification of species-specific regions by comparison of
newly isolated 16S rRNA sequences with published 16S
rRNA sequences (9), the number of which is constantly
increasing,isrelatively straightforward. Toestablishtheuse
of rRNA-derived oligonucleotide probes todifferentiate
be-tween closely related taxa, we haveinvestigated the genus
Mycobacterium as a model system. The results presented
here indicate that there are small but consistent sequence
differences in certain regions of therRNA molecule which
allow the definition ofhighly specificoligonucleotides, i.e.,
at the genus, group, or species level. We were able to
identify a highly variable region which is extremely useful
for definition of species-specific probes in mycobacteria.
Thusfar,thefeasibilityof thisregion fordeveloping species-specificDNAprobeshas beenexperimentally demonstrated
with M. tuberculosis, M.avium, and M. intracellulare.
Our aimwas to outline a strategy which shouldbe
com-parable in specificity and sensitivity to traditional culture
techniques in mycobacteriology, i.e., a method which can
detectvirtually anymycobacterial organism present in low
numbers in asample irrespective ofthe species affiliation.
Forthis purpose, we selectedregions in the mycobacterial
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.1
B
c
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FIG. 7. Extraction of nucleic acids, cDNA synthesis, andamplification. (A) Extractionof nucleic acids from M.tuberculosis,resultingin chromosomalDNAandintactrRNAbands.(B) cDNAsynthesis witholigonucleotideGGC AGT CTC TCA (correspondingto E.coli16S rRNApositions 1162 to 1151) asprimer;anethidiumbromide-stained agarose gelisshown. Lane 1, cDNAsynthesis (theappropriateshift of the 16SrRNAbandis indicatedby an arrow);lane 2,inputnucleicacids with the 16Sand 23SrRNAbands.Numberson theleft indicate sizes inkilobases. (C) Autoradiogram made when[aL-32P]dCTPwasincorporated during thecDNA synthesis reaction. Thearrowpointsto the newlysynthesized cDNA strand; numbers on the left indicate sizes in kilobases. (D) Inability ofoligonucleotide GGC AGTCTC TCA tofunctionas aprimer in the PCR. The PCR was performed as described in Materials and Methods withprimers246 and reverse264(TGC ACACAG GCC ACA AGG GA) (lane1) orprimer246incombinationwith GGC AGT CTCTCA(lane 2).Purified nucleic acids(100ng)from M.avium wereusedas atemplate. The molecularmassmarker is the1-kbladder(GIBCOBRL).
rRNA that are useful as target sites for genus-specific
oligonucleotides. The selection was based on the extent of
sequence variability as revealed by comparison with
pub-lished 16S rRNA sequences (9). The characterized
oligonu-cleotides exhibitedgenus specificity in a hybridization
for-mat(Fig.5) anddirectedthesynthesis of theappropriate16S
rRNA genefragmentwhen usedasprimersinthe
amplifica-tion reacamplifica-tion preferentially when amplification was carried
out on mycobacterial DNA (Fig. 6). Following generic amplification of mycobacterial nucleic acids after optional synthesis ofcDNA, a DNAprobe wasused to detect and
identifytheamplifiedproductatthespecieslevel. We show
in this paper that samples containing fewer than 10 intact
mycobacterialcells cangive positiveresults (Fig.9).
Theultimateuseof this proceduretodetectmycobacteria inclinical specimens will depend on factors which were not evaluated inthisstudy, i.e.,handling,versatility, sensitivity,
andspecificity inclinicalpractice. Forexample,becauseof
the favorable kinetics of oligonucleotides when compared
with cloned probes, hybridization and washing times have
reportedly been reduced to 15 to 20 min and 2 to 3 min,
respectively (15). We expect that the application ofDNA
probes coupled with specific amplification of target
se-quenceswill contribute to a more rapid diagnosisof
myco-bacterial infections.
Insummary, wehaveprovidedevidence thatamplification
of rRNAsequences allows the identification and
differenti-ation ofmycobacteria at a genus, group, or species level.
Based on rRNA as a general structure which permits the
rapid generation of specific DNA probes for eubacteria
without any cloning procedures (3, 10), asimilar approach
would probably succeed with many other bacteria, i.e., it
would determine the presence of genera of bacteria in
clinicalspecimens by the PCR followed by identification at
a:
FIG. 8. Sensitivityof thedetectionofmycobacterialnucleic acidsfollowing amplificationwithprimers246 and reverse 264. Thetwogels
ontheleftshow ananalysisofamplifiedDNAbyelectrophoresis on a0.8%agarosegel with 10 ,ulof theamplification reactionmixture. A stocksolution ofpurified nucleic acids fromM.tuberculosiswasdilutedtogivethefollowingamountsin thesamples: lane1, 10ng; lane2, i ng;lane3,100 pg; lane 4, 10 pg; lane 5, 1 pg; lane 6, 100fg;lane 7, 10fg;lane8, 1fg;lane9, 10pg; lane 10, 1 pg; lane11, 100fg;lane12, 10fg; lane 13,1fg;lane 14, 0.1fg;lane15, 0.01fg;and lane 16, no DNA. Reversetranscriptionwasperformedonthenucleicacidsin lanes 9 to 15beforethePCR. Themolecular mass marker is the 1-kb ladder(GIBCO BRL). The two panels on the rightillustrateanexperiment inwhich20-,ul samplesof the amplifiedDNAwere appliedtonitrocellulose andhybridizedtoACC ACA AGA CATGCA TCC CG. (A) Purified nucleic acidswereuseddirectlyinPCR;(B) reversetranscription ofpurified nucleicacids into cDNA withGGC AGT CTCTCAas
theprimerwasperformed first.
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FIG. 9. Sensitivityof the PCR. ThePCR was performed with primers 246 and reverse 264. On the left is an analysis of amplified DNA by electrophoresison a0.8%agarosegel.Nucleic acidswereextractedfromsamplescontaining5 x 104(lane1), 5 x103 (lane 2), 5 x 102(lane 3), 5 x 101(lane4), 5(lane5), 3x103(lane6), 3 x102(lane7), 3x 10' (lane8), 3(lane9),or 3 x10-1(lane10) M.tuberculosis bacilli. Reverse transcriptionwasperformedon thenucleicacidsin lanes 6 to 10beforethePCR. Lane 11 is the negative control. On the right,20-,ulsamples of theamplifiedDNAwereappliedtonitrocelluloseandhybridizedtoACCACA AGACAT GCATCCCG. (A) Reverse transcription of the extractedrRNAinto cDNAwasperformedfirst, withGGCAGT CTC TCAastheprimer(B)Extractednucleic acidswereuseddirectlyin thePCR.
the desired taxon level with appropriate taxon-specific
probes.
ACKNOWLEDGMENTS
We thank W. Heikens for synthesis of oligonucleotides; K. Schroder, B. J0rgensen, and J. Kazda for providing strains; S. Maibom for typing the manuscript; and D. Bitter-Suermann for continuousencouragement.
This workwassupportedbyagrantfrom the Bundesministerium fur Forschung undTechnologie(01Ki 89117) to E.C. Bôttger.
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