CA2190090A1 - Materials and methods for the detection of mycobacteria - Google Patents

Abstract

The present invention is directed to oligonucleotides useful in detection, e.g., by ligase chain reaction (LCR), of target DNA from bacteria of the genus Mycobacterium, substantially without detecting bacteria of other genera. The present invention is also directed to methods of detecting target DNAs from bacteria of the genus Mycobacteria using the ligase chain reaction.

Description

woss/31s7l ~.I/U~ ~ '

2~ qOOqO
MATERIALS AND METHODS FOR THE DET~CTION
0~ MYCOBACTERIA
FIELD OF T~IE INVENTION
The invention relates to nl L;. ~ vLi. ~. ~ useful for detecting bacteria of thegenus Mycobacteriur~. This invention also relates to methods useful in amplifying and/or detecting bacteria of the genus l~cv~u~, BACKGROUND OF THE INVENTION
After decades of decline in the incidence of ~ an alarming increase in new cases is occurring. Infection with M. tuberculosis, the causative agent of tuberculosis, most commonly occurs by inhalation of droplets contaiiiing only a few live bacilli. The llly vub~l~,t~ replicate in lung tissue to form a primary focus of infection and from there enter the local ~ymphatic system. The infection then ' widely through the body via the blood and Iymphatic system. The initial lesions usually heal to form tiny granulomas that may harbor viable tubercle bacilli indefinitely. Post-primary Lul~luulo~ iS the most common form of clinical i L ' cic and is usuaDy pulmonary.
The disease can occur many years after initial infection and is thought to be due to a temporary loss or ~ of cell-mediated immunity (due to, for example. increasing age, illness, m~ )n or alcoholism) leading to reactivation of dormant tubercle bacilli in lesions. Acquired T ~ r y Syndrome (AIDS), increased poverty and its attendant ' are now imporLant factors lea~iing to the increased incidence of thedisease.
A tentative diagnosis of i L ' or other Ill~vUbv~ diseases may be made on a basis of clinicai grounds, radiology, and on the finding of acid-fast bacilli in smears of sputum, blood bronchial-lavage, gastric-washing, urine, or cerebrai spinal fluid. Smears of these specimens typicariy are stained by the Ziehl-Noelsen technique or by fluorescent rhodamine-auramine dye and examined by microscopy. However, 2 5 ~ uv~,u~Jil,al~y positive sputum sarnples are found in only about 30 to 50 percent of pulmonary ~u1v~ .,ulo~.v patients and thus. cultures must always be performed. Cultvlring for the presence of M. lub~, vulv~siv and other IllyUUl)vvlSvlidl species is a time-consuming and difficult process because some lllJ vUbavUvlial species are very slow-growing and have fastidious nutritionai lCU,UiUCll.vllt~. While some samples may be inoculated directly onto

3 0 cultv~re medium, most specimens reciuire, ' with, for example, strong alkali.
Specimens are then typically inoculated onto egg-based media such as Lowenstein-Jensen mediurn and/or defined media such as Middlvl~luu~ 7H9 broth in the presence of antibiotics such as peniciliin to inhibit growth of other bacteria. Cultv~res are then Wo 95131571 r~
2l9a090 2 incubated at 35 C to 37 C for 1-7 weeks. However, even culture techniques are only about 70% Gffective.
Other techniques have also been developed foI the detection oF M.
tub,, ~/U~D. These methods include nucleic acid llyblivir~Liull assays using probes s directed to nucleic acid sequences found in M. tuberculosis. For example, PCT
Application No. WO 91/19004 entitled "SpecrFlc Detection of My~
Tul~,, ' " by Guesdon and Thierry published December 12, 1991 teaches the use of llu.lvvLidc primers ~.UII~ -r ' v to those found between bases 1-30; 250-275;
1029-1058;1200-1229;1260-1289;1263-1294;1735-1764and 1772-17960fIS 6110, an insertion element present in multiple copies in many M. tuberculosis strains as described in Thierry et al. Nucl. Acids Res. 18:188 (1990). The application claims sequencesderivedfromlS6110andsequencesatleast80%1,.~ r.v.,~therGto. Tbe probes described in that application were used for the detection of M. ~ub~, ' using the polymerase chain reaction.
In PCT Application No. WO 88/03957 published June 25, 1988 by Hogan, et al. a method is described for the detection of non-viral organisms including M.
u~,, ' and M. i/llr " ', e using nucleic acid llyblillir~Liull techniques. The method comprises uu.~_ v an cLv ' ' that is sufficiently ~ I ' y to hybridize to a region of ribosomal RNA (rRNA) selected to be unique to a particular non-2 o viral organism or a group of non-viral organisms sought to be detected. Tbe target rRNA
is selected by compaling one or more variable region rRNA sequences of the non-viral organisms of interest with one or more variable region rRNA sequences from one or more non-viral organisms sought to be ~ ' Probe sequences which are specific for 16S rRNA variable ,,~ of A~y~ùb~,L, aviu~n, IlSy.ùl,a.t~, intraceflulare, 2 s and the Aly~ùba.t~, l ubL ~ ~,.losis-complex bacteria and which do not cross-react with nucleic acids from each other or any other bacterial species under proper stringency are identified. A probe specific to tbree 23S rRNA variable region ~ b,~ c from theubu l~/ tub~ ' complex bacteria is also disclosed as are rRNA variable region probes useful in llylvlivi~liull assays for bacteria of the genus ~ cobl~3 o (15S,23S rRNAspecific); for ll~j~uvact~, i, aviurn intheregionuullc r ' vto bases 185-225ofE. co~i 16SrRNA;RNAof ~llycuba. itli~ irtracellulare intheregion to bases 185-225 of E. coli 16S RNA; to rRNA of the species included in the ~Sy~ubu~t~ .. tuberculosis complex in the region ,ull~ r l- v the bases 185-225 of E. coli 16S RNA; to rRNA of the species included in the Mycobacteriwn tuberculosis complexintheregion~ullcr '- vtothebases540-575,1155-1190,and2195-2235Of E. coli 23S RNA; to RNA of the genus ~ Ubu~tl ~ in the region ~u~ Julldi..v to 1025-1060 of E. coli 16S RNA; and otbers.

Wo 9~/31571 P~l/u..,~
2f 90090 U.S. Patcnt No. 4.851,330 by Kohne issued on July 25, 1989, addresses the use of nucleic acid }I,~IJlil]i~.a~iUl~ to detect and quantify non-viral Illil,lU' .. ~
More particularly the patent describes the preparaion of cDNA probes which are ~.1 ,., 1l ,1.., .. ~ - y only to ribosoma~ RNA ~l~b~ known to be conserved in an s organism, category or group of organisms. By way of iLustration, the patent describes the production of a cDNA probe ~, .. 1 ~l 1- 1. .. 1' - ~ y to ribosomal RNA from 11~
hominis but which was not f,r.l 1,~,!. ..Il. f - y to human ribosomal RNA. The paunt alleges that the M. hominis probes are useful in the detection of M. hominis in human tissue cultures and in other cultures of mrf mm~ ceLs.
o U.S. Patent No. 5,168,039 to Crawford et al. is directed to an isolated purified repeitive DNA sequence for use in detecting M. tul~l culu5~5 complex in clinical material. The patent describes the cloning and sequencing of a repetiive element found in M lub~. ~u~u~ u~ui~u~ DNA and further describes the use of these cloned repetitive dements as probes and primers for the detection of IC~ iV~ strains of the M
.'..~.:, LUIL..~ complex.
U.S. Patent No. 5,183,737 to Crawford et al. (divisiona'f of U.S. Patent No. 5,168,039 described above) teaches the use of the dlul~ Liull~.d repetitive elements as primers for the detection of bacteria of M. thfoerc~losis complex using the polymerase chain reaction. The patent afso teaches the use of the repeitive elements as probes for the 2 0 detection of bacteria of the M. tubL, L~u5i~ complex using a membrane based nucleic acid l-yfJIiufi~Liull assay.
PCT Application WO90/15,157 by Lane et al. published December 13, 1990, addresses "universa." nucleic acid probes for eubacteria and methods for the detection of bacteria. The app ication describes nucleic acid probes and probe sets which 25 hybridize under specific conditions to the ribosoma, RNA molecules (rRNA), rRNA
genes (lDNA), and certain .~ - and in ~itro ~ ,. products thereof but which do not hybri&ze under the same conditions to rRNA or rDNA of eukaryotic ceLs which may be present in test samples. More specificaLy, the probes described in this applicationarespecifically-l-...l,1F~ - ytocertainhighlyconservedbacterial23Sor 3 0 16S rRNA sequences. The probes were selected using a computer algorithm operating on aligned sets of 1 6S and 23S rRNA sequences to identify regions of greatest similarity among the eubacteria. Nucleic acid probes so derived hybridize most widely among&verse bacteria, species. Probes found hul.~ùlotu~., among the bacteria species were a so assessed for differences with non-bacterial rRNA sequences using a computer a'fgorithm.
~s Ultimately, 41 probes were selected based on these ana ysis; 22 targeting 23S rRNA and 19 targeing 16S rRNA. T}fe 16S ..I.l;ril -~ primers described in that application include primers which detect most eubacteria, Borrelia and spirochetes, the enterics, Deinococcus,C~,.",!o1:~a.,." andthe Fusobacteria and Bacillus species. Severalofthese _ WO 9S/31571 P~
90 ~4 p}obes are assertedly capable of detecting Alyccrb~ct~, ia kansasii and M. bovis. The application describes the use of sandwich type hybridization assays for the detection of llyblid;~a~iUII between probes and rRNAs in samples suspected of containing bacteria and also describes the use of the polytncrase chain reaction tPCR) directed to 16S rRNAs s using probes as derived above. The probes described in that application (both 16S, and 23S rRNA probes) were assertedly able to detect a wide variety of bacterial species.
PCT application WO90/12,875 by Hance et al. published November 1, 1990 discloses nucleotide sequences of au~illullly-,~ ,s and their application to the synthesis or detection of nucleic acids found in ~, ' The application discloses 0 â 383 base pair pol~ coding for the 65 kD (kilodalton) Ill.y~ulrd~ idl antigen which has homologs in 8 species of Ill.r~ùb~ id including M. ~ub~ osis, M. avium, M.fortuitum, M. p, u~ul~ ulosis, BCG, M. kansasii. M malmoense, and M. marinum.The use of these probes for the detection of DNA and/or the products of ~ c~ of these bacteria is described. More patticularly, an oli,~ l.,ulid~ comprising bases 397 ~s through4160fthe65kDantigenisdisclosed(See,Shinnicketal.lnfect.andlmmun.
56:446-451(1988)). Another sequence UUIIC r '' " to bases 535 through 554 of thesame gene coding for the 65 kD antigen is also disclosed. The application proposes the useofthe~r...G,... ~ ' asprirnersinthepolymerasechainreaction directed toward the detection of IIlJ_ulra~ ial species.
European Patent Application No. 0 395 292 by Barry et al. published on October 31, 1990, describes a method for generating DNA probes specific for various il,ll , Specific probes are disclosed for Aeromonas hydrophl'la, Aeromonas .-n^i~/~! Clostrld~umdifficile, Al~ tli. bovls,Al~ob~r~ ,.tuberculosis.
Mycobacterium avium, Salmonella ~ ' ' , and other bacteria. A DNA probe for 2 S M. a~ium was obtained from a variable intergenic region ' the genes coding for 16S ribosomal RNA and 23S ribosomal RNA. DNA probcs forM. bovis were obtained from a variable intergenic region " the 16S ribosomal RNA gene and the 23S
ribosomal rRNA gene and from the V6 variable region of the gene coding for 16S
ribosomal RNA. Similarly, a DNA probe for M. ~ulr~....,'~sis was obtained from the 3 o variable intergenic r~gion ' the 16S and 23S ribosomal RNA genes.
The nucleotide sequence of Protein Antigen B gene (pab gene) of M,~obuclr~ lu~ r~i~ was described by Anderson and Hanson, Inf. and Immun.
57:2481-2488 (1989). The pab gene is 1993 nucleotides in length. The deduced amino acid sequence of the pab gene reveals 30% homology to a phosphate-binding protein 3 s tPs+S) from E. coli. Protein antigen b was selected for analysis because of its association with virulent strains of M. lubc. ~1 and, to a lesser extent, with M. bovis BCG and because BCG has been used with some success as a vaccine âgainst ~ub~,..,ulu~;~ See, W0 95/31571 P~ ,>'. ~ 6 e.g., Hart, P. D. and Sutherland, I., British hledical Tuberculosis Journal 2:293-29 (I 977) Baird et al. J. Gen. Microbiol. 135: 931-939 (1989) have cloned and sequenced the 10 kD antigen gene of ~Iy.obecl~, .. tuberculosis. The 10 kD antigen has s been implicated (by analogy to the 10 kD protein of M. bovis) in the induction of T-cell mediated delayed type lly,u~ l.,;Livi~y. The 10 kD antigen gene was isolated using a DNA
probe ~,UII~,~IJ 1' ,, to the N-terminal amino acid sequence of the M. bovis 10 kD
antigen. This probe was selected because of the ~ 'og;.,~l cross-reactivity between the 10 kD M. bovis antigen and the 1 OkD antigen of M. tuberculosis.
Cloning and sequencing of the M. tubercu~osis 10kD antigen gene revealed a coding sequence of 300 nucleotides which Gncode 99 amino acids having an aggregate molecular weight of 10.7 kD. The sequence was shown to be hnm~ mc to two procaryotic heat-shoclc proteins and additionally to have heat-shock like promoter sequences upstream from the initiation codon.
Rogall et al. Int. J. Syst. Bact. 40:323-330 sequenced and analyzed the 16S rRNA genes of M. tuberculosis, M. bovis, M. bovis BCG, M. ~Ubt'~Lh~US~S H37, M.
marinum, M. kansasii DSM 43224, M. simiae ATCC 25275, M. s., U,/UlUCta~. ATCC
19981,M.szuigaiATCC25799,M.gordonaeATCC14470,M.xenopiATCC19250, Mflavescens ATCC 14474, M. avium DSM 43216, M intracellulare ATCC 15985, M.
2 0 p.. l u~ul~, .ulosis ATCC 19698, M. gastrae ATCC 15754, M. malmoense ATCC 29571, M. n,,..~" ~ . ATCC 19530, M. terrae ATCC 15755, M. chelonae ATCC
14472, M. smegmatis ATCC 14468, M.fortuitum ATCC 6841 and Nocardia asteroides ATCC 3306. The data obtained from this analysis revealed the phylogenetic . ' betwoen the bacteria. The data showed that the fast growing lUycc~b~lc~t~ M.
fortuitum, M. chelonae, M. smegmatis and M.flavescens formed a distinct group separate from all of the other ~ ub~,L~ia tested. All of the slow-growing ~ uv~L~i~ species were highly related having sirnilarity values greater than 94.8%.
Hermans et al. Inf. and lmm. 59:2695-2705 (1991) determined the sequence of the single copy insertion element IS987 from M. bovis BCG. The sequence 3 O of IS987 was noted to be virtually idenvical to the insertion sequence IS986 from M.
tuberculosis and to reside in a region of the M. bovis BCG ~ ' Ul~U:IVIII~; containing 20 identical copies of a 36bp direct repeat, each separated by 35-41bp of spacer DNA. The data also indicated that IS987 is nearly identical to IS6 11 0 from M. tuberculosis, differing in that IS987 has an open reavding frame (ORF) designated ORFa in a single ORF whereas :~ s IS6110 contains ORFa composed of two different ORFs. These open-reading frames may play a role in the expression of a putative i , The data also show that the size and ~ of the direct-repeat contarning region of thG M. ~UZ~ IVS-I~
' UIllOSV.,~ is puly.-lu~ , and that these repeats were not found in nine other , wo 95/31S7 21q0090 6 ~lly~ulJ~ ,.ial species tested. The authors also suggested that the poly--lulL,lli~--- seen in the IS insertion sites aIlow the easy typing ûf strains ûf M. tuberculosis cûmplex by restriction fragment length ~olylllull ' analysis.
Thierry et al., Nucl. Acids Res. 18:188 (1990) clûned and sequenced the s insertion sequence IS6110 from M. ~u~ ulv~.. ,. The DNA sequence of 1361 nucleotides showed ~ of insertion sequence (IS) inclu&ng inverted repeat sequences which are (28 bp in length with 3 ' ' bps and direct 3 bp repeats at each end ofthe IS elemen~ The authors suggest that this IS element will be useful as a probe fûr the r~ of the M. ~uverculûsis complex.
As noted above, nli~ probes are used for the detecùon of valious IlliCIvul~., by means of nucleic acid llyblidi~lliUII using techniques such as dot blot, slot blot, Southem blots, solution l~yblidi~lliUII, in situ llyblilli~Liull and others.
Alternaùvdy, the polymerase chain reaction may be used which amplifies the target DNA.
U.S. Patent No. 4,683,195 to Mullis et al. describes the details of the poly~nerase chain 15 reactiûn.
Of interest to the background of the invention, is an altemate mechanism for target An~rlifir ~i nn known as ligase chain reaction (LCR). In LCR, probe pairs are used which include two primaTy probe partners (first and second probes) and two secondary probe partners (third and fourth) all of which are employed in excess~ The frrst 2 o probe of a probe pair hybridizes to a frrst segment of the target strand and the second probe of a probe pair hybridizes to a second segment of the same target strand, the first and second segments being conùguous so that the primaly probes abut one another in 5' phosphate-3' hydroxyl relaùonship and so that a ligase can covalently fuse or ligate the two probes of the pair into a fused product. In addition, a third (secondary) probe can 2s hybridize to a portion of the first probe and a fourth (secondaly) probe can hybridize to a portion of the second probe in a similar abutting fashion~ Of cûurse, if the target is iniùally double stranded, the secondaly probes will also hybridize to the target- .,1 in the first instance. Once the fused strand of primaly probes is separated from the target strand, it will hybridize with the third and fûurth probes which can be 3 o ligated to form a r ~ " . q ~ - y, secondaly fused product. It is important to realize that the fused products are r ly equivalent to either the target or its ~ By repeated cycles of llyl,l;di~_Liu" and ligation, A~ `ri~ of the target sequence is achieved~ This technique is described more completely in K~ Backman, et al EP-A-320 308 published June 14, 1989 and K~ Backman, e~ al, EP-A-439 182 published July 31, 1991.
A potential problem associated with ligase chain reaction is background signal caused by target i...1~ ligation of the rrobes. Since the third probe hybridizes to the f~rst probe and the fourth probe hybridiæs to the second probe, the WO95/31571 I_l/. ~ 16 2~ sao~o 7 ~ ';
probes, which are added in excess, can easily form duplexes among tbemselves. These duplexes can become ligated i~ y of the presence of target to form a fused product which is then ' ~ ' ' ' from the desired amplified target, yet which is still capable of supporting further Alll~ .. Although target i.~ blunt-end s ligation of these duplexes is a relatively rare event, it is snfficiently common to cause undesirable high background signals in diagnostic assays.
Some attempts to overcome this background problem have been published.
For example, WO 90/01069 (Segev Diagnostics) and GB 2 225 112 A (Imperial Chemical Industries Plc.) describe versions of a ligation-based ,.~ I ;r~l l scheme which includes a puly~ mediated gap-filling step prior to ligation. In addition7 EP-A~39 182 to K.
Backmanefal.publishedJuly31, l991,teachesvariationsofLCRthatreduce k~. ' One such variation involves gap filling and ligation.
In the gap-filling ligation method described nn EP-A439 182 to Backman et al. published July 31, 1991, instead of using probe pairs capable of forming blunt-ended duplexes, at least one probe of a probe pair initially includes a "modified" end which renders the resultant duplex "non-blunt" andlor not a substrate for the ligase catalyzed fusion of the probe pair. A "modified" end is defined with respect to the ponnt of ligation rather than with respect to its ~ - y probe. A "modified end" has omitted bases to create a "gap" between the terminus of one probe of a probe pair and the 2 0 terminus of the other probe of a probe pair. Other " ~ c include mismatches between a probe and a t~rget sequence. 'lcorrectionll of the l ~ .... is then carried out to render the probes ligatable. "Correction" refers to the process of rendering (in a target dependent manner), the two probes of a probe pair ligatable. Thus, only those probes hybridized to t~rget, target ~ ' orpul~llu.,lcuLiJ~ sequences derived 25 therefrom, are corrected. "Correction" can be: . ' ' ' in a variety of ways depending on the type of modified end used.
There continues to cxist a need in the art for new reagents and methods for the rapid and sensitive detection of M. ~, ' and for bacteria of the genes Afj~uZn~t~, .~,..

WO g513 15 7 1 1 ~, I I IJ "' A C
2~ 90390 SUMMARY OF THE INVENTION
The present invention is &rected to ~ v ' ' probes useful for detection of target DNA from a plurality of l~qycobacteri~vn species. Such an vl;vullu-,lcOlid~ probe is from 10 to about 50 nucleotides long and possesses sufficient y or homology to the sequences shown in SEQ ID NOS. 34, 39, 44, 49, s 54, 59 and 64 to hybri&ze with such sequence or its ~ul~ under hybridizing con&tions, as defined herein. Sufficient c", ,~ ;Iy or homology generaDy requires about 80% to 100% . . " . ~ y or homology preferably 90% or more. Shorter probes typically require higher percentage ranges, while longer probes typically are useful with lower percentage ranges. Preferred are probes in e range of 15 to 40, usually about 0 20-25 nucleotides in length. Such an ~ r probe detects at lest two species of ~yLuva~ ;. .., preferably three or more, and ideally all species, while not cross reacting substantially with other related organisms including closely related organisms. Preferred examples of such ~-l v ' ' probes of the invention are the probes of SEQ ID NOS.35-38, 40-43, 45-48, 50-53, 55-58, 60-63, 65-68, 69-72, and 73-76.
Another aspect of the present invention includes probe . . .~ useful in detecting target DNA from ~l~ycObu.,~ .. species including: probe set 8 (SEQ ID
NOS. 35-38); probe set 9 (SEQ ID NOS. 40-43); probe set 10 (SEQ ID NOS. 45-48);
probe set 11 (SEQ ID NOS. 50-53), probe set 12 (SEQ ID NOS. 55-58), probe set 13(SEQ ID NOS. 60-63), probe set 14 (SEQ ID NOS. 65-68), probe set 15 (SEQ ID NOS.2 o 69-72), and probe set 16 (SEQ ID NOS. 73-76) as set forth in Figure 2A-2C and ~mhin~ nc thereof.
Other aspects of the present invention include methods for detecting target DNA from bacteria of the genus ll~j~v~u.~, using the ligase chain reaction using labelled ~ probes as provided by the ~ u~ of the present invention~
2s The methods of the present invention generally comprise provi&ng a sample suspected of containing said target DNA; provi&ng one or more probe sets according to the of the present invention wherein at least one probe of said probe set has a label capable of detection; and provi&ng one to three v.,v~y ' ' i L ~ . ' , apolymerase. and a ligase. The following cycle is then performed at lea3t once: mixing 3 o said probe set with said sample suspected of containing said target DNA; denatuling said mixture of said probe set and said sample suspected of containing said target DNA;
hybri&zing said denatured probe set to said denatured target DNA thereby creating hybridized probes; correcting said hybri&zed probes in a template dependent manner thereby creating adjacent probes; ligating said extended "corrected" adjacent probes using 3 s said ligase to form I~UI 1, ' ' probes; detecting said label in said , ' probes.
"Correction" as used herein is used in the same sense as in EP 439,182.

WO 95/31S71 r~l,.,... . 16 For the above mentioned ~,1:,. " .". 1. . .~ ;. 1. , probe UUlllUU~i~iU..~ and methods, bacteria from nûn~ ,ub~i genera are substantially not detected, while multiple species and/or strains within the My. ~Il . ... i.. .., genus are detected.
Additional aspects of the invention include kits useful for the detection of s M y~ub~Yc~, , the kit comprising suitable containers containing one or more probe sets according to the present invention, and a ligase reagent. Additional kits of the present invention include a ligase reagent, a polymerase reagent, one or more deo~y.lu~l~v~l~
1~ r I , one or more probe sets and wherein at least one probe from said probe set has a label.
1~
BRIEF DESCRIPTION OF THE FIGURES
Figure lA, lB and lC illustrate M. ~ ulu.,is species-specific target DNAs and n~ i. Ir probes aligned with their respective targets.
Figure 2A, 2B and 2C illustrate llry~ù~t~ genus-specific target DNAs and ~ ' ' probes aligned with their respective targets.
. In the figures the bases are designated as follows: A = adenine; T -thymine; G = guanine; C = cytosine; and I = inosirle. In figure 2A, the bases shown in lower case are ' with respect to target. Also in the figures the haptens are designated as fûllows: CZ = carbazole; AD = ;~ "..". nl, F = fluorescein; B = biotin.
Tyac~DNAsaredoobles~ded,~ho~lgh:u~h~wnllsodyasmgles~ggd.

wo 95/3157~
21qO0qO 10 DETAILED DESCRIPTION OF THE INVENTION
The . ~ f v~ , sequences of the present invention are derived from the gene coding for protein antigen B from M. tuberculosis, Anderson et al., Infect~o~ and Immunit~ 57:2481-2488 (1989); from the direcl repeats reported along with insertion element IS987 from M. bovis, Hermans et al., Infection and Immunity 59:2695-2705;
s from the insertion-like element IS6 110 from M. ~ al~sis, Thierry et al., Nucleic Acids Res. 18:188 (1990), from the 16S ribosomal RNA gene of M. ~ul,., ' - according to Rogall et al. (Int. J. Syst. Bact. 40:323-330 1990), from the gene coding for 65kD heat shock protein of M. tuberculosis according to Shinnick et al. (Infect. and lrrlmun., 56:446-451), and from the gene coding for the 10kD heat shock protein of M.
0 tuberculosis according to Baird et al. J. Gen. Microbiol. 135.931-939, (1989).
The modified ligase chain reaction (LCR) utilized in the present invention uses two pairs of probes herein designated A, B (primary probes), and A', B' (secondary probes). Probe pairs as used herein refers to two prQbes which are directed to the same target strand and which will ultimately be ligated to one another after annealing to the target. At least one prQbe of one of the probe pairs initially includes a "modified" end which renders the resultant duplex "nonblunt" and/or not a suitable substrate for a ligase catalyzed fusion of the two probe duplexes. A "modified end" is defined with respect to the point of ligatiQn rather than with respect to its .,, ~ ' y probe. A "modifled end" has omitted bases to create a "gap" between one probe terminus and the next probe 2 o terfninus when the probe pair is annealed to a target sequence. Other modified ends include a base ,..;~ " ~ h- I with the target sequence.
In most ~ ' of the present inYention, a modified end is referred to as a "recess", the recess being the gap between two primary or two secondary probes after hybridizing to the target. The presence of these modified ends reduces the falsely 2s positive signal created by blunt-end ligation of ~ y probe duplexes to one another in the absence of target "Correction"ofthe-,.~ l....is ' . '~/carriedouttorenderthe probes ligatable. As used herein "correction" refers to the process of rendering, in a target dependent manner, the two primary probes or the two secondary probes ligatable tQ their 3 o partners. Thus, only those probes hybridized to target, target ~ or AUul~ l. vLi~ sequences generated therefrom are "corrected." "Correction" can bea, ~ f~l by several procedures, depending on the type of modified end used. Gap-filling and nick translation activity are two correction methods described further in the examples.
3 5 As used herein, "point of ligation" or "intended point of ligation" refers to a specific location between probe pairs that are to be ligated in a template-dependent manner. It is the site at which the "corrected" probe lies adjacent its partner in 3'-WO95/31571 1~ a.~r~ 16 21 9009û 11 -hydroxyl 5'-phosphate ~ iu...l~ . For each set of four LCR probes there are two "points of ligation", a point for the primary probe pair and a point for the secondary probe pair. In CUI~ iulldl LCR the two points of ligation are opposite one another, thus forming blunt ended duplexes when the probe pairs hybridiæ to one another. In the LCR
s method used in most ClllbUI ~' of the present invention, the points of ligation are not opposite one another; but are displaced from one another by one or more bases by virtue of the gaps. The exact point(s) of ligation varies depending on the secluences chosen and, thus is further defined in the context of each;
Each of the probes comprise J~AYI ~ ' acid (DNA) which may be routinely synthesized using CUIl~ iulldl nucleotide 1 ' . ' ' chemistry and the incnllmf n-Q available from Applied Biosystems, Inc, (Foster City, CA): DuPont, ~'Wilmington. DE); or Milligen, (Bedford, MA). rh , ~ yL~iu~ of the 5' ends of the appropriate probes, is necessary for ligation by ligase, and may be a. f . ",,1,l: ',..I by a kinase or by commercial synthesis reagents, as is known in the art~
In general, the LCR methods useful in the practice of the present invention comprise repeated steps of 1. - ~ of the target DNA and (a) hybridizing the modified probes to the target (and, if double stranded so that target c~ is present, tothetarget~ ) (b)correctingthe,...,l;i;~-l;..,.inatargetdependentmanrler (e.g. filling the gap) to render the probes ligatable; (c) ligating the corrected probe to its 2 0 partner to forrn a fused or ligated product; and (d) dicQnnio~in~ the fused product from the target and repeating the llylJIid;~liull, correction and ligation steps to amplify the desired target sequence. Steps (a), (c) and (d) are essentially the same for all of the ~ u. l , and can be discussed together. Step (b) varies depending on the type of ' employed, but only gap filling and "exo" variations are discussed herein.
2s "HyLi liL~I~iull" or ''~I,yblilliLillg" conditions is defined generally asconditions which promote nucleation and annealing. It is well known in the art, however, that such annealing is dependent in a rather predictable manner on several parameters, including ~. ., .1~ . ,,1. " ~, ionic strength, probe length and G:C content of the probes. For example, lowering the i r of the reaction promotes annealing. For any given set 3 o of probes, melt t~,.ll~J.,.~ILllC, or Tm, can be estimated by any of several known methods.
Typically, diagnostic ., r ~' ~ utilize 1- ~ ~lid;L~iUII i , ' which are slightly below the melt i , . Ionic strength or "salr' ~ ,- . also impacts the melt t~ r, since small cations tend to stabilize the formation of duplexes by negating the negative charge on the 1 ' . ' ' backbone. Typical salt, depend on 3s the natrlre and valency of the cation but are readily understood by those skilled in the art.
Similarly, high G:C content and increased probe length are also known to stabilize duplex formation because G:C pairings involve 3 hydrogen bonds where A:T pairs have just two, and because longer probes have more hydrogen bonds holding the probes together. Thus WO 9S/31571 r~ 6 219~0q(~ 12 a high G:C content and longer probe lengths irnpact the "llyblidiL~Liu.~ conditions" by elevating the melt temperature.
Once probes are selected for a given diagnostic application. the G:C content andlengthwillbeknownandcanbeaccountedforin-l~....-";.,;,l~preciselywhat s "lly~lidi~Liull conditions" will encompass. Since ionic shrength is typicaDy optimized for enzymatic activity, the only parameter left to vary is the t,~rnrl~r~hlr~ For improved specificity, the llyblill;L~LLiull temperah~re is selected slightly below the Tm of the probe;
typically 2-10 C below the Tm. Thus, obtaining suitable "hybridization conditions" for a particular probe set and system is well within ordinary skill of ûne practicing this art.
For LCR, the probes are added in ~J,UIU~dllld,i~y equimolar ( since they are expected to react ~I "i, 1, ;. ." ,. ~ ly Eæh probe is generaDy present in a ranging from about 5 nanomolar (nM) to about 90 nM; preferably from about 10 nM to about 35 nM. For a typical reaction volume of 5011L, this is equivalent to adding from about 3 x 101 1 to about 1 x 1012 molecules of each probe; and around 5 x 1011 molecules per 50 IlL has been a good starting point. The optimum quantity of probe used for each reaction also varies depending on the number of cycles which must be performed and the reætion volume. Probe ~.. . , ~I;- ~ ~ can readily be determined by one of ordinary skill in this art to provide optimum signal for a given number of cycles.
FoDowing provision of the probes, the next step in the LCR medhûd utilized in dhe present invention is the specific correction step followed by the ligation of one probe of a probe pair to its adjacent partner. Thus, each corrected primary probe is ligated to its associated primary partner and each corrected secondary probe is ligated to its associated secondary partner. An "adjacent" probe is eidher one of two probes llyblidiL~
widh dhe target in a contiguous orientation, one of which lies widh its I ' ~ ' y' ~ 5 ' end in abutment widh dhe 3' hydroxyl end of the partner probe. "Adjacent" probes are created upon correction of dhe modified end(s) in a target dependent manner. Since enzymatic ligation is the preferred medhod of covalendy attaching two adjæent probes, the term "ligation" will be used d~roughout the application. However, "ligation" is a general term and is to be understood to include any medhod of covalendy attaching two probes.
3 o The conditions and reagents which make possible dhe preferred enzymatic ligation step are generaDy known to dhose of ordinary skiD in the art. Ligating reagents useful in the present invention include T4 ligase, and prokaryotic ligases such as E. coli DNA ligase, and rhermus aqua~icus DNA ligase available from Molecular BiologicalResources (Catalog Nos. 107001 and lO7002, Milwaukee, WI). A lh- - - ,~ ligase is 3 5 presently preferred for its ability to maintain activity during the thermal cycling of LCR.
Absent a thermally stable ligase, the ligase must be added again eæh time the cycle is repeated. Also useful are eukaryotic ligases, including DNA ligase of Drosophila, reported by Rabin, et al.. J. Biol. Chern. 261:10637-10647 (1986).

wo 95/31571 f ~.lIV~ 6 2f 9009~ 13 Once ligated, the fused (Iwl,. ') probe is dissociated (e.g. melted) from the target and, as with Cu~ iul.al LCR, the process is repeated for several cycles.
The number of repeat cycles may vary from I to about 100, although from about IS to abûut 70 are preferred presently.
s It is desirable to design probes so that when hybridized to their , J (secondary) probes, the ends away from the point of intended ligation are not able themselves to panicipate in other unwanted ligation reactions. Thus, ligatable sticky or blunt ends should be avoided. lf such ends must be used, then 5~ terminal phosphates should be avoided, eliminated or blocked. This can be ~ rl either through sy.ltl.~ illg ~ ' ' probes (which normally carry no 5~ terminal phosphate groups), or through the use Of r ~ , - enzymes to remove terminal phosphates (e.g. from, ' O ' ' - generated throu~h restriction digests of DNA).
Alternatively, ligation of the "wrong" outside ends of the probes can be prevented by blocking the end of ât least one of the probes with a "hook" or marker moiety as will be described in detail below. In the absence of one of the above techniques, the outside ends of the probes can be staggered so that if they are joined, they will not serve as template for exponential ~., ,1 .l: ~i, ," i, Following ~.I.q.l;l';l _1;",~, the amplified sequences can be detected by a number of l,UII~, '' I ways known in the art. Typically, detection is perfommed after 2 0 separation, by J~ the amount of label in the separated fraction. Of course, label in the separated fraction can also be determined ~ulJ~ ,ly by knowing the total amount of label added to the system and measuring the amount present in the, . ' fraction.
Separation may be r , '' ' I by Cl~,.,LIU~lv~ ;S, by ~,Iu~ or by the preferred method described below.
2s In a particularly preferred r~nnfi~llrrlfinn haptens, or "hooks" (also referred to as labels), are attached at the available outside ends of at least two probes (opposite ends of fused product), and preferably to the outside ends of all four probes. A "hook" is any moiety having an affinity to a binding partner. Typically, the hook(s) at one end of the fused product (e.g. the 5' end of A and the 3' end of A') comprises an antigen or 3 0 hapten capable of being i.. , .1.;1;,.. 1 by a specific binding reagent (such as anvibody or avidin) coated onto a solid phase. The hook(s) at the other end (e.g. the 3' end of B and the 5' end of B') contains a different antigen or hapten capable of being recognized by a label or a label system such as an antibody-enzyme conjugate. E~emplary hooks include but are not limited to ~IUUIIIO~ IIS~ cat~lysts such as enzymes, I ~ ~
3 5 1 .1~ r . . cv r - - ~ raoioactive elements such as 32p, biotin, fluorescein, digoxin, ~ ,v~ y~ ' " dansyl, 2~uLI~, ' 1, modified nucleotides such as bromouracil and others, ~ . .,, ,1.l. . ". .,: - y ' ' . Iectin/~a l,u~.y. ' pairs, en~ymes and their co ~nrr~,?n~ others known in the art. Other emplary hooks include . . .

WO95/31571 ,~,1lU.. .. ~! ~6 21 90090 ~

d. I ' ~ acetic acid as described in U.S. 07/808,508 (by Mattingly, P.G., entitled, "Haptens, Tracers, I ,, and Antibodies for 3-phenyl-1-: ' Acids") and carbazole and di~ uru~ derivatives as described in co-owned, co-pending U.S.Patent Application Serial No. 071808,839 (by Fino, J.R., entitled, "Haptens, Tracers, s T" ~ r,~ ~ and Antibodies for Carbazole and Di~ r, . . ~ " Derivatives", both filed December 17, 1991.
A method for adding a hapten to the 3'-end of an, ~ ' is disclosed in co-owned, co-pending U.S. Patent Application SeriaT Numbe} 07/630,908, filed Decembe} 20, 1990. Othe} methods (e.g. Amino Modifie} n, Clontech, Palo Alto, Califomia) are known and ,,, ll~, available for labeling 3' and 5' ends. The method fo} adding a hapten to the 5' end is through the use of a I ' I ' " }eagent as described in Thuong, N.T. et al., Tet. Letters, 29(46): 6905-5908 (1988), o} Cohen, J.S. et al., U.S. Patent Application SeriaT Number 071246,688 (NIIS o}de} no. Pat-Appl-7-246,688 (1988)). Thus, exemplary ligated "lir,."..,. l. v~ may have a carbazole at one end and an ,q~ rqn~ at the other end for the detection by the IMx~ instrument (Abbott T qhnr. ~ril~5~ Abbott Park, n,) using the Illil lu,u~u liCIC enzyme y (MEIA) technology. The assay p}otocol is similar to that used in the ~nmrn~r~ y available alpha-fetoprotein assay, with the following adaptions: (I) the anti-alpha-fetop}otein antibody coated ~ u~u li"l~ are }eplaced with anti~rbazole antibody coated .. i.,lu,u~ ,s, and 20 (2) the conjugates of anti-alpha r~, uu-u~.-l antihnfli~c ql' ~ r are replaced with the conjugates of anti-3-phenyl- 1-. ' acid, ~ ,- l. l r' 1' The p}otocol for the IMx(!9 MEIA assays is furthe} desc~ibed in K.
Backman etal., EP-A-439,182 published July 31, 1991. In brief, the p}otocol is as 25 follows. A 100 IlL of the }eaction mixture which has been amplified by LCR is pipetted into the sample well. 30 IIL of this sample is then pipetted into the incubation well, the b~uiulc antibody coated ..ul,lulu~ s are added to the well. An appropriate period of incubation follows which allows the formation of a comp~ex consisting of antibodies and nucleic acid sequences with the carbazole ends. After the incubation, the 3 0 mixture is pipetted onto the glass fibe} captr,re matrix of the IMxt!~ reaction cell, and antibodies conjugated to alkaline ~ r are added. This leads to a ll~lu,ual~ enzyme complex which will stay on the surface of the glass fibe} captrlre matrix. Afte} the removal of excess reagent in a wash step (throughout this protocol, the blotter beneath the glass fibe} captu}e matrix abso}bs }eagent solutions which s would otherwise overflow the glass fibe} captu}e matrix), the glass-fibe} capture matrix is treated with 4~ lu~b~,lli~yl phosphate (MUP). The surface-bound enzyme converts the IlUllrlUUI Uo~ MUP to 4-1-l~ lullL~,llirel~ (MU), who~e r~i .. r~ can be measured. Th~e numerical values given in the following examples are the rate }eads of this W09SB1571 r~ l~U~
2 1 9~090 process, expressed in /3~,c/~,c (c/s/S). The amount of ligated probes is direcdyrelated to this rate. This concept of MEIA readout of labeled, ' ~ ' ' is described in European Patent Application, publication No. 357,011, published March 7, 1990, "Deoection and ~ ;.... of Target Nucleic Acid Sequences," to Laffler, T.G., et al.;
s and elsewhere.
In the illustrative examples which follow, probe pairs are labeled with a "~ " hapten and a "biotin" hapten or with a "carbazole" hapten and an - acid ("~ ,e") hapten. Typically, "fluorescein" and "biotin" are used together and "- ' - " and "carbazole" are used together in accordance with the description aboYe, although any . . " . .I .;, - -~ ,. " . of virtually any haptens would be possible.
Preferably, each member of a probe pair has a different label.
In most of the examples, results were read in an IMx@) instrument. This is o nmm~rei~lly available from Abbott T ~l~nr~t~ (Abbott Park, Illinois) and is described in EP-A-288 793 and in Fiore, M. et al Clin. Chem., 34/9:1726-1732 (1988). It should be noted that the IMx~D instrument typically generates "machine" noise or background in the range of 5-12 /~.,/~c. Other equally suitable methods of detection useful in the practice of the present invention include ELISA, EIA, and ;,.u ..~,o"lu ul.~a~utld~l.y and nucleic acid ll.yl/dL~a~iull techniques including southem blotting, dot blotting, slot blotting, solution ll~ idi~a~iull and others well known in the art.
2 o Ouantities of polymerase are expressed in units, defined as follows: I unit of enzyme equals the amount of enzyme required to incorporate 10 nanomoles of total nucleotide into acid-insoluble material in 30 min at 70~C. Units of ligase enzyme are defined herein as: I mg of 95% purifid Therm~s .,~ ' DNA ligase has a specific actiYity of about I x 108 units. While this is not precisely ~ - I;, ., l and may vary by 2 s as much as 20%, ~.l,1; ,: ~l ;. ,., is within the skill of the routine l The invention wiTI now be describd further by way of examples which are illustrative of the invention and are not intended to limit it in any way. For example, sequences of specific length are listed. It should be understood that sequences covering the same map positions but having slightly fewer or greater numbers of bases are deemd 3 o to be equivalents of these sequences and fall within the scope of the invention, provided they will hybridize to the same positions on dhe target as the listd sequences. It is also understood that sequences having homology to the target sequences of about 80% or more also fall within the scope of the present invention. T?refcrably any base ~ - - in the sequences of the present invention lie 3 or more nudeotides away from the modified ends.
3 s Target sequences and probes were selected so as to irlclude a "stop base"
as taught in EP 439 182 by K. Backman et al. published July 21, 1991 to temlinate gap filling extension precisely at the point of ligation so that the extendd probe abuts its probe partner and can be ligated to it.

wo g~/31~71 2~ qO090 16 For the purposes of the following examples line diluent (LD) is a standard IMxX buffer reagent used to detect machine noise in the absence of target DNA and probe. All data are expressed as IMx~ rates of counts~ ,u--d/~ ' (c/s/s).
s Example 1: Detection of M. tuberculosis Using Probe Set I
(SEQ ID NOS. 2, 3, 4, and ~) Probe set I (SEQ ID NOS. 2, 3, 4, and 5) was selected to detect a target sequence in M. tuberculosis u~ ul~-lu g to nucleotides 347-390 (SEQ ID NO. I ) of the protein antigen b (pab) gene of M. Iuv~, ulu~s. LCR reaction mixtures contained 50 mM
EPPS, 30 mM MgC12, 20 mM K+ (from KOH and KCI) (LCR Buffer), 10 ~LM NAD, 1.7 laM dATP. and 1.7 ~LM dCTP (gap filling nucleotides), I x 1012 molecules of each ~1;L- ~ Ir probe, 18,000 units of Therrnus ~hermophilus DNA ligase, 2 units Thermus polymerase, (Molecular Biology Resources, Milwaukee, Wl, cat. no. 1070.01) 2 llg human placental DNA, and target DNA as set out in Table I in a final volume of 200 1. Cycling was performed on a Perkin-Elmer model 480 ~ ,y~ ,l at the followir~g settings: 93 C for I second; 65 C for I second; then 68 C for I minute 15 seconds for a total of 40 cycles. Probes were labelled with carbazole and - ' as described above. Following ~ ligation products were detected using a sandwich y using an Abbottautomated IMx~ analyzer as described above.
2 o Table I shows the results of LCR using probe set I with 10, 25, and 100 molecules of target DNA derived from M. ~ub~, .ulv~.5 Erdman. Table 2 also shows the results of LCR using target DNA, derived from M. avium, M. in~l L~Ll~lluLI I~, M. kansasii, and human placental DNA.
Table I
Tar~et DNA IMx~ Rate(c/s/s) M. tr- S~ . ' ~ Erdma 10 molecules 432.9 25 molecules 1149.8 100 molecules 1636 9 M. avium 107 molecules 6.5 M. al ~ ~ T~1 7~re lo7 molecules 5.8 M. kansasii 107 molecules 5.8 Cont~ols human placental DNA (2~Lg) 71 The results of these ~ ; " ~- t~ show that probe set 1 (SEQ ID NOS. 2,2 s 3, 4, and 5) was capable of detecting as few as 10 molecules of DNA derived from M.

WOg5/31571 ~ t ~ O O ~
tub., cul~sis and showed no cross-reactivity with as many as 107 genomes of DNA
derived from M. avium, M. intracellulare, and M. kansasii.
Example 2: Detection of M. tuberculosis Using Probe Set 2 s (SEQ ID NOS. 7, 8, 9 and 10) Probe set 2 (SEQ ID NOS. 7, 8, 9 and 10) was selected to detect a target sequence in M. tuberculosis UUllU~.U ' ~ to nucleotides 350-387 (SEQ ID NO. 6) of the pab gene of M. tuberculosis. LCR was performed as described in Exarnple I except that human placental DNA was present at 330 ng/rxn, and cycling was performed on a Perkin-Elrner model 480 Ll~ u~yul~,l at the following settings: 94 C, I second; 55 C, I second;
60 C for 55 seconds, for a totai of 40 cycles. M. ~ u ~u~ target DNA was present at IO I~ ~Liull, other target DNAs were present at l x los ~ J~
Probes were labelled with carbazole and ;~ " ~ f and detected as above. Results are shown in Table 2.
Table 2 T~r~t DNA x(!l) c/sls M. tuberculosis 201 26~.0 M. kansasii Florisse ~.:~
M.marinum 11564 7 M. simiae 25273 6 `.J
M. szulgai 23069 .~
M. xenopi 19971 ~.:
M. malmoense 29571 7.
M. fortuitum 6160 7.~
M. chelonae 35752 7.,-M. avium Ser.#l 7.
M. intracellulare Howell-P42 7.' M. ir~l/ ~" ' e P-54 7.~
M. intracellulare Ser. 21 7.~, Ç~
human placental DNA (330 ng) 7.2 Results indicate that probe set 2 (SEQ ID NOS. 7, 8, 9 and 10) was capable of detecting as few as 10 1 ~ /~GU~LiUn of M. tuoerculosls DNA while other target DNAs present at I x 105 ll-ul~ ,s/reaction gave little if any detectable signal.
20 E%ample 3: Detection of M. tuberculosis Using Probe Set 3 (SEQ ID NO. 12, 13, 14, and 15) Probe set 3 (SEQ ID NOS. 12, 13, 14, and 15) was selected to detect a target sequence in M. ~ul,., .~sl~ CUIIG~.U~ " ~ to nucleotides 2544-2593 (SEQ ID NO.
11) of the direct repeat sequences around IS987. Reactions were perfor~ned as described .... .... ..... . .. .. _ . _ .. _ .. _ . , . ., . , _ _ _ . . . .

WO 9!i/31571 ~ 16 in Example 1 except that the gap filling nucleotides were dCI'P and dTTP. Target DNAs and their ~ are shown in Table 3. In addition, LCR was performed using both I x 1012 molecules and 2 x 1012 molecules of each ~ r probe~ LcR
cycling was performed on a Perkin-Elmer model 480 thermocycler at the following s settings~ 93 C, I sec; 67 C, I sec; 70 C, I min 15 sec.
Probes were labelled with carbazole and ~ as described above and data was obtained using an automated IMx~ analyzer as described above.
Table 3 Tar~et DNA IMx~ rate (clsls) 1 x 1012 mt~l probe ~~ 2 mol. probe M. tuberculosis Erdman (25 molecules) 348.8 418.9 M. tubercu~osis Erdman (lOOmolecules) 1174.7 41g.1 Ç~
humanplacentalDNA (211g) 19.7 12.7 These data illustrate that probe set 3 (SEQ ID NOS. 12, 13, 14, and 15) was capable of detecting 25 and 100 molecules of M. lul,~, 1,..~05i~ target DNA usrng either I x 1012 molecules of probe/rxn or 2 x lol 2 molecules of probe/rxn.
Example 4: Detection of M. fuberculosis Using Probe Set 4 o (SEQ ID NOS. 17, 18, 19, and 20) ~ Ir probe set 4 (SEQ ID NOS. 17, 18, 19, and 20) was selected to detect a target sequence in M. tuberculosis UUll~ to nucleotides 535-578 (SEQ ID NO.16) of the IS-like element IS6110 of M. tuberculosis. In these , reactions were performed as described in Example I except that dCTP and dTTP were used for gap filling and cycling was performed in a Perkin-Elmer Model 480 lu~yul~,~ at the following settings: 94 C, I second; 65 C, I second; 70 C, 55 seconds for a total of 40 cycles. Target DNAs and their quantities are sbown in Table 4.
Reaction products were then cooled and analyzed as described in Example 1. Probes were labelled with carbazole and - ' as described above.

WO 95/31571 ~ 5,'~- 16 Tabie 4 TarPet DNA I~x~ Rate (c/sls) Comment on Data SPread M. ~ub~ lu~l~ Erdman (10 molecules) 607.2 M. i/~ 1419 (106 molecules) 8.7 (all 10 samples less than 10) M. avium CSU Ser. #l 6 molecules) 54-5 (I sample at 471.4 and 9 samples less than 10) M. kansasii 1203 (106 molecules) 15.7 I sample at 84, and 9 samples less than 10) (I sarnple each at 526.5, 509.7 human placental DNA 25.9 and 358.4, (2~g) and 77 samples less than 10) LD 6.6 These data iDustrate that probG set 4 (SEQ ID NOS. 17, 18, 19, and 20) is capable of detecting as few as 10 molecules of M. ~ target DNA. With the exception of an apparent "outlier", Probe set 4 (SEQ ID NOS. 17, 18, 19, and 20) gave s no signal when tested against 106 molecules of target DNA derived from M. i/.h ~. " ' 1419, M. avium CSU Ser #1 and M. kansasii 1203. Human placental DNA (211g) gave an average IMx~) Rate of 25.92 c/sls which is artificaDy high due to 3 outliers.
Example 5: Specificity of Probe Set 5 for M. tuberculosis (SEQ ID ~OS. 22, 23, 24 and 25) Oli~ probe set 5 (SEQ ID NOS. 22, 23, 24 and 25) was selected to detect a target DNA ,u~ ' ,, to nucleotides 585-627 (SEQ ID NO. 21) of the pab gene of M. ~ul,~ osis.
Each reaction was preformed in LCR buffer and contained 5 x 10 1 1 molecules of each probe/rxn, 3400 units of ligase, 0.5 units polymerase, 50 ng of human placental DNA, 2000 copies/reaction of target DNA, all in a final volume of 50~LI. The gap-filling nucleotide was dCTP. Reactions were overlaid with mineral oil and cycling was performed in a COY ~ lu~ at the following settings: 85 C, 30 sec; 55 C, 20 sec, for 45 cycles. The 50~1 samples were then diluted to 200 111 with IMx~ line diluent 20 to provide adequate volume for IMx~ operation. Labels were biotin and fluorescein.
Results are shown in Table 5.

WO 9513157~

Table 5 T;~r~ t DNA IMY'~ ~- tr~ (C/S/S) M. tuberculosis 02 :. ~3 ',0 1 _ ~0.1 37 _~3.
:37RV ~' .' 0.
, dman .~
M. scrofulaceum _ ~130 ' ~. ' .
,,02 .:
M. for~uitum 47 . ~
M. szulgai CAP .7 M. oovis '10 16 .8 '01 17 0.7 ` ,CG (Glaxo) 19 8.4 M. avium _R107 > 2.0 _R163 > 0 M. i,.. , " ' ~ LR158 >10 LRl05 >1 M. chelonae 1343 .
1 8_. ~
M. gordonae 1318 .J
M. terrae CAP .~
M. phlei 1516 25 ~.
M. Icansasii 1214 ).
M.marinum 1218 ~3 The IMx~9 rate for M. scrofulaceum LR130 in this experiment is suspect because of the wide spread rn the data (1687.5 clsls and 416.1 c/s~s) and in light of the low average seen for M. s.,~lac~,.". 1302 which gave an average of 8.1 c/s/s. This is possibly due to a ~ of the LR130 sample. Similarly a single, spuriously highreading accounts for the unusu~lly high average IMx~ rate seen for M. chelonae which gave IMx~ rates of 317.8 c/s/s and 47.5 c/s/s while M. chelonae 1343 gave IMx~ rates of only 8.3 c/s/s and 8.4 c/s/s. Similarly, M. phlei gave an unusually high average IMx~
0 rate by vrrtue of the fact that one of the reactions gave an IMx~D rate of 510.5 c/s/s opposed to a rate of 9.1 c/s/s for its duplicate and an average rate of 8.7 c/s/s for M. phlei 1516.
Example 6: Detection of M. ~7 ' '- Using Probe Set 6 (SEQ ID NOS. 26, 27, 28, and 29) A mn-1;fin~inn of the LCR method described above was utilized to detect a target sequence ~Ull~i_r ' to nucleotides 585-627 (SEQ ID NO. 21) of the proteinantigen b (pab) gene of M. tub~, ' using probe set S (SEQ I:D NOS. 26, 27, 28, and 29). The LCR method used in this example differs from Example 1 in that it utilizes two 2 0 sets of blunt-ended probes wherern the 5' end of probe B has a mismatch with the target W0 95/31571 s ~
2 1 90~90 corrected prior to ligation by 5' to 3 "-Y~ ' activity of the DNA polymerase used in the reaction. This method, variously known as the "exo" or "nick translation" method, the basis for which is described in more detail in co-pending, co-owned U.S. Ser. No.
07/925,402, filed August 3, 1992.
s Reaction conditions were those described in Example 1. However, reactions were run using both 2 units of DNA polymerase and 4 units of DNA polymerase in an attempt to determine the effects of increasing polymerase ' on the efficiency of this variation of LCR. This specific LCR method requires the presence of only one gap-filling nucleotide i r ' 1 ~ ', in this case dCTP. Target DNA was present at 25 molecules per reaction. Cycling was performed in a Perkin-Elmer model 480 thermocycler at the following ~settings: 93 G I sec.; 62 C, I sec; 65 C, I min. 30 sec.
for 50 cycles. Probes were labelled with carbazole and Al ~ Ligation products were detected using an automated IMx~ analyzer as described above. F, ' using 2 units and 4 units of polymerase gave nearly the same average IMx~) rates of 1745.7 C/S/S and 1734.3 c/s/s, respectively. Control l,A,U~.lilll~,llLs using only 2 llg of human placental DNA/rxn and 2 and 4 units of polymerase gave nearly the same IMx~ rates of 11.32 c/sls and 12.44 c/s/s, ~
Example 7: Detection of M. ~ubercl~losis Using Probe Set 7 (SEQ ID NOS. 30, 31, 32 and 33) ~ probe set 7 (SEQ ID NOS. 30, 31, 32 and 33) was selected to detect a target DNA ~UII~ ' ,, to nucleotides 585-633 (SEQ ID NO. 21) of the pab gene of M. t~b~ usls. LCR was carried out as described in Example I with the following exceptions: human placental DNA was present at 300 nglrxn, dGTP and dTTP
2s were used for gap filling, and cycling was performed in a Perkin-Elmer model 480 lu~ ,l at the following settings: 93 C, 1 sec; 62 C, 1 sec; 65 G 1 min. 10 sec for 40 cycles. The probe set was labelled with ~ ' and carbazole as described above.Ligation products were detected as described in Example 1. Table 7 shows the results of the assay.

WO95/~1571 I_I/LI.. ~ ' Table 7 Tii~rvrt DNA IMx(!~ Rate (cls1s) M. tuberculosis 371.7 s (10 molecules) M tuberculosls 1143.4 (100 molecules~
SQ~
human lacental DNA 10.8 (330 n3 These data show that probe set 7 (SEQ ID NOS. 30, 31, 32, and 33) was capable ofdetecting as few as 10 molecules of target DNA from M. ~UIJ~
Example 8: Detection of Bacteria of the Genus Alycobacterium Using Probe Set 9 (SEQ ID NO. 40, 41, 42, and 43) t~ ' ' probe set 9 (SEQ ID NOS. 40, 41, 42, and 43) was sdected to detect a target sequence f . ", r~ to nucleotides 721-760 of the 16S
rRNA gene of M. tuberculosis (SEQ IP NO. 39). LCR was performed as described in Example I using a Perkin-Elmer Model 480 i' y~h,l except that cycbng was performed at the following settings: 94 C, I sec.; 55 C, 1 sec.; 60 C, 40 sec. for a total of 2s 40 cycles, human placental DNA was present at 330 ng/rxn, and each probe was present at 7.5 x 101 1 111OI~Ul~,.7/~C~lhJll. Gap filling nucleotides were dCTP and dlTP. Probes were labelled with fluorescein and biotin Ir~ "ly. Table 8 shows the results of these assays.

WO95131571 .~~ 816 21 900qO 23 Table g IMx~) Rate (c/s/s) Target DNA 100 mol. tar~et 1000 mol. tarFet s M. tub.~ulo~;s ~' 7RV ^,9'.' 90, .7 :''08 ~,7,._ 84~.5 M. avium 3 : 7' . 3J~.5 _ ~147 ' 0~., 9~ .9 M. ~r~ 120 ,5''.~ 60~.6 0 1~03 ~61.9 4 0.5 M. scrofulaceum L-~195 ,.~5.. 5:~6.9 1,~121 ~2.~ 964.7 M. kansasii 01 :.~0.~ 583.4 :_17 .,J_. 946.5 5 M. chelonae ' 08 ':,.0 411.8 :9977 ' --.2 1 160.3 M.fortuitum :545 ~:. ,.7 557.9 :529 ' :,~.7 671.6 5Q~ , human placental DNA (330 ng) 79.0 These results show that probe set 16S 721-760 is capable of detecting numerous species and strains of the genus A~y-,ubu~t~; ,...
2s EYample 9: Detection of Bacteria of the Genus Mycobacterium Using Probe Set 11 (SEQ ID NOS. S0, 51, 52 and 53) ' probe set 11 (SEQ ID NOS. 50, 51, 52, 53) was selected to detect a target sequence CU~ to nucleotides 244-286 of the 65 kD heat shock gene of M. 1~l .ulu,..~ (SEQ ID NO. 49) with the aim of using the probe set to detect 3 0 bacte}ia of the genus A~y~ul,ac ~ LCR was performed as described in Example 5 except that the gap filling nucleotide was dTTP. Unless otherwise indicated in Table 9, Illyl olJ~l~,t~iidl target DNA was present at 10 pg/rxn which is the equivalent of about 2000 copies of a typical 1--1~ul,d~,t~ 1 genome. The final reaction volume was 50 111. Cycling was performed in a COY thermocyder at the following settings: 85 C, 30 sec; 55 C, 20 3 5 . sec; for 40 cycles. Labels were biotin and fluorescein. Reaction products were analyzed as described rn Example 1. Results are shown in Table 9. The data indicate that probe set I l (SEQ ID NOS. 50, 51, 52 and 53) detected all of the target DNAs tested except target DNA derived from M.fortuitum and M. terrae.

wo 95/31571 2 1 9 0 0 9 0 r~l,o.,,~ 16 Table 9 Tar~et DNA IMx~ Rate (clsls) M. tuv~l culvsis 1243.5 (s.o pg) M. avium L,~' 07 93'.3 L~' 6' 37~.9 M. scrofulace~m _`~1';0 lrl4~ s M. intracellulare . ~1~8 . 7:.6 _ ~105 '. .3 ,~. bovis 7 '.6 .~. tuberculosis 1507 7 M. hemophilus 1OJ7~~
M. szulgai 8' 8. ' ,~.fortuitum . 2.t I.lusis 8~ 0. ~
.~. marinum 10~ ., ,k'. scrofulaceum 7: ., .k'. kansasii 7''. '.~
u~ r~. l M. phlei ', 7.7 ,k'. bovis 1'-7.5 M. gordonae ,0 '.1 M. chelonae ~2-.6 M. terrae ~-9 M. malmoense 70 -.6 M. bovis BCG Glaxo 151lJ.3 Example 10: Detection of Bacteria of the Genus Mycobacterium Using Probe Set 12 (SEQ ID NOS. 55, 56, 57 and 58) Oli~ 1 V~ probe set 12 (SEQ ID NOS. 55, 56, 57, and 58) was selected to detect a target secluence ~ to nucleotides 405452 of the 65 kD
heat shock gene (SEQ ID NO. 54) of M. tub~, .ulv, i~ with the aim of using the probe set as a probe for bacteria of the genus ll~j~vvu~c~ LCR was performed as described in Example I except that the gap filling nucleotides were dATP and dG7~`P, human placental DNAwaspresentat330ng/rxn,eachprobewaspresentat6xlO12probes/rxn,and cycling was performed on a Perkin-Elmer model 480 ~ u~,yul-,l at the following settings: 94 C, 1 sec.; 55 C, I sec.; and 60 C, 55 sec., for a total of 40 cycles.
Probes were labelled with biotin and fluorescein l~.*,~i~,l~.
Reaction products were then analyzed by the automated IMX~9 procedure described in Example 1. The results of these ' -l~ ,., ,,l~ are shown in Table 10. Target DNAs and their amounts are listed in Table 10.

WO 95131571 ~ o16 21 900~0 Table 10 T~rs~t DNA ( lo4 lllu~ ul~ a~liull) ~X R ! ' (clsls) M. lu/~, ~ulosi~ 2ûl 495.6 s M. avium LR163 123 M.i/--i " ' t' LR158 213.2 human placental DNA 10.2 LD 9.9 The data indicate that probe set 12 (SEQ ID NOS. 55, 56, 57, and 58) was capable of detecting 10,000 molecules each of genomic DNA from M. tu~erculosis, M.
avium, and M. i,.~, " ', ~.
Example 11: Detection of Bacteria of the Genus Mycobacterium Using Probe Set 13 (SEQ ID NOS. 60, 61, 62 and 63) Oli~;- ,.1. .:.1;.1~ probe set 13 (SEQ ID NOS. 60, 61, 62, and 63) was selected to detect a target DNA CUI~C* ' ~ to nucleotides 477-524 (SEQ ID NO 59) of the 10kD heat-shock protein gene from M. tuberculosis. LCR was performed as described in Example I except that the gap filling nucleotides were dTTP and dGTP, probes were 2 0 present at 2 x 101 l molecules each, and cycling was performed on a Perkin-Elmer model 480 L;~ ùuy~ at the following settings: 94 C~ I sec.; 60 C, I sec.; and 65 C, 55 sec., for 40 cycles. Probes were labelled with carbazole and ~ ,ly. Reaction products were then analyzed by the automated IMx~ procedure described in Example 1.
Target DNAs and therr amounts arc listed in Table 11. The results of these, 25 are ~",.,." -,,. .1 in Table 11.

WO 95/31571 ~ ol6 Table ll IMx(D Rate (c/s/s) TarL~et DNA 1000 mcl- cules 100 molecules M. tuberculosis H37RV ~2~- .7 1632.4 35801 : 9',~.6 i237.2 M. avium 113 4~~.7 ~ 6 .0 146 :9 11l7~.4 M. i,,~ 120 : 8 ~ ; .2 1430 4 1.:, ;`.. 1 M. 195 '0 3. 1 ' ~.8 s."~ '~ 121 ~ 0.~ 1,(7 M. kar~sasii 1201 ~~3_~ 735.8 1217 --~ 9Q9 M. chelonae 108 ~~.', 177.8 19977 1~0~' 396.1 A,l. fortuitum 1545 1~ 649.1 1529 1,.3.. 646.8 Controls human placental DNA (330 ng) 93.5 LD
These data indicate that probe set 13l 54 (SEQ ID NOS. 60, 61, 62, and 63) was capable of detecting as few as 100 molecules of target DNA derived from the S ~ ub~iu~iol species and strains tested. In addition, the probe set gaYe little or no signal when used in reactions contarning human placental DNA.
Example 12: Det~ction of Bacteria of the Gerlus Mycobacterium Using Probe Se~ 14 (SEQ Il) NOS. 65, 66, 67 and 68) (lli, I ' probe set 14 (SEQ ID NOS. 65, 66, 67, and 68 were selectedtodetectatargetDNAcull~ r 1- " tonucleotides 1059-1098 (SEQIDNO.
64) of the M. tuo~ lvs,~ 16S ribosomal RNA gene. LCR assays were performed as described in Example 1 except probes were present at 5 x 101 1 molecules each and the reaction contained 330 ng human placental DNA. Gap filling nucleotides were dATP and 15 dGTP. Probes were labelled with biotin and fluorescein c*~ ly. Cycling was performed in a Perkin-Elmer Model 480 Tl~ llu-,yuh,- at the following settings: 94 C, I
sec: 57 C, I sec; 62 C, 25 sec; for a total of 40 cycles. Reaction products were analyzed as described in Example 1. Results are shown in Table 12.

WO95/31571 2 ~ 90090 .~1111 16 Table 12 Tar~et DNA (1000 Molecules) IMx R te clsls M. tuberculasis H37RV 7 2.7 35801 3 7.4 M. avium 113 6.3 LR147 ~0 M. intracellulare LR120 ',~3.1 1403 52.5 M.scrofulaceum LR195 ~22.:
LR121 '95.f M. kansasii 120 ~79.' 12-7 '60.7 M. chelonae 10' ''6: .2 19-7 104'.6 M.fortuitum 15~5 37'.5 1529 33,.6 Controls LD 9.7 human placental DNA (330 ng) 30.8 The results indicate that the probe set 14 (SEQ ID NOS 65, 66, 67 and 68) s set was capable of detecting 1,000 molecules of all of the target DNAs tested.
Example 13: Detection of Bacteria of the Genus Myco~6 l."
Using Probe Set 15 (SEQ ID NOS. 70, 71, 72 and 73) O-'~ ' ' ' probe set 15 (SEQ ID NOS. 70, 71, 72, and 73) was used to detect a target DNA corresponding to nucleotides 690-732 (SEQ lD NO. 69) of the 16S
ribosomal RNA of M. tu1,~, ,..~i~. LCR was carried out as described in Example Sexcept that the gap-filling nucleotide was dATP, and 2 x 10l2 molecules of each probe was used. Probes were labelled with biotin and flllnn-c~in Reactions were o~erlaid with mineral oil and reactions were cycled in a COY ~ ~y~ ,. at the following settings: 85 C for 30 sec; 40 C for 30 sec; for 45 cycles. Reaction products were analyzed as described in Example 1. Results are shown in Table 13.
Table 13 T r~et DNA (I ~) Jl\/lxtg) Rate (c/sls) M. tu~erculosis ' 5 01P 487.2 M. ~ 7' 94 ~ 11.7 M. tuuerculosis ,5 01E ' ~0.6 M tu.~., L~l~)si.~ ' 5~81 ., ~1.7 M. avium L ~113 '"5.7 M. avium L~150 11''9.1 Mavium 2.'291 2.6 M. smegmatis 9420 1l l2.0 M.fortuitum 12478 2t 4 5 ~Qn~
Calf thymus DNA (100 ng) 2.5 E. coli DNA (50 ng) 7.4 . _ ~

Wo 95/31571 2 1 9 0 0 q O ~ Jv r These results indicate that probe set 15 was capable of detecting target DNA from at least four species from the genus M~cobacL, i.~,...
s Example 14: Detection of Bacteria of the Genus Mycobac~erium Using Probe Set 8 (SEQ ID NOS. 35, 36, 37, and 38) ('71i,, ' J~ide probe set 8 (SEQ ID NOS. 35, 36, 37, and 38) was selected to detect a target DNA, . ", ~ to nucleotides 690-732 (SEQ ID NO 34) ofthe 1 6S ribosomal RNA gene of M. ~ub., .ul~,sis (and others). LCR was carried out as describedinExample5exceptthatprobeswerepresentat2x l012~lule~uL,~ iull, and cycling was performed at: 85 C, 37v sec; 55 C for 30 sec; for a total of 45 cycles.
Reaction products were analyzed as described in Example 1. Probes were labelled with biotin and fluorescein, respectively. Results are shown in Table 14.
Table 14 Target DNA (5 p~) IMx t~ R ;e (clsls) M. in~l " " f,' LR158 5 .5 M. gordonae 1318 1 ' .0 M. r~.almoense 802 2' .l M. terrae CAI7 : .~.0 M. avium LR107 4~1.1 M /-- -p7 17~7i7 40 0 .V't. kansasii 1214 '~7-.V.t. s~,u~ulacL~.. 1302 , '.~
~Vl7. rnarinum 1218 t .W. tuberculosis 102 _,~, IV17. szulgai 9'.. 1 human placental DNA ( 1 ~g) 9.7 These results show that probe set 8 was capable of detecting target DNA from several species of bacteria of the genus IlJy.7~ba.~, However, this probe set failed to detect target DNA from M. terrae.
Example 15: Detection of Bacteria of the Genus Mycobaclerium Using Probe Set 16 (SEQ ID NOS. 73, 74, 75 and 76) ~ V~ probe set 16 (SEQ ID NOS. 73, 74, 75 and 76) was selected to detect a target DNA of the 16S ribosomal RNA gene of various .11 7 ~,uhv/v.~
25 LCR was performed as described in Example I using dATP as the gap-filling nucleotide, and 2 x 1012 molecules of each probe. Tl-~,.-l~u~yl,lill~ was performed in a Sutter Dunker at the following settings: 85 C for 8~ sec; 62 C for 120 sec; for a tota. of 40 cycles.
Reaction products were analyzed as described in Example 1. Results are shown in Table WO95/31571 2 1 9 0 0 9 0 1 IIUL~'._ ~ol(i Table 15 Tar~et DNA IMx@~ Rate 100 Molecules 1,000 Molecules of T lr~- Qf T l~et M. chelonae 8 9.. 13 1.5 M. aviam t 3.', ;3.5 M. terrae 9' Cl.: 13 n.0 M. phlei 9~'. 13~: .0 M. tuberculosis ,'.9 3C' .1 M. avium ~.2 1: .1 M. i,." " ' ~ .5 20'.6 Con~ol LUDrnan plaCental DNA (330 ng) 259 c)7 These data indicate that probe set 16 (SEQ ID NOS. 73, 74, 75 and 76) was capable of s detecting a subset of target DNAs from bacteria of the genus .1lYL()~UL~/
Example 16: De~ection of Bacteria of the Genus Myc~l~al I, with Probes from Probe Sets 8 (SEQ NOS. 35, 36, 37, and 38) and 16 (SEQ ID NOS. 73, 74, 75 and 76).
Probes from rllig.~ irlr probe sets 8 (SEQ ID NOS. 35, 36, 37, and 38), and 16 (SEQ ID NOS. 73, 74, 75 and 76) were selected to detect a target sequence UUll~*/l '' ,, to the 16S ribosomal RNA gene of various ~ ulJ~t~ . LCR was performed as described in Example I except that cycling was performed in a Sutter dunker at 85 C for 55 sec. followed by 62 C for 85 sec. for a total of 40 cycles. Reaction products were analyzed as described in Example 1. Probes were labelled with carbazole and ' as described previously. Hurnan placental DNA was present at 330 ng/reacùon.
The following groups of probes were used:
Group 1: Probe A (SEQ ID NO. 35), and A' (SEQ ID NO. 36) from Set 8 (GSMIA);
Probe A (SEQ ID NO. 73), and A' (SEQ ID NO. 74) from Set 16 (GSMIB).
Group 2: Probe B (SEQ ID NO. 75), and B' (SEQ ID NO. 76) from Set 16 (GSMIB).
Group 3: Probe A (SEQ ID NO. 35), probe A'(SEQ ID NO. 36), probe B (SEQ ID
NO. 37), probe B' (SEQ ID NO. 38) from Set 8 GSMIA.
Tablel6showsthe-.. ,,.,1,;,--~;""ofprobesused,andtheirc.",.~ i"

WO95/31571 1~1/. 16 Table 16 Trial Group I ~Q~ G}oup 3 2.5 x 1012/rxn 2.5 x 1012/rxn 2 3.5 x 1012/rxn 2.5 x 1012/rxn 3 4.5 x 1012/rxn 2.5 x 1012/rxn

4 2.5 x 1012/rxn 3.5 x 1012/rxn

5 3.5 x 1012/rxn 3.5 x 1012/rxn

6 4.5 x 1012/rxn 3.5 x 1012/rxn

7 3.5 x 1012/rxn

8 4.5 x 1012/rxn

9 5.5 x 1012/rxn s Table 17 M tuh, ' - - M. chelonae hurnan 7lacental DNA
I~l C~ L3 -0~- 3 C/~1~ 7 2~, 9 3, ~ ~.4 : ,J.4 4, ~.0 S. ,~ , g 6 ~ 9.5 '`.iJ~.4 7~40. 2. . I
8'5 1 .7 24.4 9~54.~ 9.5 Results derived using each of these ..., . ,l ,;" - ~ ;....; are shown in Table 17, and illustrate that the combined probe sets of group I and 2 detected both M. ~ul,~, and M. chelonae.
Example 17: Detection of Bacteria of the Genus MJ ~L ' ' Using A
Mixture of Probe Set 8 (SEQ ID NOS. 35, 36, 37, and 38) and Probe Set 16 (SEQ ID NOS. 73, 74, 75 and 76) ()lil ' ~ probe set 8 (SEQ ID NOS. 35, 36, 37, and 38) and oL~ ' ' probe set 16 (SEQ ID NOS. 73, 74, 75 and 76) were used together to detect a target DNA l,Ull~,~JUlldi.g to the 16S ribosomal RNA gene of various 111~ .,Ubcu,u,li~. LCR was performed as described in Example S except that all 8 probes were present at S x 101 1 l.lol~ iu~l, and cycling was performed at in a COY
2 0 ~ II Io~ .l at the following settings: 55 C, 30 sec., 85 C, 20 sec. for a total of 45 cycles. Reaction products were analyzed as described in Example 1. Probes were labeUed with biotin and fluorescein. Results are shown in T-~le 18.

W0 95/31571 2 1 9 0 0 9 ~ ol6 Table 18 Tar,eet DNA (5 p~) IMx(~) Rate (clsls) M. avium 444 9 M. gordonae 1318 3^9.2 M. malmoense 802 7~.4 M. terrae CAP 1~' ~.5 M. chelonae 1 ':: .6 M. ~ .2 M. kansasii 1214 ~ 6.. 6 M. scrofulace~m 302 . 7 .5 M. marinurn 1218 )1 .6 M. phlei 1144.7 M. szulgai C 563.5 Control human placental DNA 52.1 These results show that the ", ' ' of probe sets 8 (SEQ ID NOS.
s 35, 36, 37, and 38) and 16 (SEQ ID NOS. 73, 74, 75 and 76) was capable of detecting target DNA from all of the species of bacteria of the genus ll~j~aL~ ,, tested.
Example 18: Detection of Bacteria of the Genus Mycr:l- ' With Probes From Probe Set 8 (SEQ ID NOS. 35, 36, 37 and 38) and 16 (SEQ ID NOS. 73, 74, 75 and 76) As can be seen in Figure 2A, the three differences between GSMlA (Probe Set 8) and GSMlB (Probe Set 16) reside at two locations in A/A' and one location B/B'.
As was suggested by the previous examples, the two probe sets detect different subsets of the IllyuulJ~ L Therefore, in an attempt to better detect both subsets, probes from GSMlA and GSMIB were combined in different ratios and used as probes for the detection of a wide variety of IlI.~Uba~ i.ll species.
In a preferred method, the A/A ' pairs of GSM 1 A and GSM 1 B were combined in a ratio of 2~ Iy, with the .., .. ,~ ;..., of total A/A ' = BIB '.
LCR was performed as in Example 1 except that the amount of nlig ' ~ ILid~ probes were as follows: GSMIA A/A' was 2.67 x 1012/rxn, GSMIB
A/A' was 1.33 x 1012/rxn, GSMIB B/B' was 4 x 1012/rxn, and human placental DNA
was present at 330 ng/rxn. Cycling was performed in a Sutter dunker at 85 C for 85 sec., 62 C for 85 sec., for a total of 40 cycles. Reaction products were detected as described in Lxample I . Myl,ub~t~ ial target DNA was present at 100 copies/rxn. Non-~., y .,ùl,.~
target DNAs were present at 10 ng/rxn (about I x 106 molecules). Probes were labelleo with carbazole and a ~ as described above. Results are shown in Table 19.

WO 95/31S71 2 ~ 9 0 0 9 0 P~ 6 Table 19 Tar~et DNA IMx~ ^ (c/sls) M. tuberculosis 201 278.
M. chelonae 170 sM. aurum 3~.
M terrae M. phlei ~ .
M. gordonae ' ~.~
M. ~., vJi~lac~ J.~.0 0M. marinum ,; .5 M. fortuitum ",'7 4 M. malmoense : ~^.0 M. szulgai 7-.1 M. bovis :, .9 5M. scrofulaceum 1302 ,: :"2 M. hn, , 1, ~,9 M. smegm~tis ~~4.0 M. kansas i 7.2 M. avium _R107 5.9 20M. i,~ LR158 ':0.5 M~ - " ' ,e LR105 ~ ' 2 M. tuv~ 102 : ~ .7 M. bo-~is 410 ~ !-., MBCG Glaxo . I L.
2sM. avium LR163 ~' ~.J
Citrobacterfreundii ~: .:
ul~h, cloacae '.6 _,. coli ' 7.0 ~almonellainterindus ~ .1 3 o . 'higella sonei ' ' .7 .. 'lebsiella l ~. . . J.7 . '. '.. aeruginosa ).6 Corynebacterrenale : '.9 N. dassonvillei 3sN.asteroides ~- f N. brasiliensus ~.
Stu .~cvc~ aureus ~ J.
S~ivco~Lu~ pyroge~es ~ .5 human placental (330 ng) ;.5.
40LD 9.1 Theseresultsshowt71latthe~ ",1,;"-~;"..ofprobeset8(SEQIDNOS.35,36,37~and 38) and probe set 16 (SEQ ID NOS. 73, 74, 75 and 76) was capable of detecting target DNA from a wide variety of bacteria of the genus ~ vba~
The foregoing examples are presented by way of illustration and are not 4 s intended to limit the scope of the invention as set forth in the appended claims. For exa7nple, sequences of specific length are listed. It should be understood that sequences covering the same map positions but having slightly fewer or greater numbers of bases are deemed to be equivalents of these sequences and fa l within the scope of the invenvion, provided they Wi711 hybridize to the same positions on the target as the listed sequences.

WO9~/31571 2 ~ 9 0 0 9 0 ~ 16 SEQUENCE l.ISTING
( 1 ) GENERAL INFORMATION:
i) APPLICANT: Solomon, N.
Leckie, G.
KrA t o chvi l, J .
O ' Donnell, D .
(ii) TITLE OF INVENTION: MateriAls _nd Methods for the Detection of My~ hA~ t,.ri A
(iii) NtlNSER OF SEQUENCES: 76 (iV) ~Ul~ UNll~N~' ADDRESS:
(A) ADDRESSEE: Abbott Labor_tories ~ -(B) STREET: One Abbott P_rk Ro~d (C) CITV: Abbott P~rk (D) STATE: Illinois (E) COUNTRY: USA
(F) ZIP: 60064-3500 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC comp~tible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Wordperfect (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NtlMBER:
( B ) PILING DATE:
(C) CLASSlFICATION:
( vi i i ) ATTORNEY/AGENT INFORMATION:
~A) NAME: ThomAs D. Brainard ~B) REGISTRATION Nl~MBER: 32,459 (C) REFERENCE/DOC~ET NHMBER 5371.PC,0l (ix) ~rRT~T~ nMMTTNTcATIoN INFORMATION:
(A) TELEPHONE: 708/937-4884 (B) TELEFAX: 708/938-2623 ( C ) TELEX:
( 2 ) INFORMATION FOR SEQ ID No: l:
( i ) SEQUENCE CLARACTERISTICS:
(A) LENGTH: 44 b~se pAirs (B) TYPE: nucleic ~cid (C) S~rR~NnT~ilN~cc double ( D ) TOPOLOGY: l inear ( i i ) MOLECULE TYPE: DNA ( genomi c ) (vi) ORIGINAL SOUROE:
(A) ORGANISM: MycobActerium tuberculosis (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
AACCTGTGGG ~ ~WI~ TCACGAGAGG TATCCGAACG TCAC 44 ( 2 ) INFORMATION FOR SEQ ID NO: 2:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base p~irs (B) TYPE: nucleic _cid (C) S'rR~NT~T'n~RCC: single WO 95/31571 2 1 q O 0 9 0 r~ ol6 (D) TOPOLOGY: line~r (ii) MOLECULE TYPE: synthetic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
AACCTGTGGG l~ L l T 21 (2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CEARACTERISTICS:
( A ) LENGTH: 1 8 o~s e p~Li r8 (B) TYPE: nucleic ~cid (C) STRANnR~ CC: single ( D ) TOPOLOGY: linezlr (ii) MOLECULE TYPE: synthetic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
rr~rrr~-:Arrr CACAGGTT 18 (2) INFORMATION FOR SEQ ID NO:4:
( i ) SEQIJENCE CBARACTERISTICS:
(A) LENGTH: 20 bflse p~irs (B) TYPE: nucleic ~cid (C) s~R~NnRnNR~c: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic DNA
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

( 2 ) INFORMATION FOR SEQ ID NO: S:
( i ) SEQUENCE C~ARACTERISTICS:
(A) LRNGTH: 23 b~3e p~irs (B) TYPE: nucleic ~cid (C) S'rRANnRnNRCC: slngle (D) TOPOLOGY: line~lr (ii) MOLECULE TYPE: !~ynthetic DNA
( Xi ) SEQUENCE DESCRIl~ION: SEQ ID NO: S:

( 2 ) INFORMATION FOR SEQ ID NO: 6:
(Omitted aince it is ~ duplication of SEQ ~D No 1) (2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CB`ARACTERISTICS:
(A) LENGTH: 18 b~se p~irs (B) TYPE: nucleic ~cid (C) STR~NnRnNRcc single (D) TOPOLOGY: line~r (ii) MOLECULE TYPE: synthetic DNA
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
~,L~ iWb . ~ CGGCCTTT 18 (2) INFORMATION FOR SEQ ID NO:8:
(i) SEQIJENCE ~`~TAR:~r~rRRTCTICS:
(A) LENGTH: 15 b~se p~irs (B) TYPE: nucleic ~cid (C) sTRANnRnNRcc single ( D ) TO POLOGY: 1 ine~r (ii) MOLECULE TYPE: synthetic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

W09S/31571 2 1 9 0 0 9 0 1~ Sol6 rrrrr.r~rr CACAG 15 ( 2 ) INFORMATION FOR SEQ ID NO: 9:
( i ) SEQln~TCE CHARACTERISTICS:
(A) LENGTH: 17 b~se p~irs (B) TYPE: nucleic ~cid (C) sTRAr~n~nlv~cc: single (D) TOPOLOGY: line~r (ii) MOLECULE TYPE: synthetic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE rHAR~rT~RrcTIcs:
(A) LENGTH: 20 b~se p~irs (B) TYPE: nucleic ~cid ( C ) STR ~DrlnNRsc: 9 ingle (D) TOPOLOGY: line~r (ii) MOLECULE TYPE: synthetic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

(2) INFORMATION FOR SEQ ID NO:11:
( i ) SEQUENCE CBARACTERISTICS:
(A) LENGTH: 50 b~se pairs (B) TYPE: nucleic ~cid ( C ) S~R Al~lD~nN~ c c: doub l e ( D ) TOPOLOGY: l ine~r (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE:
(A) ORGANISM: M. Bovis BCG
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
CTTGACGCAG TCGTCAGACC r~ rrrr, ArAr.rr~rr~ GAAACTCGAC 50 (2) INFORMATION FOR SEQ ID NO:12:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 b~se p~irs (B) TYPE: nucleic ~cid (C) STR~nrlnl~Ecc: :~ingle (D) TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:

( 2 ) INFORMATION FOR SEQ ID NO :13:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 b~se p~irs (B) TYPE: nucleic ilcid (C) sTRA~DrlnN~cc single (D) TOPOLOGY: line~r (ii) MOLECULE TYPE: synthetic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:

(2) INFORMATION FOR SEQ ID NO:1~:
( i ) SEQUENCE CHARACTERISTICS:

WO 95/31571 2 1 9 3 0 9 0 A ~ ~
~A) LENG~ 21 base p~irs ~B) TYPE: nucleic ~cid (C) STRANnTnNRc.c: single ~D) TOPOLOGY: Line~r (ii) MOLECULE TYPE: synthetic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
r.ArAr~r~rAr GGAAACTCGA C 21 (2) INFORMATION pOR SEQ ID NO:15:
( i ) SEQUENCE rpAR~r~RTcTIcs:
(A) LENGTH: 25 base p~irs (B) TYPE: nucleic acid (C) STRANn~nNEcc: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: ~ynthetic DNA
(xi~ SEQUENCE DESCRIPTION: SEQ ID NO:15:
GTCGAGTTTC U~.iL~_U~X, LC'l' CGGGG 25 ( 2 ) INFORMATION FOR SEQ ID NO :16:
( i ) SEQUENCE CE~ARACTERISTICS:
(A) LENGTE~: 4~ b~se pairs (B) TYPE: nucleic acid (C) STRANnRnNRcc double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
GCGAGCTGCG CGATGGCGAA r~r~Ar~r~ r ACATCAGCCG CGTC ~4 ( 2 ) INFORMATION FOR SEQ ID NO :17:
( i) SEQUENCE C~RACTERISTICS: : :
(A) LENGTI~: 20 base p~irs (B) TYPE: nucleic ~cid (C) sTR~NnRnNRcc single (D) TOPOLOGY: linear ~ii) MOLECULE TYPE: synthetic DNA
~xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
GCGAGCTGCG rr~ATr~r~rr~ 20 (2) INFORMATION FOR SEQ ID NO:18:
( i ) SEQUENCE C~RACTERISTICS_ (A) LENGT~: 18 bdse pairs (B) TYPE: nncleic ~Lcid (C) STRANn~nN~Cc: single (D) TOPOLOGY: line~Lr (ii) MOLECULE TYPE: synthetic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:la:

(2) INFORMATION FOR SEQ ID NO:l9:
(i) SEQUENCE rPARA~ Ll~:i (A) LENGT~: 21 base p~irs ~B) TYPE: nucleic ~cid (C) STRANnT~.nN~C.C: single (D) TOPOLOGY: line~I
(ii) MOLECULE TYPE: synthetic DNA

WO95/31571 2 1 9 ~ O q O i~

(xi) SEQUENCE DESCRIPTION: SEQ ID NO~

(2) INFORMATION FOR SEQ ID NO:20:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base p~irs (B) TYPE: nucleic Acid (C) STRANnFnNEC~: single (D) TOPOLOGY: line~r (ii) MOLECULE TYPE: synthetic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
GACGCGGCTG A; ~ 'L' TGAG 2 4 (2) ,INFORMATION FOR SEQ ID NO:21:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 49 b~se p~irs (B) TYPE: nucleic ~cid (C) STR~NnRnNE.cc: double ( D ) TOPOLOGY: 1 iner r (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycob~cterium tuberculosis (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
TGAACGGAAA AGTCCTGGCG GCCATGTACC ~r~C~t~C~T CAAAACCTG 49 ( 2 ) INFORMATION FOR SEQ ID NO: 22:
( i ) SEQUENCE CHAFACTERISTICS:
(A) LENGTH: 21 bqse p~irs (B) TYPE: nucleic ~cid (C) STR~NnEnN~CC: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: synthe~ic DNA
(xi) SEQUENCE L)~:iC~ llOI\l: SEQ ID NO:22:

(2) INFORMATION FOR SEQ ID No:23:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 bese pqirs (B) TYPE: nucleic ~cid (C) s~RANn~nNEcc single ( D ) TO POLOGY: 1 ine~r (ii) MOLECULE TYPE: Synthetic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID No:23:

(2) INFORMATION FOR SEQ ID No:24:
( i ) SEQUENCE CBARACTERISTICS:
(A) LENGTH: 20 bqse p~irs (B) TYPE: nucleic ccid (C) STR~NT)EnNECC: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Synthetic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:

(2) INFORMATION FOR SEQ ID NO:2~: ~

W 9513 1 ~ . . 16 ( i ) SEOUENCE C~RACTERISTICS:
(A) LENGTE~: 21 base pAirs ~B) TYPE: nucleic acid ~C) s~R~Nn~nNRcc: single ~D) TOPOLOGY: linear ~ii) MOLECULE TYPE: Synthetic DNA
~xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:

2 ) INFORMATION FOR SEQ ID NO: 2 6:
i ) SEQUENCE CBARACTERISTICS:
~A) LENGTB: 21 b~se pairs (B) TYPE: nucleic ~cid (C) C~R~Nn~nl~ECC: single ( D ) TOPOLOGY: linear (ii) MOLECULE TYPE: Synthetic DNA
~xi) SEQUENCE DESCRIPTION: SEQ ID NQ:26:

~2) INFORMATION FOR SEQ ID NO:27:
i ) SEQUENCE CBARACTERISTICS:
~A) LENGT~: 21 b~se pairs ~B) TYPE: nucleic acid ~C) S'rR~Nn~nNRCC: single ~D) TOPOLOGY: linear ~ii) MOLECULE TYPE: Synthetic DNA
~xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:
ACGCCAGGAC 'LLLL~(~ C 21 ~2) INFORMATION FOR SEQ ID NO:28:
i ) SEQUENCE CBARACTERISTICs:
~A) LENGTR: 23 b~se pairs ~B) TYPE: nucleic acid ~C) S~D~nllRC.C: single ~D) TOPOLOGY: linear ~ii) MOLECULE TYPE: Synthetic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2~:
ACATGTACCA r~rrr,rarr~r Cl~A 23 (2) INFORMATION FOR SEQ ID NO:29:
(i) SEQUENCE rll~R~`qq:RTCTICS:
(A) LENGT}~: 22 base p~irs (B) TYPE: nucleic ~cid (C) s~R~Nn~nN~cc single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Synthetic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:

( 2 ) INFORMATION FOR SEQ ID NO: 3 0:
(i) SEQUENCE rT~Rp~rTER~cTIcs:
(A) LENGTP: 2~ b~se p~irs (B) TYPE: nucleic acid (C) s~rR~Nn~nl~Ecc: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Synthetic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:

WO 95/:~1571 2 1 9 0 0 9 0 . ~"~ 16 ( 2 ) INFORMATION FOR SEQ ID NO: 3 l:
( i ) SEQUENCE CH~RACTERISTICS:
(A) LENGTH: 21 base pAirs (B) TYPE: nucleic _cid (C) s~pR~NnRnNRcc: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Synthetic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:
rr~r~ L L L l ~_~ .; L L-- A 21 (2) INFORMATION FOR SEQ ID No:32:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 bAse pair6 (B) TYPE: nucleic zLcid (C) s~pR~NnpnNRcc: single ( D ) TOPOLOGY: 1 inear (ii) MOLECULE TYPE: Synthetic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID No:32:
~-c2~ 0~C CATCAAAACC TG 22 (2) INFORMATION FOR SEQ ID No:33:
( i ) SEQUENCE CBARACTERISTICS:
(A) LENGTH: 25 base pairs (B) TYPE: nucleic _cid (C) SlrR~NnRnNRcc: single (D) TOPOLOGY: line~r (ii) MOLECULE TYPE: Synthetic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:
CAGGTTTTGA l~iLiL~ v GTACA 25 ( 2 ) INFORMATION FOR SEQ ID NO: 3 4:
(i) SEQUENCE f9l~R~ rRRTcTIcs:
(A) LENGTH: 44 b_se p~irs (B) TYPE: nucleic Acid (C) S~R~NnRn ~C double (D) TOPOLOGY: line~r ( i i ) MOLECULE TYPE: DNA ( genomi c ) (vi) ORIGINAL SOURCE:
(A) ORGANISM: My~ hA. ~l~ri (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:
TGTTCGTGAA ATCTCACGGC TTAACTGTGA ~ GATA 44 ( 2 ) INFORMATION FOR SEQ ID NO: 3 5:
( i ) SEQUENCE CHARACTERISTICS -(A) LENGTH: 22 b_se pairs (B) TYPE: nucleic ~cid (C~ S~R~NnRnNRcc: ~ingle (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Synthetic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:

( 2 ) INFORMATION FOR SEQ ID NO: 3 6:
( i ) SEQUENCE r~ R~r~T~.RTCTICS:

WO 95/31571 1 ~~
21 903qO

(A) LENGTH: 21 base pairs ~B) TYPE: nucleic ~cid (C) sTR~NnRnNRcc single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Synthetic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:
GCCGTTCAGA TTTCACGAAC A 2 l (2) INFORMATION FOR SEQ ID NO:37:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic ~cid (C) s~TR~NnRnNl:cc: single (D) TOPOLOGY- line~r (ii) MOLECULE TYPE: Synthetic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:
CTGTGAGCGT r,rr,~rrr.~ 20 ( 2 ) INFORMATION FOR SEQ ID NO: 3 8:
( i ) SEQUENCE CBARACTERISTICS:
(A) LENGTH: 22 b~se pairs (B) TYPE: nucleic ~Lcid (C) S~RZ~Nn~nNRCC: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Synthetic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:

(2) INFORMATION FOF~ SEQ ID NO:39:
(i) SEQUENCE rR~R~rl~RTcTIcs:
(A) LENGTH: 40 b~se p~irs (B) TYPE: nucleic ~cid (C) S~R~ nNRcc double (D) TOPOLOGY: line~!Lr (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SO~7RCE:
(A) ORGANISM: Mycob~cterium tuberculosis (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:
b~ iA ~Arr~r~r~r~ CT~GAGTACT r.r~rrr.r2-r.~ 40 (2) INFORI~ATION FOR SEQ ID NO:40:
(i) SEQUENCE rtl~R~r'rRRTCTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic ~cid (C~ S~ Nn~nNRcc single (D) TOPOLOGY: line~r (ii~ ~OLEC~7LE TYPE: Synthetic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:
.WA ~Arr~,r.rz-r.~ 20 (2) INFORMATION FOR SEQ ID NO:41:
( i ) SEQUENCE r~R ~ r~RR T CTICS:
(A) LENGTH: 17 base p~irs (B) TYPE: nucleic acid (C) s~TR~NnRnNRcc single (D) TOPOLOGY: line~r (ii) MOLECULE TYPE: Synthetic DNA

Wo 9s~ls7l 9 0 1 ~

~xi) SEQUENCE DESCRIPTION: SEQ ID No:~1:

( 2 ) INFORMATION FOR SEQ ID NO: 42:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 b~se pairs (B) TYPE: nucleic ~cid (C) s~TR~Nl)~nNRcc: single (D) TOPOLOGY: line3r (ii) ~OLECULE TYPE: Synthetic DNA
(xi ) SEQUENCE l/E~:n~ oN: SEQ ID NO: 42:

(2) INFORMATION FOR SEQ ID NO:43:
(i) SEQUENCE r~R~r~rRRTCTICS
(A) LENGTH: 20 b~se pairs (B) TYPE: nucleic Acid (C) S'TRZ-NrlRnNRCC: gingle (D) TOPOLOGY: line~r (ii) MOLECULE TYPE: Synthetic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:~3:

(2) INFORMATION FOR SEQ ID NO:44:
( i ) SEQUENCE rTl~R ~rlrRR T cTI CS:
(A) LENGTH: ~0 base pairs (B) TYPE: nucleic acid (C) S~RANr)RnNT:CC: double (D) TOPOLOGY: linear (ii) ~lOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:
rnrrr.~r GGTGGGTACT AGGTGTGGGT LJC~ ,'L 40 (2) INFORNATION FOR SEQ ID NO:45:
(i) SEQUENCE rT~R~r'rRRTCTICS:
(A) LENGTH: 20 b~se p~irs (B) TYPE: nucleic ~cld (C) s~TR~NnRnNRcc: single (D) TOPOLOGY: linear (ii) ~OLECULE TYPE: Synthetic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:
rr,rrr,~ r GGTGGGTACT 20 ( 2 ) INFORMATION FOR SEQ ID NO: 4 6:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs (B) TYPE: nucleic ~cid (C) s~TR~Nr)Rn~ c: single (D) TOPOLOGY: linear (ii) NOLECULE TYPE: Synthetic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:

(2) INFORNPTION FOR SEQ ID NO:47:

WO 9~/31571 2 l q O O ~

( i ) SEQUENCE CKARACTERISTICS:
(A) LENGTK: 17 b~se p~irs (B) TYPE: nucleic ~cid (C) S~R~ RnNEcc: single (D) TOPOLOGY: line~r (ii) MOLECULE TYPE: 5ynthetic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:
l. CTTCCTT _ 17 (2) INFORMATION FOR SEQ ID No:48:
( i ) SEQUENCE rB ~R A~'TRR T.cTICS:
(A) LENGTK: 20 b~se p~Lirs (B) TYPE: nucleic ~cid (C) STRANnRnN~C.C: single ( D ) TO POLOGY: l in e~r (ii) MOLECULE TYPE: Synthetic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID No:48 AA~r~AA~ ACCCACACCT 20 (2) INFORMATION FOR SEQ ID NO:49:
(i) SEQUENCE rK~RA~'r~Rr.CTICS:
(A) LENGTK: 43 b~se pairs (B) TYPE: nucl~ic ~cid (C) s~R~ND~nNRcc double (D) TOPOLOGY: line~r (ii) NOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE-(A) ORGANISM: Mycob~lcterium (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:
ACTTCGCAAT r~ A~ ATTGCGTACG Af~ r~ c CCG = ~3 (2) INFORMATION FOR SEo ID NO:~0:
( i ) SEQUENCE CKARACTERISTICS
(A) LENGTK: 21 base prlirs (B) TYPE: nucleic ~Lcid (C) s~RAND~nNRcc single (D) TOPOLOGY: line~r (ii) MOLECULE TYPE: Synthetic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:SQ:

(2) INFORMATION FOR SEQ ID NO:S1:
( i ) SEQUENCE CKARACTERISTICS:
(A) LENGTK: 19 b~se p~irs (B) TYPE: nucle~c ~cid (C) SrR~ ~nN~CC: single (D) TOPOLOGY: line~r (ii) MOLECULE TYPE: Synthetic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:
~_~ TTGCGAAGT 19 (2) INFORMATION FOR SEQ ID NO:52:
( i ) SEQUENCE C ARACTERISTICS:
(A) LENGTK: 20 b~se p~irs (B) TYPE: nucleic ~cid (C) sTRANnr~nl~lRcc single (D) TOPOLOGY: line~r W0 95/31571 2 1 9 o o 9 o P~ 16 , 43 (ii~ MOLECULE TYPE: Synthetic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:
GCGTACGACG AAr.Arr.rrr~. 2 0 ( 2 ) INFOR~5ATION FOR SEQ ID NO: 5 3:
- ( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 bdse p~irs (B) TYPE: nucleic ~Icid (C) STRANDRnNEqc: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Synthetic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:
CLi j i~l~l- CGTCGTACGC AA 22 (2) INFORMATION FOR SEQ ID NO:54:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 48 hase p~irs (B) TYPE: nucleic ~Lcid (C) STRANDRn~`lECS: double (D) TOPOLOGY: line~r (ii) MOLECULE TYPE: DNA (genor~ic) (vi) ORIGINAL SOURCE:
(A) ORGANISM: My~ h;~ ~riI
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:
L;~il~i I ~il.CA TCGCCAAGGA GATCGAGCTG GAGGATCCGT ACGAGAAG 4 (2) INFORMATION FOR SEQ ID NO:55:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 hase p~irs (B) TYPE: nucleic ~cid (C) STRANn~nNEes: single ( D ) TOPOLOGY: 1 inear (ii) MOLECULE TYPE: Synthetic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:
Wl~ l~CA TCGCCAAGGA GATC 24 (2) INFOE~MATION FOR SEQ ID NO:56:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 b~se p~irs (B) TYPE: nucleic ~cid (C) STR~IunRn~cc: single (D) TOPOLOGY: linear ( ii ) MOLECULE TYPE: Synthetic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:
rATrr~rArA CC 22 (2) INFOR~ATION FOR SEQ ID No:57:
( i ) SEQUENCE CHA,RACTERISTICS:
(A) LENGTH 21 }~se pairs (B) TYPE: nucleic ~cid ( C ) STR ANDRn~E c q: s ing l e (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Synthetic DNA
(xi) SEQUENCE DESCP~IPTION: SEQ ID NO:57:
CTGGAGGATC rrT~rr.Ar.A~ G 21 W0 95/31571 P~ 6 2~ ~30~0 (2) INFORMATION FOR SEQ ID NO:58:
( i ) SEQUENCE rHARArTERTcTIcs:
(A) LENGTH: 24 b~se p~irs (B) TYPE: nucleic ~cid ( C ) STR ANnFr~NF C ~: s ing l e ( D ) TO POLOGY: l ine~r (ii1 MOLECULE TYPE: Synthetic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:

( 2 ) INFORMATION FOR SEQ }D NO: 59:
(i) SEQUENCE rRARAr~ERTcTIcs~:
(A) LENGTH: 43 base p~irs (B) TYPE: nucleic acid ( C ) STR ANnEn~E c ~: doub 1 e ~ D ) TOPOLOGY: 1 inear (ii) NOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE-(A) ORCANISM: Mycob~cterium (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:
GGGTGACACC GTCATCTACA GGAAGTACGG. rr,rrArrr~. ATC 43 (2) INFORMATION FOR SEQ ID NO:60:
(i) SEQUENCE rTTARAr~ERTCTICS:
(A) LENGTH: 24. base pairs (B) TYPE: nucleic ~cid (C) STRANnEnl`~EC':: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Synthetic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:
GGGTGACACC r~TCATCAACA GCAA 2 4 (2) INFORMATION FOR SEQ ID NO:61:
(i) SEQUENCE CHAEACTERISTICS:

(A) LENGTH: 20 b~se pairs (B) TYPE: nucleic ~cid ( C ) S TB ANnFn~\lE c c: s i n g 1 e (D) TQPOLOGY: linear (ii) MOLECULE TYPE: Synthetic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:
CTGTGATGAC GGTGTCACCC . 2 0 (2) INFORMATION FOR SEQ ID NO:62:
(i) SEQUENCE rHARAr~ERT.cTICS:
(A) LENGTH: 22 base p=ira (B) TYPE: nucleic ~cid (C) STRANTl~nl~c~c single (D) TOPOLOGY: line~ r (ii) MOLECULE TYPE: Synthetic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:
Arr~r~rr~rAr CGAGATCAAG TA 22 ( 2 ) INFORMATION FOR SEQ ID NO: 63:
( i ) SEQUENCE CPARACTERISTICS:
(A) LENGTH: 25 b~se p~irs (B) TYPE: nucleic ~cid W~ 95/31571 2 1 9 0 0 9 0 ~5 (C) sTRl~Nn~nNRc~ sillgle ( D ) TOPOLOGY: 1 inear (ii) ~OLECULE TYPE: Synthetic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:
~AGTTTCATC I~wl~io--v-- CGTAC 25 (2) INFORMATION FOR SEQ ID NO:64:
( i ) SEQUENCE CH~RACTERISTICS:
(A) LENGTH: 40 b~se pairs (B) TYPE: nucleic acid (C) STR~NnRnNEee: double (D) TOPOLOCY: linear (ii) MOLECULE TYPE: DNA (genomie) (vi ) ORIGINAL SOURCE:
(A) ORGANISM: ~ycobacterium (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:
rA~rr.rr~G AACCTTACCT GGGTTTGACA TGCACAGGAC 40 (2) INFORMATION FOR SEQ ID NO:65:
i ) SEQUENCE CHARA~
(A) LENGTH: 20 b~se p~irs ~B) TYPE: nueleic aeld (C) STRD~`TnRnNRCC: ~ingle (D) TOPOLOGY: line~r (ii) MOLECULE TYPE: Synthetic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:
rAArrrr~AAr. AACCTTACCT 20 (2) INFORMATION FOR SEQ ID NO:66:
(i) SEQUENCE f~H~R~'TERTeTICS:
(A) LENGTH: 17 b~se pairs (B) TYPE: nueleic aeid (C) sTR~NDRnNFcc single (D) TOPOLOGY: line~r (ii) MOLECULE TYPE: Synthetie DNA
(xi) SEQUENCE DESC~IPTION: SEQ ID NO:66:

( 2 ) INFORMATION FOR SEQ ID NO: 67:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs (B) TYPE: nueleie acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ( i i ) MOLECULE TYPE: Synthetie DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:
TTTGACATGC ACAGGAC = 17 (2) INFORMATION FOR SEQ ID NO:68:
( i ) SEQUENCE rHAR ~ rTRR T qTICS:
(A) LENGTH: 20 b~se pairs (B) TYPE: nucleic ~eid (C) sTRl~NnRnNRcc: single (D) TOPOLOGY: line~r (ii) MOLECULE TYPE: Synthetic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:

WO 95/31571 1 ~ l6 46 2 1 90~
TGTCAAACCC ~ . 20 ( 2 ) INFORMATION FOR SEQ ID NO: 69:
( i ) SEQUENCE rFl ~R A rT~R T c T I cs ( A ) LENGTH: 2 0 bA s e pa i rs (B) TYPE: nucleic acid (C) sTR~Nn~nNFcc single (D) TOPOLOGY: line~r (ii) MOLECULE TYPE: Synthetic DNA
ix) FEATURE:
(A) NAME/KEY: N stands ior inosine ~B) LOCATION: positions 10 ~nd 16 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:

( 2 ) INFORMATION FOR SEQ ID NO: 7 0:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: la base pairs (B) TYPE: nualeic Acid (C) STB~ nN~qS: single (D) TOPOLOGY: line~r (ii) MOLECULE TYPE: Synthetic DNA
( ix ) FEATURE:
(A) NAMEIKEY: N stands for inosine (B) LOCATION: positions 3 ~nd 9 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:
GCNGTGAGNT TTCACGA~ , la (2) INFORMATION FOR SEQ ID NO:71:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 bese p~irs (B) TYPE: nucleic ~cid (C) sTRDNnpnNEcc single (D) TOPOLOGY: line~lr (ii) MOLECULE TYPE: Syntheti~ DNA
( ix) PEATURE:
(A) NAME/KEY: N stends for inosine ( B ) LOCATION: pos i t ion 6 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:
-I .r .( ~ ~ 2 0 (2) INFORMATION FOR SEQ ID NO:72:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base p~irs (B) TYPE: nucleic ~cid (C) sTR~NDRnN~ss single ( D ) TOPOLOGY: 1 inear (ii) MOLECULE TYPE: Synthetic DNA
(ix) FEATURE:
(A) NAME/KEY: N st~nds for inosine (B) LOCATION: position 15 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:
TATCGCCCGC Arn~rNr~r~n TT 22 (2) INFORMATION FOR SEQ ID NO:73:
( i ) SEQUENCE rl~R~rT~RTcTIcs: . _ _ WO 95/31571 2 1 9 o o 9 o ~ 16 (A) LENGTH: 21 base p~irs (B) TYPE: nucleic acid (C) STRANl)T'nN~CC: single (D) TOPOLOGY: line~r (ii) MOLECULE TYPE: Synthetic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:

(2) INFORMATION FOR SEQ ID NO:74:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 b~se p~irs (R) TYPE: nucleic rCid (C) sTRANnT~nNEcc: single (D) TOPOLOGY: linerr (ii) MOLECULE TYPE: Synthetic DNA
' (xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:
GCTGTGAGTT TTC~CGAACA 20 ( 2 ~ INFORNATION FOR SEQ ID NO: 7 5:
(i) SEQUENCE rH:~R~rTFRTSTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STR~Nn~nNFCC: single (D) TOPOLOGY: line~r ( i i ) MOLECULE TYPE: Synthetic DNA
(xi) SEQUENCE DESCF~IPTION: SEQ ID NO:75:
WuiL rrr.r~rr~T~ 20 (2) INFrJRMATION FOR SEQ ID NO:76:
( i ) SEQUENCE CPARACTERISTICS:
(A) LENGTH: 21 base pAirs (~) TYPE: nucleic ~cid (C) STRAND~nN~CC: sin~le (D) TOPOLOGY: line~r (ii) MOLECULE TYPE: Synthetic DNA
(xi) SEQUENCE L)hS~:Kl~ : SEQ ID NO:76:
~CC~:CA rarrr~r~

Claims (19)

We Claim:

1. An oligonucleotide probe useful in the detection of target DNA from a plurality of Mycobacterium species, said probe characterized in that it is from 10 to about 50 nucleotides long and possesses sufficient complementarity or homology to the sequences shown in SEQ ID NOS. 34, 39, 44, 49, 54, 59 and 64 to hybridize with such sequence or its complement under hybridizing conditions, and in that it detects a plurality of Mycobacterium species while not cross reacting substantially with other organisms.

2. An oligonucleotide probe according to claim 1 wherein said probe is selected from the group consisting of probes designated herein as SEQ ID NOS. 35-38, 40-43, 45-48, 50-53, 55-58, 60-63, 65-68, 69-72, and 73-76.

3. A method for detecting the presence of target DNA from bacteria of the genus Mycobacterium in a sample, the method comprising the steps of:
a) providing a sample suspected of containing said target DNA;
b) denaturing said target DNA;
c) hybridizing an oligonucleotide according to claim 1 or 2 to said target DNA in said sample, said oligonucleotide being conjugated to a label, said label capable of generating a detectable signal;
d) determining the presence of said target DNA by detecting the signal generated.

4. The method according to claim 3 further comprising at least one target amplifying step in addition to said hybridizing step.

5. The method of claim 3 wherein said amplifying step is a ligase chain reaction.

6. A Mycobacterium genus-specific composition, said composition selected from the group consisting of probe set 8 (SEQ ID NOS. 35-38), probe set 9 (SEQ ID NOS. 40-43), probe set 10 (SEQ ID NOS. 45-48), probe set 11 (SEQ ID NOS.50-53), probe set 12 (SEQ ID NOS. 55-58), probe set 13 (SEQ ID NOS. 60-63), probe set 14 (SEQ ID NOS. 65-68), probe set 15 (SEQ ID NOS. 69-72), probe set 16 (SEQ ID
NOS. 73-76), and combinations thereof.

7. A method for detecting the presence of target DNA from bacteria of the genus Mycobacterium in a sample, said method utilizing the ligase chain reactioncomprising the steps of:
a) providing a sample suspected of containing said target DNA;
b) providing one or more probe sets according to claim 6, wherein at least one probe of said probe set has a label capable of detection;
c) providing a ligase;
d) performing the following cycle at least once:
i) mixing said one or more probe sets with said sample suspected of containing said target DNA;
ii) denaturing said mixture of said probe set and said sample suspected of containing said target DNA;
iii) hybridizing said denatured probe set to said denatured target DNA thereby creating hybridized probes;
iv) correcting said hybridized probes in a template dependent manner creating adjacent probes;
v) ligating said adjacent probes using said ligase to form reorganized probes;
vi) detecting said label in said reorganized probes; and wherein steps i) and ii) may be performed in reverse order.

8. The method of claim 7 wherein step c) further includes providing one or more deoxynucleotide triphosphates and a polymerase.

9. The method of claim 7 wherein steps ii-iv are repeated from about 10 to about 50 times.

10. The method of claim 7 wherein said ligase is a thermostable ligase.

11. The method of claim 7 wherein said polymerase is a thermostable DNA polymerase.

12. The method of claim 7 wherein said label comprises a specific binding partner.

13. The method of claim 7 wherein said probe set has two labels and wherein probe partners have different labels.

14. The method of claim 12 wherein said probe set has two binding partners and wherein probe partners have different binding partners.

15. The method of claim 12 wherein said labels in said reorganized probes are haptens and are detected using a sandwich immunoassay.

16. A kit useful in the detection of target DNA from bacteria of the genus Mycobacterium said kit comprising one or more suitable containers containing:
a) one or more probe sets wherein said probe set is selected from the group consisting of probe set 8 (SEQ ID NOS. 35-38), probe set 9 (SEQ ID NOS. 40-43), probe set 10 (SEQ ID NOS. 45-48), probe set 11 (SEQ ID NOS. 50-53), probe set 12 (SEQ ID NOS.
55-58), probe set 13 (SEQ ID NOS. 60-63), probe set 14 (SEQ ID
NOS. 65-68), probe set 15 (SEQ ID NOS. 70-73), probe set 16 (SEQ ID NOS. 75-78), and combinations thereof;
b) a polymerase reagent; and c) a ligase reagent.

17. The kit according to claim 16 wherein at least one probe of said probe set has a label capable of detection.

18. The kit according to claim 16 wherein probe pairs have two different labels.

19. The kit according to claim 16 wherein the polymerase and ligase are thermostable.