CA2055302A1 - Specific microbial strain detection method - Google Patents

Abstract

ABSTRACT

A method of detecting a particular microbial strain amongst closely related microbial strains and other strains present in vast numbers is described. Following transposition a unique DNA sequence comprising an endogenous transposable element and its flanking DNA
sequence is generated. This new sequence is an acquired characteristic, diagnostic for that strain and its progeny alone. In one example of this method, DNA primers are chosen from a region which includes the uniquely located transposable element. These primers are used to amplify the DNA for its subsequent detection, and hence the detection of the particular microbial strain.

Description

z~
SPECIFIC MICRDBIAL SrR~IN DEIECTION METHOD

~is invention relates to a ~ethcd of detecting a novel microo ~ ism strain am~gst closely related strains an~ other microbial straLns. More specifically t~is invention r~lates to a methcd for de~ecting a unique sequence of DN~ in microorganisms, result ~ frcm ~he r~ndcm transposition of a sequence of DN~ in the ~icrobial genome.

Back~ro ~ and Prior Art Historically the detection of microorganisms has depended upon morphological and biochemical analysis. More rec~ntly micro~rganisms have been de~ected by the use of specific DN~ probes and specific an~ibo~y probes.

U.S. Patent 4,35~,535 issued t~ FaIkow describ~s the use of specific DN~ probes for diagnosis of infecti~us ~ise~ses.

Antibodies u~ed in the detection of particllæ
microoryanism can include both pvl~clonal antibodies and monoclonal an ~ ies. For an example of such diagnoetic detection methods using monocl~nal an~ibodies, see U.S.
Paten~ 4,376,110, to Hybritech Inc~rp~ra~ed.

A pro~l~m inherent in kokh the DNA and a~tibody diagnostic technique is that samples may c~ntain a relatively small numker o~ mic~obes and the methods are not.
sensitive encuqh to detect ~hem. mis probl~m has been addressed in Canadian Patent 1,237,685. Ihis patent discloses a me~lcd of ampli~ying nucleic ac~d se~uances, which is commonly known as polymerase chain reaction (PCR).
Using PCR two primers are chosen which are substantially complemen~ary to di~ferent nucleic ~cid strands from a specific sequence. qhe extension product synthesized f.rom one prim~r, when it is separatad frcm i~s complement, can serve as a temyla~e f~r the s~nthesis of a complementary strand as the extension prcduct of the other pr ~ . 5eparating the primary extension produc~ from the template on which it was 6ynthesized and re-synthesizing ne~ pr ~ extension product~ con~inues for many cycles.
m e cycle is repeated for as mhny t.umes as it takes to increase the target or nucleic acid segment to a concentration where it can ~e detect0d. Using ~his method detection of ~he target 'microorganism DN~ sequences will be accomplished by the use of DN~ stains, agarose gel electrophoresis or DNA p~obi~g. Iherefore PCR is a pcwer~ul method which can be used to increase the sensitivity of the detection o~ specific micro~rganisms, l~e me~hod disclosed in Canadian Patent 1,237,~85, however does not address the pro~lem of sp3cificity. Uslr~J
methods known in the æ t it is very difficult, i~ not impossible, to distingui~h between tw~ closely related microorganisms. This is the problem addressed in the present invention. By ~he careful selection o~ prImers, it is possible to amplify a novel sequence of DN~, resultin~
from the insertian of an endogenous seguence of DN~ at a novel site. m e PCR method can be used to ampli~y this novel sequence which can be later detected by procedures known in ~he art. Ihe no~el seguence of DN~ resulting from the aforementioned insertion will be an inherited characteristic diagno6tic for that strain and its progeny a.lone.

Summary of In~ention According to ~he present inven~ion there is provided a method for detecting a unique seguence of DN~ in a microorganism, said sequence comprising a transpcsable element and flanking DN~ sequence, comprisin~ the steps of:

(a~ seles~ting a site a~ ~ich the ins~tion of the ~b) s~31ect~n~ at lea~t one primer wh~rein said prime~
is substantially canplemen~7 to a nu~.leic acid strand of the uniq~e s~quewe to be dete~d;
tc) usir~ the primer, of step (b), to ~li~y the unique se~ence ~f D~ to be da~ted; and (d) detecting the pres~nce of the ~nplified sequence of DN~.
In a fl~rther ~inant of the pres~ invention there is provided pr~s ælected ~rom a trar~osable elemen~, naturally o~lrrin~ in a microbial strain.

l~rief_Des~iptior~ ~f the l~rawinc~s Fi3ure 1 shc1ws the nucleotide sequ~ of t:he in~tion sequence I~ and tlle ~lar~ing D~ result.ing frcQn the in~tion of I~ to pr~uce the novel st~ain of l~izobi~n melll~ti, 825. Ihe E~im~3rs ~ich can be used, accordin~ to this prese~t invention are E~ha~ in the boxes.

Figure 2 sh~ws the nucleotide s q ence O:e the in~ertion sequence I5Rma, which is ~n addition example of a transposable element which can be used aocording to the present invention.

Figure 3 sho~s the detection of the unique sequence generated by ~he spontaneouæ transposition event which producsd and distin~uisheæ R. msliloki, strain 825 from its parent strain SW7.

Detailed Descripkion of the Invention ~he present mven~ion involves a method for detectLng a microorganism c~nd itæ progeny wherein said microorganism arose from the trc~spDsition o~ c~n endQgenaNs sequence o~ DN~. This transposition event will resNlt in the creation of a navel sequence of DNA which ~ill be c~n inheritable characteristic for that microorganism alone and its prcgeny. There~ore this present invention provides a me~hod for the de~ecti~n of a specific micrOD ~ sm cYmongst closely related microo ~ sms and ~kher microo ~ sms present Ln a mix0d population. Iherefore m using the present inven~ion th~ problem of lack of ~ cificity, inhe~ent in the prior art de~ectio~ methcds, is overcome.

m e sequence of DNA which inserts itself to creatP
the novel seguence in the genomic DN~ o~ the microorganism can be any inbigenous DN~ ~equence capable of random transposition. In the e=todiments of the present mvention the indigenous DMA sequence capable of transposition are transposable elem2nts. Transposable elements in~lude insertion seguences and transpo60ns.

Transposable elenents are widely known and studied in microorganisms~ r~ransposable elements are sequences of DN~ which are apparently mobile since they can replicate independen~ly and insert a oopy o~ the~selves in the DNA
at another pcsition. Thus/ the transpcsition e~ent results in an addition~l copy of the transposable element in the genome.

The present invention applies to an~ transposable element. For examples of bacterial insertion sequences, see 'q~obile DNA", ~ds. Dou~las E. Berg and Martha M. H~we, American Society of Microbiology, Washinyton, D.C., 1989.
In the present invent.ion, the hacterial insertion sequenc~
ISRml and ISRm3 are given as typical examples. Trar~c~ons can include for example the transposons an~ trarE~son-like sequences of the Tn3 family. Tnl, Tn2, Tn3, Th801, Th802, Tn901, Tn90~, Tn401, Ihl701, Tn2601, ln2602, ar~ Tn2660 are a short list of typical transpo~able elements from the Th3 2~5~
fam~ly.

Ihe microor~anisms ~ich can be used ac~rdir~ to the present invention in~ l~de both prcikaryotic and eukaryotic stxains. Bacterial stra~ns, yeasts arxl othe~
fur~i, are particularly am~able to this type of detection method. ~ansp~le elemen~s are wid~3ly knc~ in the~;e microo~ms. ~en these sequ~nces are transpos~l acpies are insarted in~ he ger~n~c D ~ of the microt~rganis that a ur~.que s~quence of DNA comprising the transposable element and the flanking DN~ sequences is created, whi~h can be detected us ~ the me~hod of the present invention.

A short list of ~ypical bacterial strains which can be used ~cc~rd ~ t~ the present .L~Vention include:
Rhizobium spp., Pseudowonas spp., Alcal~enes spp., Aarobacterium spp., Enterobacter s~., Thiobacillus SpE~, Aæ~tobacter spp., Flav~ka ~ ium spE~, Xanthomonas spp~, Staphlococcus spp., Strep~cmyces sEe~~ Bacillus spp., Mycobacterium s~p., and Rhodoccccus spp.. Typical examples of yeast species, which can be used according to the present invention include: SaccharcmYces spp., ~ zosaccharomyces spp., Candi~a ~p. and RhodGkorulaspp..
Examples of other fungi, whic~ can be used according to the present invention include: Penicillium spp., Asper~illus spp., Puccinia sPP., Pythium sp~, Phy~phthora ~pp. and hlsari~n spp..

In the sp~cific examples, of the present invention the bacterial strain R. meliloti, contaimng the insertion sequence I~æml, is used to dem~nstrate the present inverltion. Alternatively, the insertion sequen oe ISRm3 from R. meliloti can also be used. Rhizobium bacteria fLx nitrogen within rout nodules of legumes. m ey are used extensivel~ as seed and soil inoculants for legume crops to decrease. the reqlirement for nitrGgen fertilizer. For example R. meliloti is used commerc.ially to inoculate alfalfa crops.

About 80% of R. me_iloti s*rains cDntain 1 to 11 copies of th2 insextion ~e~lence ISRml in their genomes ~Wheatcroft and Watson J. ~en. ~icrokial 1~4:113-121, 198~a). Ihis insertion sequence is 1319 n~lcleotides long and, in common with all active insertion s~quences, is capable of limited æ lf-multiplication and dispersal, or transposition to different locations in the genome in which it oocurs. All the copies of an insertion s0quenc , present a characteristic pattern or "DN~ f mgerprint"l for a particular strain and its cl~sest relatives. me patterns are reveal0d ~y the hykridization o~ endonucle~se restriction ~ragments to insertion sequence prQbes and this method i5 ncw ~raquently ussd for bacterial strain identification (W~eatcroft, R., and Wa~son, R.J., 1988b; A
Positive Strain Identification Methcd f~r R~izobium meliloti. Applied and Ehvironmen~al Microbioloyy 54: 574--.
576).
R. meliloti, strain SU47 contains 8 copies of the insertion sequence ISRml. ~. neliloti, strain 825 dif~ers from SU47 Ln that it bas acquired an additional copy o~
ISRml by transposition to a no~rel and ~ que loca~ion in the gencme. me genomic DN~ in this new regi~n oonsists of a strain-specific nu d eotide seguence where the cop~ of ISRml adjoins its flanking ~ e poesibility is very small tha~ any such sequence should haYe an identical count~rpart .Ln any other strain. The identification of such stable sl~3cific segments of DNA containing novel copies of ~he insertion sequence is the basis of tbe present strain detection me~hod.

qhe first step in the prccess of ane e=bodiment o~
the present m ~ention is the selection of a novel target site at which insertion of the transposable element has occurred. qhe isolation of transposable elements is well kncwn in the art, many are now available and ~he DNA
nucl~o~ide sequence has been dete~nmined in æv~ral cases.
Therefore, these is~lated transposable elemants can be used to probe for new tr~nsposition events, which result in the crea~ivn of a unique DN~ segu~nce which includes the transposable element and its flanking DN~ sequenca. Su~h novel ~ranspo6ition eYen~s can be detected by probe hybridization o~ the bacterial gencmic DN~/ ~ollowing restriction endonuclea~e digestion and gel electrophor~sis (for example, see Wheatcrof~ R., and Watson, R.J., 1s87;
Can. J. of Microbiol. 33: 314-321)D

The novel transposition eYent will r~sult in a unique hybridization band, visually observable, follswin~
pro~e hybridization as descrihed above. O~ce the size of the restriction fragmen~ c~rresponding to t~e ~mique hybridization band has been determinRd, restriction fragments of that approximate si~e range can be harvested frcm a preparative electrophoresis gel. m e DN~ fragments can then be cloned into an appr~priate cl ~ v~ctor and transformed int~ an appropriate host o~ganism. m e resultin~ transformed ho6t organisms would be screen~d with a probe specific for the tr~nspcsable ele~ent by methods well known .un the art. Once suitable clones are identified the cloned DNA wculd be seguenced and DNA primers would be select0d from within the inser~ion sequence a~d wi~hin the ~lanking DWA ~equences.

In this particular =dbodi~ent prLmerS would be selected whereLn one primer is selected from the transposable elem2nt and the other prLmer is selected from one of the two flanking regions. Alternatively, according to the method of this particular embodiment both pri~ers could be æ lected frcm the flanking region, one from each flanking sequence~ The le~ h of the primr is not critical provid mg that the primer is long enough to prime the synthesis of the extension pro~ucts in the pre æ nce of r~ t~ ~n~
the approæriate ~gents. A typical length o the prLmers could range frcm about 7 nucleotides to akout 30 nucleotides. Preferably the primrs contain frcm abo~t 17 to about 25 nucleotides.

In the EXamples to demonstrate the present inventionr the transposa~le element is I~R~ from the bac~erial strain R. meliloki. Sui~able primers, from wi~hin the insertion seguence, ~hich can be usad ~re listed 10 } ~ wo PE3 5' - d [~ n~lArlAOC] - 3' EP~ 5' d [~ [~D~]C~-I5'l-] - 3' EP2 5' - d [cc~ccTGcEroGra~T] - 3~ and PE2 5' - d [IqGCGCCIaG~CGGTT~] 3'.
In the prese~t embodlment for the detection of R.
meliloti, strain ~5, ~r~m~rs from flankin~ DN~ se3uences, which can be used aocording to the present invention includeo XEl 5'- d ~T5C~TG~GTGACCC~TTTY OE a~C] 3' XE2 5' - d [~ L~X~IoX~A~OCClJ - 3' and PEl. 5' - d [C~ U ~ -] - 3'.

m e position of these selecked primers within the insertion sequen~e ISRml and the flanking DN~ saquences is shcwn in Figure 1.

It will be o~vious to persons skilled in the art, that other primers can be chosen, ~hich can be used according tD the present inve~ion.

At least one primer, usually pairs of these primers will be used in the PCR proce~ure to amplify-the intervening s0quence and therefore allow the ~etection of the nc~el strain of bacteria and its progeny.

In a further embodiment of the present invention there is described a ~ethod wh~rein the flar~ing sequence prim~rs are no~ required. The use o:E a single pr~r ~rcen a trar~le element, present in multiple ca~ie~ in th~
gen~nic D~ vr the use of pairs of pr~s frcqn dif:Ee~ent transp~able elements~ presan~ together in t:he s~ne genv~ne, will often allaw the d~tecti~n of rDvel ~trains without the need for flar~cing s ~ ence clon~ng, D ~ sequencin~f vr additional primer synthesis. Ih.is simplification brings dramatic savings, but gel electrophoresis is necessary to identify the dia~nostic pr~ducts of the E~ a~plification.

In this particular e~i OdImeD~ it is necessary that the tra ~ le elemen~ has inserted into the microbial DNA in close proxImi~y to another transposable element identical to itself or a different transpo6able element.
P~R will result in an extension product which spans frcm one transposable element to ~ha second in clcse pro~imity.
Consequently, a dia ~ tic region between the two transpo:able elements will be amplified by the PCR m~thod~

Thi5 e~bodim~Dt is nok as highly specific a strain detection method as the m~thod wh~n a~ least one of the primers is from the flank mg DNA sequenoe. The method o~
this embodimen~ relies on a size comparison of the amplified prod~ct to ascertain the presen~e of the novel strain. Hawever, since DN~ sequencing of the flanking region is not required it does pro~ide for a quick asses~ment, and in scme cases it nay be specific enough for a particular application.
m e detection method of the preferred embod.lment of the present invention involves the amplification of a unique sequence so tha~ it can be unambiguously id~ntified using agarose gel electrophoresis or ~N~ probe hybridization. This method relies upon the use the PCR
method of DNA amplification, which is de ærihed fully in Canadian Patent 1,237,685.

If one primer i5 chosen ~rcm within the transpo:able element and th~ 5eaond pri~eL is selec~ed from a flanking seguence, the de~ection of ~he ~mique sequence resulting fr~m the transpositicn event can be determined as the sole product of amplification by DNA sta ~ directly, for example by stainlng wi-th ethidium br ~ide or agarose gel electrophoresis. Alternatively, when ~ne pri~er is chosen fr~m each sequence flanking the tra~spo6able el~ent, the nDvel copy of the transposable element can be determined as a product o~ a~plification ar~ confirmed by res~riction ~nalysis, agarose gel electrophoresis or DN~
probe hybridization. In the latter case, the probe used to detect ~he amplified D~ is the transpoeable element i~self. The DNA probe can be a radio-active or a non-radioactive label. E~ampl~s o~ radio~active lakelled probes include 32R, 35S, or 3H. Non-radioactive labels are gainin~ wide acceptance since they avoid the use of radio activity. Such non-radi.oact.ive labels include for example digoxigenin-dUIP detected by enzyme immunoas~ay.

Early detection o~ specific bacterial strains is important Ln agriculture, in*ustry and medici~e. For example the ability to monitor specific beMeficial or harmful backeria strains resulting from the insertion of a sequence of DNA resulting in a unique D~ sequence will be ~ery usef`ul in medicine, industry or agriculture.

Ihe following examples set forth various embodiments of the invention but are nok to be constIued as limit mg.

~r~J~?
~MPLE 1 SELl~!lION AND PREPAR~II~ OF PE~S ~OR TffE AMPLI~ IICN
OF q~IE I~nl~XN~lN~ Sl?l~IEIC D~ OF R. ~RAIN

A ~relin:Lnary step, re~l~red o~oe orL~y for ea~
bact~ial straln to be detected, is the ~election of suitable D~ poly~ ~rimer sequ~::es 7rl the r~gion ~ich inclu~; the uniquely located i~ion se~e~
copy. R~triction fra~Tments E;u~tal3le for ~encing the DN~ nucleotid&s of this region w~re isolated fram ~
meliloti~ s~ain 825. Ihe basic ~chniques used for manipalla~ion o~ culæs we~e, unless irxlica~3d belaw, as described els~ere (S~roc~ , Eri~h, E.F. arxl Maniatis, T. 19~9. ~aleculæ cloning. A lab~ ory Manua:L
2nd Edition. ~old gprir~ Hæbor laboratory l~ess, N.Y., U.S.A.) .

Genomic DNA of both R. meliloti stra.Ln 825 and the parent R. meliloki, st:rain SU47 was purified frcm 5 m:L
cell cultures a~s previ~u~sly describ2d (Wheatcroft and Watson, 1988b op. cit.).

A p~elimunary digest of 2 ~g DMA of each strain was made with 4 Uhits of various restriction enzyme~
(includin~ EcoRI and XhoI) aocording to the supplier's reccmmerd~tion~s (BoehringPr M~nnheim, Darval, ~uebec, Canada). m e fragments w~re separated according to size by agar~6e gel electrophoresis in adjaoen~ lanes and transferred tD Biodyne nylo~ filters by blottin~
(Southern, E.M. 1975. ~etection of specific sequences amang DN~ fragment separated by gel electrophoresis.
Journal of Molecular Biology 98: 503-517).
The nylon bloks were probed to detect fragments containin~ ISRml, using the specific probe pRWRml3 labelled .
: for su~6equent detection ei~her with a r~dioactive label or non-radioactive ~arker (Wheatcroft, R. and Watson, R.J.
j 1987, op. cit.).
:
Aut~radiograFhy, or non~radioactive stai ~ , revealed oopies of the unique I5Rml sequence in Ro meliloti strain 825, ~hich were absent from R. meliloti, strain SU~7, in different restriction fragments aaco.rding to the enzyme used. In fact these fragments were the only known distinguishing fea~ures of the tw~ gencmes. For example, unique R~ ~ lil~ti, strain 8~5 fragm~nts of 3.9 kb and 2.1 kb for EcoRI and ~hoI respecti~èly were d~tectsd and cho~en f~r s ~ ent purification.

~els were then prepared f~r electrophor~sis using 10 ~g of R. ~eliloti, strain 825 DN~, di~ested with either 10 Units o~ EcoRI or XhoI. Restriction fragm2nts in the approximate size range, 1.5 to 4.5 kb, were harvested using N~45 nylon strips inser~ed into the gels and then eluted ~ree according to the suppliers' instructions ~Schleicher and Schuell, ~ene, NH, U.S.A.).

m e restriction fragments ~ere then ligated to a suitable cleaved p~C cloning vector with T4 DN~ ligase (Vieira, J. and Messing, J. 1982. Ihe pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencin~ with synthetic universal primers. Gene 19:
259-268)o Next the cloned fragm~nts oantaining ISRml were isolated and amplified in transformants o~ Es~herichia coli identified by colony hybridization to the ISRml probe (Grunstein, M. and Hogness, D.A. 1975. Colony hybridization: a method for the isolation of cloned DNA's that oontain a 5~cific gene. Proceedings of National Academy o~ ~ciences. U.S.A. 72: 3961-3965).

Nucleo~ide sequencm g Gf the strain specific DNA
was done after sub~cloning smaller restriction units i~to M13mp18 and 19 (Y~nisch-Perron, C~, Vieira, J. ~d Messing, J. 1985. Impr wed M13 phagP cloniny vectors ~nd host strains: nucleotide ssguences of the ~3mp18 and pUC19 vec~ors. Gene 33- 103-119). qhe Cbmmercial Seguenase kit ~U.S. Biodhemical Cbrp., Cle~land, ~H, UoS~A~ ) was used in the dideox~nucleotide se~uencing method (Sang~, F., Nickle~, S. and C~oulson, P..R. 1977. DNZ~
seguencing with cha m ~ ating inhibit~rs. Proceedings National Academy Sciences. U.S.A. 74: 5463-5467).

Suitable sequences (17-25 nucleotides long) were then chos~ or use as PCR primers ~rom bDth within the ISRml copy ~Figure 1; e.g. Prim~rs ~P2 and ~E2) and its flanking sequences (Figure 2; e.g. PrLmers PE1 and XE1) to amplify the uniyue ~NA sequences ~pecific to R.meli oti, strain 825 compris m g the insertion se~ue~ce ~SR~l and its flanking sequence in this uni~ue location.
Primer sequences were synthesiz0d us.m g a Biosearch 8750 DNA Syn~hesizer acoordLng to the manufacturer's reccmmendations.

EX~MPLE 2 AMEIIFI QTION AND D ~ ON OF UNqQUE DN~ ~EQVENC~ FRoM R.
MELILCII, STRAIN 825 FRoM PNVIK~NNENr~L ~

Environmental samples for example from soil, water or plant tissue, were prep~red in order to release the bacteria ~hey contained. For example, 100 mg of field soil was shaken for 5 min in 1 mL kuffer (2 mM Tris-HCl E~l 7.7;
5 mM NaCl, 0.1 ~M ethylenediaminetetraace~ic acicl c~sodium salt (EDIA)) co~taining 40 mg insoluble polyvinylpolypyrrolidone (PVP~.

t~
Ihe sample was heated at 100C for 5 m~n, coo].ed on ice, and c~ntrifuged for 1 nuin. The denatured D~ of the bact~ial lysate retained in 10 ,1.6l of ~he sL~:natant served as the s~te for PC~R.

Polymerase chain reaction (PCR) D~ application was carried out as descxi~d previously (Si3iki, R., Scharf, S. I Faloona, F., ~llis, K.B., Ham, (~.T., Erli~h, H.A. a~d Arr~e~m, N. 1985. Enzy~ic alTg?lification of B-globin g~namic se~uences ans3 restriction site ~ sis for diagnosis of si~kle cell an~mQa. Scienoe 230: 1350-1354~.
m e optimMm conditions of thermocy~ling for the detection of B~ lils~i~ stra m 825 in 100 ~l Taql rN~ polym2rase reaction mixture (Pr~m~ga CD~pora~ion, Madison, WS, U.S.A.) were the following: 92~C 2 ~ ; 50C 2.5 ~in; 74C 3 mun anl repeated for 30 cycles; with a ~inal round of DN~
synthesis a~ 74C f~r 15 min.

Amplified DN~ product waæ detected by either direct staLning, gel elsctrophoresi~ or probing. For Rxample~ using primers P~1 and EP2, a primer from flankin~
DNA seg~len~e and the Lnsertion sequence, respectively, a di~gnostic fra~ment of 0.8 kb w~s generated. m is fragm~nt is pre~ent only ~hen R. meliloti, straLn 825 is present in the sample (see Figure 2, gel track # 9) but not when only th~ parental strain R. meliloti SU47 is tested t~ee Figure 2, gel track # 10).

Alternatively, the use of the primers XE1 and PE1, one fram each flanking sequenoe ~Figure 1~, generated a comple~e copy of ISRml which was verifiable by probing with pR~ml3 (data not shown).

As an internal control~ ~he primers PE2 and EP2, which are b3th wi~hin the insertion sequence ISRml (Figure 1), were used Ln the PCR method. m ese prImers give a 0.5 kb amplification product whenever, and wharever in the ~ 15 -~2 genome, I ~ is present. For ~xample, in Figure 3, gel track 11 contains the strain 825 and gel track 12 con~ains the sample SU47~ Gel tra~k 3 of Figure 2 shows the m~lec~llar weight markers.

It is und~rstood tha~ the mvention has been disclosed herein in connection with certain exampl~s and embodimcnts. However, such changes~ mcdi~ications or ~ valents as can be usad by those skilled in the art are intend0d to be included. A~cordingly, the disclosure is to be construed as exemplary, rather than limiting, and suc~
changes wi~hin the principles o~ the in~ention as are obYioNs to one skilled in the art are Lntended to be included within the scape ~f the claims.

Claims (25)

1. A method of detecting a unique sequence of DNA in a microorganism, said unique sequence comprising a transposable element and its flanking DNA sequence, comprising the steps of:
a) selecting a novel site at which insertion of the transposable element has occurred;
b) selecting at least one primer wherein said primer is substantially complementary to a nucleic acid strand from the unique sequence to be detected;
c) using the primer, of step (b), to amplify the unique sequence of DNA to be detected; and d) detecting the presence of the amplified sequence of DNA.

2. The method of claim 1 wherein a first primer is complementary to a nucleic acid strand within the transposable element.

3. The method of claim 2 wherein a second primer is complementary to a nucleic acid strand selected from the flanking DNA sequence.

4. The method of claim 1 wherein a first and second primer is primer is selected and wherein each primer is selected from the flanking DNA sequence from either end of the transposable element.

5. The method of claim 1 wherein the transposable element is selected from the group consisting of an insertion sequence and a transposon.

6. The method of claim 1 wherein selecting a site at which insertion of the transposable element has occurred comprises the steps of:
a) treating genomic DNA in a microorganism with a selected restriction endonuclease to produce restriction endonuclease fragments;
(b) separating the fragments by gel electrophoresis (c) identifying DNA fragments, in which insertion of the transposable element has occurred, by probe hybridization of the separated restriction fragments.

7. The method of claim 6 wherein selecting a primer the steps of:
a) isolating the restriction fragment containing the transposable element;
b) ligating the fragment into an appropriate cloning vector;
c) transforming the resulting cloning vector into an appropriate host organism;
d) identifying clones containing the transposable element;
e) obtaining a DNA sequence of the cloned restriction fragment; and f) selecting an appropriate primer from within the unique sequence of DNA to be detected.

8. The method of claim 7 wherein the selected primer is substantially complementary to different strands of the unique sequence to be detected and wherein the primer amplifies the unique sequence by the steps of:
a) using the primer to initiate synthesize an extension product from the strands of each unique sequence to be detected;
b) using the extension products as a template for the synthesis of further extension products and;
c) repeating the synthesis so that the unique sequence is amplified to an extent where it can easily be detected in a heterogenous mixture of DNA sequences.

9. The method of claim 8 wherein detecting the presence of the amplified sequence of DNA comprises the step of subjecting the amplified sequence to gel electrophoresis and detecting the amplified DNA.

10. The method of claim 9 wherein the amplified unique sequence of DNA is detected by the method selected from the group consisting of probe hybridization and DNA
staining.

11. The method of claim 1 wherein the microorganism is selected from the group consisting of bacteria, yeast and other fungi.

12. The method of claim 11 wherein the bacteria is Rhizobium sp.

13. The method of claim 12 wherein the Rhizobium sp.
is R. meliloti.

14. The method of claim 13 wherein the insertion sequence is selected from the group consisting of ISRml and ISRm3.

15. The method of claim 14 wherein the primers are selected from the primers within the insertion sequence ISRml.

16. The method of claim 15 wherein the primers are selected form the groups consisting of: PE3, EP1, EP2 and PE2.

17. The method of claim 3 wherein the primers are selected from the group consisting of: XE1, XE2, PE3, EP1, EP2, PE2 and PE1.

18. The method of claim 4 wherein the primers are selected from the group consisting of: XE1, XE2 and PE1.

19. The method of claim 1 wherein the primers are 7 nucleotides to 30 nucleotides in length.

20. The method of claim 18 wherein the primers are 17 nucleotides to 25 nucleotides in length.

21. The method of claim 3 wherein the flanking DNA
sequence contains another transposable element.

22. A primer for use in the detection of a specific bacterial strain, wherein the primer is selected from a transposable element, naturally occurring in the bacterial strain, and are from about 7 to about 30 nucleotides in length.

23. The primer of claim 22 wherein the primer is æ selected from the transposable element ISRml.

24. The primer of claim 23 wherein the primer is selected from the group consisting of: PE3, EP1, EP2 and PE2.

25. A method of detecting a unique sequence of DNA in a microorganism, said unique sequence, comprising transposable element and flanking DNA sequence, comprising the steps of:
a) selecting a novel site at which insertion of the transposable element has occurred;
b) selecting at least one primer wherein said primer is substantially complementary to a nucleic acid strand from the unique sequence to be detected.