CA1278987C - Method for detection identification and quantitation of non-viral organisms - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE A method for determining the presence of organisms which contain RNA in a sample which might contain such organisms, which comprises the steps of: a) bringing together the nucleic acids in the sample with a probe comprising marked nucleic acid molecules which are complementary to a RNA subsequence which is conserved in all organisms; b) incubating the mixture under hybridization conditions for a specified time; and c) assaying the incubated mixture for hybridization of the probe.

Description

~89~7 _ 1 _ DE SCRIPTION

~IETHOD FOR DE'rECTING, IDENTIFYING, A~D
QUA~TITATING ORGANISMS AND VIRUSES

TEC~ICAL FIEI,D

S T~ ~v~tion relates eo a me~ d ~ru f~r deteoting, is~entifying, and ~antitatin8 orgs~ in biolo~Lcal ~1 other sa~les. lta~s, lt relate~ to a T~thod for ~pec~f~ lly and iti~rely detecting and quantitating ~ org~; c~ ~e r~os~l R~, (hereinafte~ R-R~), transfer RNA (h~eir~:~t~ t-R~A) cate~ie~ or t~Lc 3ro~p3 of ~h organis~ and pr~vi~ly ur~ org~ c~ntaining R-g~ or t-~. The method i~ capable of da~ct~ preqer~:e of ev~ c~e or~, caitainin8 R-RNA

11~ i~tia~ al30 ~lve-~ a m~t:hod for uuing spec~fically prcx~ced nucle~c asid3 ca~?l~ntary to specifi~ seq~su::e~ or ~ti4r~ of di~ferent s~e~ of the ~ clas3 ~NA, cr ~; hr~A, or sr~ or the cla~ of ~NA se~ce3 ~herelslaftec prec~so~
specific seq~:es or b~dgEaA) ~h æe pre~ent ally in the pre-cursor ~A, R~ , t-R~A, hr~ oq: ~A ~l~cule~, ant noe in re ~IA, R-}~A, t-R~,.~ c1r sr~A ~Dlecules r eo det~t, id~ti~y, ~d ~i~te ~ organi~, groups of c~gani~s, thereof can be m~re cle~ly u~c~ood and a~preciated wh~
ca~it~ed in 1~ of ~e repre~en~tit7e ba~egrould inf=~

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BACXG~O~ ART

Each of t~ cells of all life for~, except vi~ , cantain sep~ra~e ingl@ ~tr~ld E~ rrDlecules, namel~, a læg~e ~lecule, a 5 ~ un s~z~d ~lecule, and a s~all ~lealle. The t~ larger R-RNA

Ribos~l RNA i~ a d~rect ~ pro~ct znd i~ c~ or by the R~ . ~ ~A ~ce ~s used ~ a t~late ~
sgn~he~ize R-R~A mD~ e3o A sepæate ge~ or each of 10 ~he ribo3a~1 RNA s~i~. Mhlt~ple R R~ gena~ eYdst in ~st cb~i~l R~ . Pl~nt8 ar~ c~rtai~l o~r form~ caAta~
~clesr, ~itx~sl d chlaropla~t ~-R~ gene~. For slmp~lc~
o di~als ion h~re~na~, the t~ee ~ep~ate R~aA gene~ will be 15 refsrred to a~ t~e R-R~A gQ~e.

A~ut 85-90 p~c of ~ tot~ E~ i~a a typi~l eell ~ R~
A bacteria ~h ~ ~. ~li c~tain~ 104 ril:osa~ per cell a ~lL~ liver oell contaisu al~ 5 x 10~ r~o~. S~e ~0 each ri~osom2 coatai~ or~ d esch R-R~ g~ he bac~ l cell ar~ ~ 104 ar~l ~ x 10~, ~especti~7ely~ of each R-~A ~it.

25 G~ 1~ ~ti~8 o~ a p~t~ nu~ cld ~e, e~

~d~ae~ æe f~, far ~le, in ~c e~e~R of ceLl~ ~8l ~retic analy~i~ of lii~e :

_~3.~l398~

Probably the best ch~acterized and ~ct stu~ied gene ant gene product are *~e R~ A ~e and R-R~3A, and the priar art Includes use of ~ybridizacion of R~ d ribosanal geres ~n gene~ic dysis ar~ e~lution and ra~or~ classifis:atic~ of ar~ a~d r~bosa~
5 g~ne se~:e3~ Genetic dycis in~ or ~ple, the deter-m~atic~ of ~e ~ers of ribasarlal R~ gena~ in væious ar~an:~;
the dete~nat~an of the similarity bet~æen the ~ti~le ribo=al RNA genes which ~e present in cell~ ~tian of the~ rate and e~ctent of synthe~eis Of R-RNA in cell~l and ~ faceors whichO control them, E~lution ~ul eaxon~ ~udies i~}volve ca~æ~
R-Rl~ 8en~ base sequ~e ~ related and widely different ~gan~. , It i~ Icnawn ~:hat the ribo~al R~ ~ene ba~e seq~ce ~ a least p~ Llly simil~ in w~dely d~ ent organ~ms, ans!l tha~ the 15 ~ of coli bacte~al r~bow0al R~ gen~8 ~dize~ well with R-~NQ from plant~ and a w~de variet~y o~ o*~e:r bæteri~l s~c~ ti~n of the E:. col~ geae ~lch h~e9 tO
t~ other sp~cies varieq wi*~ ~he degreQ of relat~ess of the argE~. Vir~ually 211 of the R~ ~e ~ce ~rldizes 20 to R-~&~. i~ clo~ly related bsc~rial speci@~, ~hile les~
~hridize~ to ~-R~ ~r~ distanely related bæterlal spec~es, and ev~ les8 wl~
As wi~h R-RNAs, .t-E~ æe pr@~ is all liv~ cells, a~
~æll as in ~ v~ase~. t-~ genes ~e p~esen in c~os~al 25 and pl~ ~8 o~ prok~y~te3 and in ~æ ~ o e~caryotic cells, includ~ ~he ~ of the T~c~ ~it~ia and chloroplasts.
~f~rent t~ ar cne t-R~A species of~n exist in a single cell. S-R~ gene~ of alitoc~n~ria, ~n~leic ~nd chloropla~sts æe 30 ~ eo th0 viru~.

~, ~ ;~7~39~7 t-RNA molecules are direct gene.products and are aynthe3ized in the .c~ using ehe t-RNA gene a~ a templaee. The t-RNA i~ of~cen synthesized a~ part of a larger ~IA. ~olecula, and the t-RNA portion i~ ~hen removed 5 fro~ thi~ precur~or molecule. After synthesls a fractlon of the bases of the ~-~IA molecule are chemically modified by t~le cell. A typlcal t-RNA molecule contains from 75-85 ba~e ~ .
Nu~erous ~-RNA molecul~s are preRent in all cell~
10 of all life for~, and uQually abou~ 10 percent of a cell ' g ~cotal RNA i9 compo~ed of t-RNA, a typical bacterial cell contain~ng about 1.5 x 105 c-RNA molecule~
of all types. If ~ach differen~ kind of t-RNA i3 equally represented in a bacter~al cell then 2500 of each d~fferent 15 t-RNA Dlolecule i9 pre~ent in each cell. A t~ical ~ammalian liver cell contains about 108 t-RNA molecule~ or an average of about 106 copies per cell of each different t-3~NA type.
During prs~eirl ~ynthesi~ individual a~no a id~ are ali~ned in the proper order by variou~ ~pecif ic t-RNA~, 20 each amino acid being o~dered by a differ~nt t-RNA specie So~e amino acids are ordered by ~or~ than one t-RNA t~rpe.
: ~ : There are cer~ai~ viru~e~ which contain t-RNA gene~
~: ~ in ~heir genomQ~, ~hese ~ene producing ~r~ru~ -~pecific t~RNA wher~ the viru~ g~no~e ls ~ctive in a cell. ThesQ
25 t-RNA~ can al o be prc~ent ~n multipIe copieq in each ~nfected cell.

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789~37 A~ with R-RNA gene~ and R-RNA9 the prlor art discloses u~ of hxbridlzatlon of t-RNA and t-RNA
genes in gen~tic analy~i~ and evolution and taxonomic cla~sification o~ organi~s and t-RNA gene ~equence~.
5 Genetic analysis include~, for example, the deterr~
nation of the nu~ber~ of t EINA gene~ in variou~ organ-ism~; the determination of che ~i~nilarlty between the mult~ple t-RNA gene~ which are pre.~ent in cells; ~
dQter~ination of the rate and exten~ of syntheqi~ of .
10 t-RNA in cells and the factors whlch control theDa.
Evolution and ~axonomic -~tudie~ involv~ comparing the t-RNA gene base ~equence from rela~ed and witely different organi~
And a~ with R-R~ ~ene base ~equences, it ~ ~ ~nown tha~:
15 an individual t-RNA gene bsse sequence is at least partlally si~ilar in dlfferent organi3m~. Total e-RNA
~how~ this a~ type of relationship and bulk t-RNA from one ~peci~s will hybrld~ze ~ignlficarltl~ with t-RNA gene3 of a di~tantlsr r~lated o~ga~is~. Ral: D~itoehorldrial 20 leucyl-t R~ hy~r~d~z~t signiflcantly with T~itochontria `, DNA of chickell a~d yea~ tBiocheD~ ry (1975) 14, tlO.
p. 2037). t-RN~ geT~e~ ve al~o been hown to be hlghly corl~rved amon~3 ~h~ ~e:~ber~ of th~ baeterial fa~nily En~erol~-cce ~e. Bull~ t-R~A genes fro~ E. col~
~5 hybr~dlze w~ll wi'ch t-RNA i~olatet ro~ 3pee~es repre- ~
sen~ 3 different gene~ (J. Bacceriology (1977) 129, ~3, p. 1435-1439). Th~ fract~on of th~ E. coll t~RNA/gene~
~h~ch hybridizes to thes~ o her species varie~ w~th the deg2ee of relate~;e~ of the orESan~ ~8 . A large fract~on 30 of ~che E .. col:L t~R~ gene seques~ce hstb~ dize~ eo ~-RNA

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~ ;~78 from a closely related ~pecles while much le~s hybridizet to R-RNA fro~a distantly related ~pecies.
The extent of conservation of the t-RNA gene ~equences during evolution is not a~ great as that for the R-RNA gene sequence3. Nonetheles~ the t-RNA
gene sequenee~ are much ~ore highly conser~ed than the vast bul~ of the DNA ~equences pres~nt ~n cell~.
The sen~itivity and ease of detection of mem~ers of 3pecific groups of organlsms ~y utillz~ng probe specif ic for th~ R-RNA or ~-RNA of that g;roup o~ organ-isms i~ greatly enhanc~d b~ the larg~ nu~ber of both R-RNA and t R~aA snolecules which ar~ present ~n each cell.
In addleion the hybr~ dization tes~ 1~ made ~ignifiean~ly easier ~ince RNA molecules pre~ent ~n oell~ ~re ~ingle s~randed. Thu~ a denaturation 8tep, such a~ must be used for a hybridization te~t which detect~ any fraction of cell DNA, is not neces~ary wh~n the tar8et molecule i~ RNA. Probe~ specific foE other clas3es of oell nueleic acld~, be31d~ R-RN~ or t-RNA, D~y be u3ed to specif~ eally detect 9 ident$fy nd quanti~ate ~pccific group~ of organlsTIl~ or cells by nucleic scid hybr~dization. Thus, - other classe~ o~ p~okaryotic cells include messenge2~ R~A (hereinafter ~), and RNA ~equence~
- ~ which are part of a var~ ~ty of precursor molecule~ .

2~ For exa~ple R-RNA i~ ~ynthe~izet in the bacteria E. coli a a precur~or molecule about 6000 base~ long. Thi~
pr~cursor Ellolecule 1~ ~hen proce~3~d to yleld the R-RNA
subunits (totali~g about 4500 baqe~) which are lncorpor~
atet into ribo~o~ ~nd the exera R~ ~equence~ ( 1500 ~ ~ 30 bas~s in ~cot~l~ which are discardedO t-RN~ ~aolecul2~
::~ and ribo~omal 5S RNA are also synthe~ized and proces~ed uch ~ manrlerD

In prokar~otic cell-q infected by viru3e~ there i3 al90 virus pecific mRNA pre ent. The DlRNAs of certain prokaryotic v~ruses are ~lqo synthe~izet as a precursor molecule which contains eXCesQ RMA ~equence~
5 which are er~ d away and dlscarded.
Many of the prokaryotlc mRllA3 and v~ nRNA~ are present up to ~everal hunc~ed . time9 per cell ~hile thousand.~ of the excess RNA ~eque~ce3 preQent in~-RNA
or t-RNA precursor mule~ules can be present in each cell.
Eukar~otic cells al80 contain preeurYor mRNA, a~
well as precur~or R-RNA and t-RNA, molecule~ wh~ch are lar~er than the final R~ or t-R~lA. ~olecule~.
Irl contra~t to prokaryote~, many newly ~ynthe~ized eukaryotic mRNA molecule~ are ~uch lar~ser than the 15 final mRNA ~olecule and contain exe@ss RNA -~equ~nce~
which are tr~ned away and ti carded. Another claqs ~ ~ o~ RNA. present ln eukaryotic cell~ i~ heterogeneou~
nuclear RNA (hereinafter known a~ hn-~), which i8 a di~erse cla~ oiE RNA which contain~ mRNA pr~cur or 20 lecule~ (which lea~e ths nucle~ ~For the cytopla~
where proteirl ~yrlthe~is occ~r~) and a larE~e amount of RNA which nev~r leaYe~ the nucl~u3. Thi~ fraction also corl~ain3 a 8mall fract~on of double ~rand RNA. Eukar-yotic nuclel al~o contain ~all RNA molecule~ called ;~ ~5 Yalall nuclear RNA (hareinafter nRNA), varying in length froffl 100-200 ba~e~.
The al:~undance " or rlu~ber o~ copies p~r cell, of dif~erent 21~RNA molecules var1e~ eatly. Thi~ varies :; froln a cQmpleac cla~3 o~ ~RNA molecules wh~ch are pre~ent 30 only 1-2 ~ per cell, ~o the mod~rately abundant c1~3~ of RNA mol~cllles whlch ar~ pr~sen~c s~ver~l hundred ~:

~.~'789~7 times per cell, to the superabundant cla~3 o RNA
~olecule3 whlch may be present 104 or more times per cell. Many of t~e RNA sequence~ present in hnRNA
are al90 very ab~ndan~ in each cell~ The RNA sequences present in the precur30r RNA molecules for R-RNA, t-RNAs and ma~y ~RNAs are also very abundan~ in each cell.
Individual snRNA sequence~ are extremely ab~ndant ant say be present from 104 tO 106 time3 per cell.
Eukaryotic cells are al~o infected by ~iruse~
which produce ~rus sp~c~fic mRNA and in may cases virus specific precux~or mRNA moleculeY which contain RNA sequences not present in th~ ~ature mRNA ~olecule.
~he indlvldual viru~ speciflc ~RNA and precur~or RNA
molecule~ vary in abunda~ce from complex ~1-2 copies : 15 per cell) to ~uperabundant (around 104 copie~ per cell).
My invention al80 r~late~ therefore, o a method for 3pecifically an~ sen~it~ely detec~ing, identifying and quantitat~ng or~a~ism~ ~ as well a8 ~ vlru~e~ ~ presen~
in cell~. Mor~ partic~larly, the method 1~ u3eful for en~itively deteceing, identifying and quant~tating any me~ber.o~.dif~erent .~lze~ categor~e~ of organi~
~; eukaryotic cell~, viru~, and ~n ~o~ ea~e prev~ously unkn~wn organi3m~ containing ~RNA, hnRNA, snRNA or excesq RNA ~olecules present in R-RNA, t~RNA, mRN~, or hnRNA
molecules.
Thi~ invention therefore ha~ broad application to an~ area in which it i~ important ~o det~r~ine; the pre~ence or ab~ence o ll~ing organi~m~, or ~iruses pre~ene in cell~; ~he q~ate of ge~etic expre~ion of, an orga~l~, cell, virus pre~nt ~n ~ cell, or groups of ~ .o~ pr~ol~o~lc Gr e~ka~tic oru~ . Such ~3=~ inclocb ' `

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7~ 7 _g_ medicsl, ve~er~ary" and agricultural d~a~no~ics and industrial ant pha~aceutical quallty cotltrol.
The invention involves a methot for u3ing specifically producet nucleic acid~ complesllentary S to, not only R~ and t-RNA, bu~ al o to ~pecific ~equences ar popula~ions of different sequences of the RNA class mRNA or hnR~A. 09 STlEUlA or the l-La~ of RNA sequence~ (hereinafter knolwn a~ precur~or ~pecific RNA sequenceq or p8RN~) which are present only in the precursor3 ~RNA, t RNA, hnRNA or ~n~aA molecules and not in ma~ure mRNA, R-RNA, t-R~, hnRNA or ~nRNA
~lecules, to detect, identify and quantl~ate specific organisTns, group or organism~, groups of eukaryotic cell~ or viru3e in cell~, by the proc~3~ of nucleic acid hybridizstion .
My invention and the novel~y, utilicy and unknown ob~r$0usrles3 thereof can be more clearly u~der~tood a~i;d ~eciated ~ ~i~et in th~ l~ght ~ ehe additional . remesen~t~ve ~nd inform~tion hereinater set ou~, 2û comprislnE~ thi~ art;
1. n~A~, and psRNAs are preseYlt in all organi~m3 and c~ , hnRNAs a~d ~nRNAs ar~ present only in eulcaryotlc: cells. Cell orga~ell~ which contain DNA, i~clud~g mitochondria and chloropl~s 3, al~o conta~n m~UaA, p~A, R-~, ~i t-RNA.
2. A typlcal bacterial cell con~ains ~Dore thara a thousand gen~s, the ~a~ a~ orit~ of whlch cod~ for a specl~ic prot2in. A m~malian c~ll ::: cQnt~ins over~lO,ûOQ genes e~Ph of which can produce RN~. Any g~e ha~ ~he potent~al to p~oduc~ ~ultiple cop~e~ of RNA in a cell. Each specific RN~ olecule protuced :13 a direct gene produce .

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3. Many dlferent mRNA ~equenoes can be present in eaSh organls~ or cell. The individual cells of a mul~icelled organiQm may have different mRNA sequence~ pre3ent in each s:ell or in different groups of cells.
Many differerle hn~NA, p RNA, and ~s~RNA
Yequence~ can be pre32nt in each cell or group of cell~ of a eukaryotic organl~m. '.
Cells infected wlth a ~peciflc: v~ru3 can ..
have pre~ent withln the~D a varlety of dif~erent typeY o viru~ speclfic mRNA and p~RNA

4. The number of copie~ l:hereinaf~er eha abundance) of a 3pecific ~RNA in a prokaryotic cell varies from z~ro to se~reral hundredr The abundance of a ~peci~lc psRNA qequerlce ~ a prokaryotic organi3~ or cell can be 10 to 20 tin~e3 hi~5her.
The abundance of a speclfic ~RNA ~olecule in a eukar~rotic cell ran~es fro~ 1~2 ~o greater than :; 10 per c~ll.
The abus~tance of a pecif ~ c hnRN~ ~equence in a eukargotic cell range~ :~ro~ 1-2 eo greater than 10 pe~ cell.
abund~ce of a ~pec~fic s~A mol~cule in a eukaryotic cell ~rarie5 fro~ 104 to 106 per cell.
The abu3l~ance o~ a specific p5RNl~ sequenc~ in ~; a ~ukar~otic cell var~e~ ~rom 1-2 to ove2 104 pe~
eell.

5. In ~an~r e~kar~otes, RP~ of ~variou~ type~ ~ ~
produced fro~ tlie repeated ~equence frac~ion~ of : th~ 1)~. Thl~ can resul~ in a populat~on of abu~dant R~ ~olecules who~e ~eque~ce8 are ~i~lar bu~ not identical to one another. A probe co~le-,~

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1;~7B9~7 meneary to one of ~hese RNA molecule~ wlll, however, hybr,~d~ze w~ th all o the other 9imilar RNA molecule~.

6. Th~ gesle sequence~ which code for the various individual mR~As. psRNAs, hr~7As and snRNA~ of vi:c'u5e~i and living organiqmo, hav~ beell con~erved to varylllg degreeY through evolution. The vast ma~ority of thece sequence~ are much.le~s.cOnserved than toRNA qequence~. SOme of the sequenc~s, h~wever, are hlghly conservet. For exar~ple the . gene which code~ for histon~ mRNA i~ very h~ ghly con~erved through evolution and the hi~ton~ ger~e ~equence is quite ~imilar irl w~dely differerl~ organisms.
l~he lack of conservae~on in th~ DNA 3e~uence3 1~ of ~any of the e RN~s all~w~ the production of :~ probes whi ch can readlly distingui~h between clo~ely rela~ed organ~ sm~ or ~iruse~.
A large number of s tudie s have been done on var~ ous mRNA8, hlRN~g, snRNA~ and p~7A~ (see Gene Expr~sios~ by B. ~ewln, in r~fer~nces). Thes~ inciude hybridiz~tlon of these RNAs i~ ~:ud~e~ ~n genetic analy~is ~ regulatio~
and evolution, ~n prokax~o~ and çul~aryotic organ~ and viru e~.

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:~ ~78987 Prior Art Hvbrldization Procedures Two basic nucleic acid hybrldization procedure~
are disclo~ed in the prior art. In one, in solution hybridizatlon, both the probe and sa~ple nuole~ c acid 5 moleculeR are free in ~olution. With the other method the ~ample i~ immobllized on a ~olid ~upport and the probe i~ free in solution. Both o~ the~e methods are w~dely u~ed and well documented in the literature. An ex~le of ~ olucion m~ presented ~lereinaftOE ~I th~
10 eaE~sple~ o, in ~ article by ~ et al., Proc. ~atl. Acad.
Sc$. USA ~1980), 77, p. 520, i~ an ex~le of the im~bilized ~d.
.. _~v . ... .. . . ... . .
The basic co~pon~n~s of a nuclelc acld hybridizat~on te t are:
1. Prob2 - A market ~ingle 3~saIad nucleic acid ~equence which i~ comp~ementary to the nucleic acid qequences tG be deeected (that i~ the tar~et sequen-ce~). A~ u~ed herein, the target sequQnce ~ ~ the ~cotal sequence or a sub-~equence of R-RNA, t-RNA, or other RNA.
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The prob~ lengt~ ca~ vary fro~ S ba~e~ to ten~ of thou~ant3 of b~e~, and will depend upon the specific te~t eo be doll~. Only part of the p~obe ~olecule need 25 be complementar~ 'co ~he nucleie acid sequence to be de~cected thereinafter the targe~ qequenc2s). In addition, the comple~entarity between the prob~ abd the target sequence ne~d not b~ perect. Hybrldi2atiosl does occur be~een i~p~rfectly co~plementary ~olecule~ wie~ the 30 result thae a e~ri:ain fraction of the ba~e~ in the h~bridized region are:no~ paired with the proper comple-~n~ary ba~. A p~obe may.be compo~ed of either RNA
or DNA. The ~orn~ o th~ nucleic acid prabe 2~ be a mar~ed sl~glQ ~trant T~lol~cule of 3ust one polarlty or

7~3~387 marked Qingl~ strand mol~cule having boeh polarit~eQ
pre~ent. ~he form of the probe, like ~t~ length, will be determlned. by t~e t~pe o~ hybridization test to be done .
2. Sample - The ~ple ~ay or may not contain the targee molecule ( i . e . the organi~ ~f intere~t). The sample may take a variety of for~, including liquid such as water or seru~, or ~olld such as dust, ~oil or tis3ue sa~ple3. The s'ample nucleic acid must be made available to contact the probe be~re any hybridizatlon of probe and targat molecule can occur. rhu~ the or-ganisD~I 3 RNA must be free from the cell and placet under the proper corldielon~ before hybridiza~cion can occux. Prior art method3 of in solution hybrldiza~ion neces-~Itate the pi~I~f the RNA
in order to be able to obtais hybridization of the ample R~
~ with the probe. This ha~ mea~
:~ 25 that to utilize th~ in ~olution method for det~ctinE~ targët sequence3 in a ~a~ple, the nucleic acid~ c~f the ~a~ple Dlu~e f iS~t be purified to el~ina~e proteir~, llpid~, and other cell component~, and then con~cact~d with the probe ~: ~der hybridizati on condition~ . The purlficatlon~ o~ the ~ample nucleic acid take~ at lea~t several hours and can take up to a day ~ dependir~g ~; : on the nature and quantity of the sample .
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: 3 . Hybrid~ zat~orl M~hod - Probe and ~a~ple fflU~'C be mixed lmder ~: 40 condition~ whieh will per~ nucleic ......... ... ............. ........ ... ... . ac~d hybrit~z.tio~ n~rol~re-~
con~acting th~ prob~ a~d 3ff~l~ in the pre sence of an i~organic or ~` or~anic ~alt under the proper concen-: eration arld te~pera~e condit~ on prob2 and ample nucelic acid3 DW~ b~ ln contact for a long enough ~, , ': :

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tlme that any possible hybrid-ization beeween the probe and ~ample nucleic ae~t may occur.

The concentration of probe or target ~ the mix~ure will determine the ti~e necessary for hybrid-ization to occur. The hlgher the probe or target concentration the ~horter the hybrldization lncubaeion time ne~ded.
A nuclelc acid hybridlza~on incub~ation mixture composed of pro~e and ~ample ~ucleic ac~ds mu~t be incubated at a specific tempera~ure for a long enough t$m~ for hybridization to occur. The lengeh o~ time ~ece~sary for hybridiza~ion to complete depend3 upon the concentration of the probe nucleic acid, the concentration o~ ~he ~ample nucleic acid which i~
; ~omple~entary to he probe, and a ba~ic rate of hybrid-iza~ion wh~ch i~ ch~race~ri~tic of the hybridiza~ion condieion~ u~ed. The ba~lc rate of hybridization is ~de~er~ined by th~ ~pe of ~alt pre~nt in ~he incubation mlx, it~ concentxatio~, and th~ temperature of incubatio~.
Sodiu~ chloride, sodlu~ pho~pha~e and 30dlum citra~e are : the sal~ mos~ frequen~ly u~t for hybr~tization and ~h~ ~alt conee~tration u~ed i8 rarely above 1 M and :~o~e ime~ a~ high as 1.5 - 2 M. The salts mentioned ~25: abo~e yield co~arable ra~es of nucleic acid hybrldi-~ ~ zatio~ when u~ed at the ~ame concentration~ and te~pera-:~ hur~s, a~ do the co~parable po~as~ , lithiun, rubidiu~, and CQ~iU~ ~alt~ Britte~ et al. ~1974~ (Method~ in Enz~mology, Volu~e XXI~, pa~ E., ~d. Gro~sman aad ~olda~e; Acade~ic Pres8, N~w Y3rk, page 364~ and ~etmur s~d David~on (1968) tJ. ~olecuLar Biology, Vol. 31, page , .

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gL;~78~387 349) present data wh~ch illustrate~ the 3tandard basic rate~ of hybridization attained in commonly used salt3. The hybridlzation rates of DNA with RNA
vary somewhat from ~ho~e of DNA hybridizing with DNA.
The magni~ude of the ~ariation i~ rarely over tenfold and varie~, depending for example, on whether an excess of DNA or RNA is used. See Galau et al. ~1977) (Proc.
Natl. Acad. Sci. USA,. Vol 74, ~, pg. 2306).
Cert~ln conditio~s re3ult in the acceleration of DN~;~NA hybridization. An emul3ion of phenol and al pro~otes the very rapid hybrid~zaeion of DN~ when the ~xture 1~ agitated. Rate inerea~es ~everal thou~and ti~e~ fa~ er ~han standard DNA hybr~dizati4~ rate~ are attalned with .hi~ sy3te~ (Xohn~ et al.> Biochemi~try 15. ~1977) Vol. 16, p. 532a). DNA hybridizaeion rate acceleration fo 50 to 100 fold over the standard rate~
haY al~o been ob~erYed when neutral and anionic dextran polymers w~r~ ~ixed with slngle ~trand DNA in ~olution (Wetmur, B~opolym~rs (1975) Vol 14.-p. 2517). Nei~her :~ 20 of the~e DNA ace~leratet rate condtion wa$ reporter ~o accelerate the hybridlzatio~ ra~ of DNA:RNA hybridi-z~tlon~ no~ a~are of any prior art which documents a condltion for accelerating th~ r~te of RNA:D~A
hybritiza~ o~
4. ~ybrid~zation A~a~ - A procedure i3 need to detect ~he pre~enc~ of probe molecules hybridized to th~ ta~g2t molec~le-~.
- ~ Such a ~ethod depend~ upon the abilit~r to ~eparate probe which 19 h~ridiz~d to targ~t molecule~ fro~
prob~ whlch i~ not hybrid~zed to ~arge~ ~olecule~. Prior are pro-cedure~ or a~sa~ g in ~olu~ion hybridization D~ cures ha~i done on ~ample nuGleic ~cids which ar~ st purified a~d then oontact~t wlth the pro~e in the hybridization Lncutl~t lo= mi~cturl! .

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~L~7~9~37 Hydroxyapatite (~) h~s been u~ed a~ a standard method for a~ayln~; in ~o~ution hybridization mixtures for ehe presenee o~ hybridized probe. Under the proper conditions HA selectively bind~ hybridized DNA probe 5 but does not blnd probe which is not hybridizPd. Other methods are available to a~say for hybridized probe.
The~e include Sl nuclease as3ay wh~ch depend~ on the ability of a ~pecific enzyme to degrade non-hybridized prob~ to small subunit~ while the hybridizet probe i not 10 degrad~d by the enz~e and remains large. Th~ ~cgraded probe can the~ be ~eparated from the hybridized probe by a ~ ze separation eechllique . Variou~ method3 for a~saying for in soluelon hybridized nucleic acid3 are pres~nted in Britten ~e al. (1974) ~upra.
The immobili::e~ ~ample nucleic acit hybridization method~ haYe the hybridization assay bullt lnto the hybridization ~ethsd. The~e method inYolve fixing th~ 3amp1e nucleic: ac~d onto an iner~ c~upport and t'nen h~bridizing thi~ i~nmobilized nucleic acid with a marked probe which i~ free i~ solutlon. Xybridization of any probe with the i~mobilized ~ample ~ucleic acit results th~ bi~din8 of the prob~ to ~he ~a~npel nucleic acid ~d ~here~ore the attach~Qe~t of ehe probe eo the iner~
support. N~n~hybr~d~zed probe rema~Lns free ~n solu~ion ~: 25 and can be ~ hed away fro~ th~ inert ~upport and che hybritized probe. Such a method requ~re~ at least ~everal hours to prepar~ th~ sample for nucleic acid hybridizatios~ and one to two hour3 of wa3hing and utilizeq large a~o~t3 of probe~ An advar~tage of ~hi~
E~thod is the capab~lity to place multipl ~ ~ample~ on ~: th~ ~e lner~ suppor~ and to hybFidlz~ and process all th~ le~ a~ os~e time. Examples or' ~uch an im~obilized 3a~1e ~ethot i~ pr2s~nted i.n Analytical Biochemi~ry ~ ;~78'3 -17^

(1983j Vol~ 128, p. 415, ant J. of Infectlous Disease (1982~ Vol. 145, ~,6, p. ~63.

Ma~cing Nucleic Acid~ Available for HYbridization In ~ nuclelc ac~d hybridization methods 5 have alwayq utilized nuclelc ~cids which have been purifled away from other cell components. Nucleic acid3 in cell~ and viru ~ are normally tightly ~
co~plexed wlth other cell componen~, uQually p~otein, and, in thi~ form are llot available for hybridi~st~on.
~0 Simply breaking ~he cell or v~rus open to releas~ the cont~nts does not r~nt~r the nucleic acid~ available for hybridization. The nucleic aci.d~ remain coslplexed to other cell or viral components even though rele~ed fro~ the cell, and ~ay in fact beco~e exten~ively 15 degraded by nucleQses which also maybe relea~d. In addition a mar~ced probe added to such a mix may beeome complexed to "~tirky" cell or ~iral components and be rendered ~navailable f'or hybridiza~cion, or the probe may be degraded b r nuclease action.
A variety o prior are ~thod exi3t for purifiying nucleic acld~ ant se~eral of these are described in Maniati~ et ~1., 8upra. These method~ are all time consu~ing - ~one talking an hour i~ re8arded as very rap ld- -and require ~ltiple manipulation~.
. Insofar as I a~ aware, there is no pr~or art method or performlng in ~ ution nucleic acid hybridization which doe~ not . require the use of ~ome sort of prepuri-ficat~on s~ep ~o^~lke the nucleic ac~d~ available for hybridlzatio~ .
The i~aoblliz@d mlcleic acid hybridlza~ion methods ~avolve fi~inl3 t~e ~ ple nucleic ac~d onto an inert suppor~ and then hybridizing thi~ imeaobilized nucleic ~.~

~.~78~387 acid with marked probe whieh is free in solution.
The proces3 of flxin& ~he nucle~c acid~ on ~he intert support provid2s a pur~fication 3tep effectlve enough to make the bound nucleic acid~ available for hybridl-5 zatlon. Mo~t of the non-nucleic acid cell or viral componen~c3 do ns~t bind to the iner~ support, and those which do bind do ~o at a different location than the nucleic acid~. Such a method require~ several hours, at a minimum, to prepare ~he sample nucleic acid for hybrid~zatlon. An advantage of thi~ method i9 the ability to place ~ul~iple ~a~ple~ on the i~er~ 3upport and proce3~ the~ all tcgether through the hybridization and ehe hybrldizatio~ a~say step~. The hybridization a~say con~t~ of remo~in8 the inert ~uppore from the hybridlzation m~xture. Prove which i~ hybridized to the fixed 3ample re~ain~ with the intert ~upport while non-hybridized probe remain~ free in ~olution.

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31 ~7~9~7 TFIuq, while the pre3ence of organlsms can be detected by any one of a large variety of prior art method~, none s~f the~e 1~ entirely satiqfactory for one rea.~on or another. Such method~ include , e . g., growth 5 n:ethot~J opeical detection methodQ, ~erolog~c and im~nunoche2~ical method~, and biochen~ical method~, as shown below: -Growth Tes t ~:
A large number of different growth te . t~ exis~, lQ each useful for the growth of a speciflc organi~m or group of organism~ . G:rowth tese3 ha~ e th~ potential ~ensitivi~y to detect one organi Dl. In practice, however, many organls~ are difr'icul~ or impo~ble to grow. These te~t are usually lengthy, taking fro~ one day, to ~on~h~, 15 eo co~2plet e. In add~tion, ~ ~ery large n~ber of tests would be needed to detect the pre~ence of any me~ber of a large group of organis~s ~e . g ., all bacteria~, assuming that the grow~h conditions for all members of the ~3roup are kxlown.
20 OpticaLl Detectioa Method~:
M1 cro~copic aaaly is coupled wlth different~al ~t~ining ~ethod~ i~ very po~7~r~ul, and in znany ca~es, ver~ rapid detectio~ ~!thodO A ma~or problem w~th thi~ approach i~ the derect~on of specific organl~ in the preQence 25 o~ large quantltie~ of other organi3ms, for e~cample, the iderltificstlon of a 3pecifle typ~ of graTn negative rod ~haped bac~er~a, irl ~he pre~nce of many d~fferent kindQ
of gra2ll nega~ive rod shaped bactaria. In addi~cion, a large number of te9t~ would be needed ~o det~ct the0 presence of ~11 me~nbe~s of a large group of osganis~ns ch a8 the group o~ alI bacteria).

.

- 20~ 789~7 Serolog~c and I~munochemical Me~hods and Biochemical Te~ t 9 -_ _ _ A larg number of different types of the3e te~ts exist. They are usually qualieative, not very se~3itive ant often require a growth step. A great many of these teqts would be required to detect all members of a large group of organi~m~.

* * * * *

U.S. Paten~ 4,358,535 to Falkow et al. di~clo~es a m~thod for the detection of genet~.c material, l.e., G2ne~ or Genome~. In this patent a clinical sample or ~solate su~pect~d of containlng a pathogen is transferred onto an inert pvrou~ Yupport, ~uch a~ a nitrocellulose fllter, and treated in ~uch a way that ehe cell~ are localized. The cell~ are th~n treated ~n such a way as to release their DNA and cau~e it to eoupl~ on~o the suppor~. Subsequent treatmen~ cau~e~ a separa~ion of the individual DNA ~trand~ of the geno~e. The strand~
are then contac~ed with labeled prob~ ~peciflc for the characterl~tlc polynucleotide ~equence under hybridiza~ion condltion~. Hybridiza~ion of the probe to the ~ingl~
~ ~tranded polynucleotide~ fro~ he pathogen i~ de~ected : by mean~ of ~he lab~l.
The method of ~hi3 paten , for detectlng gene~ or g~nom~ ke ~h~ oth~r m~od~ ~ntio~ed abo~ does not have the ~pecif~city, sen~itiYlty, rapidity or ea3e o~
performanee o t~a~.o~ ~y ~n~en~ion. A sum~2ry of com~ari~on~ o the Falkow et al. m~thod as di~clo~ed in ~he pa~ent and tha~ of applicant ' ~ method, a~ h~rein dlsclo~ed, i~ ~et out below:
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~.2~89~37 1. Method of doing hybridization FAI~W ET AL. METHGD APPLICANT'S METHOD
Im~obilized ~ethod only In Solution ~ethod emphasi ed.
Im00bilized method can be used.

2. Class of nucleic acid to be detected FAI~~W ET AL. METHDD APPLICANT'S METH~D
, Geneeic ~aterial (i.e., Detection of a primary gene Genes or Genom s). In product (RNO cnly. RNA is cellular organisms the not present as genetic ~sterial genetic ~aterial is always in cellul~r organisms.
D~A.

3. Abundance (copies per cell) of nucleic acid sequences to be detected FAI~ ET AL. ~T~D A~PLICANT'S MEl~lDD
Vi~tually all microorganism ~ of R-RNA are pres~nt chranosc~al genes ~e pre- per bactcrial cell. A~out 2 x ~: ~ se~t anly one ti~la per cell. 133 capies of each t-R~ is rouDscmal gene are present lsl each bacterial cell.
ususall~ presene 1-3 time~ Ten to 200 of each specific 1~. Ribosa~al RNA ~IA molecule is present are pre~ent 3-6 ti~ p~ bac~erial cell. The : ~ per cell. ~ are ger~ally hi~her in e~cæyo~c cells.

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-2~- ~L~'7 ~ 9 ~7 4. Ability of hybridization ~ethod to quantitate nucleic acids FALK~W ET AL. MEIHDD APPLICANT'S METHOD
None disclosed Excellent ability to quantitate nucleic acids, both DNA and RNA.

5. Ability to determine and quantita~e the state of genetic expression of a cell F ~ ET AL. ME~ D APPIICANT'S MEXXOD
Genetic expression cannot be Genetic expre.qsiQn c~n be deter-detersined by deteeting ~Qncd and quantitaeed by usLng genetic material. probe~ ~ich detect the pr gene pr~ducts or RN~s.

~: 15 6. Relative probability of detecting a false positi~e during diagnosis FALKOW ET AL. ME~H~D APPLICANT'S hETHDD
High (deteets only specific Low ~*~n emphasis is ~n genes). detec~ing RNA~.
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20 7. Relative sensiti~ity of detection of n~cleic acid~
ALI~W El ~L. ~ {~3 APPLICANT'S METHDD
Good. Nuclei~ acid hy~ridi- Hi ~ y sen~itive. From 20 za~ion test axe ln general to 104 times more sensitive ~: ~ 25 ~ qui~e sensitive. than possible with the approach outlined i~ Falkcw. RNA is almost ;~ alway~ mDre abundant than the genes : : which ~akR i~. The in solutisn ne~hod also ccnfer~ extra ,., ~ 3a ~ sensitivity oNer th~ imIobilized , ~ :

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8. Pre3aration of s~le for ~ybri~izatian te~t F~ Er AL. MEl~D APPLICANr'S ~ DD
Takes fr~ 2 - 10 h~s co Takes 1 - 5 minuteS to make im~bilize sallple rTucleic sa~le available for hybridi-acids and ~ake th~ avail- zation. RNA in cells is alrea~y able for hybridizatiasl. singl~ stranded. All of the Includies a step for corl- s~le ~ucleic acid is capable verting D~A to si~le strand of hybrid~zing.
form. Not all the sarrple T~cleic a~ ~e capable of l~ybridization .

9. h~nt of pro~e needet F~ E'r AL. ~ APPLICANI 'S ~ET~D
Us~lly taked 0.01 ~o 1 Need 10 ~to 10 6 micrograms of microgra~ of probe in probe per sa~l~.
~ybridization m~cture.

10. Ti~e needed far ~ybridization to oc ~; 20 FA~W ET AL. ~l~W APPLICANI'S MET~DD
2 - 20 h~s 0.2 - 0.6 hours , ' .

, 1~ 7~7 I am not aware of any prior art which teaches my method of detectin~ th2 presence or ab~ence of R-RNA, or of e-RNA characteristics of a particular group of organism~ utilizing nucleic ac~d hybrid~zation wherein i~ used a selected mearked nucleic acid molecule comple-mentary to a subsequence of R-RNA from a particular source. Nor am I aware of any prior art which discloses my method for detect~ng the presence or absence ~ R-RNA
in general, or of ~-RNA fro~ a particular source, by 19 nucleic acid hybridiza~ion u~ing a marked nucleic acid molecule complementary to all of ~he R-RNA, or t-RNA
subsequence fro~ a specific sourc~.
Nor a~ I aware of any prior art whi~h teache3 my ~ethod of detectlng the pre~ence or ab~ence o~ specific ; 15 ~equences or populations of different Qpecific sequences ~:~ of mR~A, psRNA, hnRNA or sn~NA to detect, identify and quantitate specific organis~, group~ of organis~ groups : of eukaryoti cell~, or specific viru~e~ in cell~ or a group of speeific viru~e~ i~ cells, by nulceic acid : 20 hybridization wherein ~ u~ed selected marked nucleic acid molecule~ com~le~entary-to a sub~equence(~), a ~equence~ or ~ population of ~equence~ or ~ubsequence3 `~: of ~R~A, hnR~A, ~nR~A or p~RNA fro~ a particular source.
Nor ~ I awar~ of any prior art which teaches my ~ethod of detec~ing the pre~ence or a~sence of a nucleic ~; acid charac~eri~tic o~ a particular group of organis~
: or virus~; or of rap~dly m2kl~g a~ailable foriA solution nucleic acid hybrtdization wi~h a spec~fic marked :~ ~ probe, the nucle$~ a~ids of a partleular group of organisms : 3Q or viruses for any purpo~e; or of utilizing ~n in solution ~: nucleic acid hybridization method which eombine~ a rapid ' ,: :

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7~3~7 me~hod for making the nucleic acids of specific groups of organi~ms available for hybridization with a specific complementary probe, with a method for detecting an or~anls~ nucleic acid by greatly accelleraeing the 5 rate of in solutlon hybridiza~ion of the nucleic acids of an organi~ or viru~ and the marked probe complementary to ~he organism' ~ or virus ' ~ nucleic c~d; or of determining the anti.microbial agent sen~itivity or an~iviral agent sen~itivity of a particular group of 10 organis~n~ or viru~e~; or of assaying for the prëQence of antiDIicrob~al or antiviral sub~tance~ in bloot, urine, other body ~lu~ds or ti~sue~ or other qa~ple~; or for dete~Qining the sta~e of growth of cell~; or of detecting microorganisDI or viru3 infection-~; or of rap~dly assaying l~S for the presence, ~n a hybridizaeion ~nixture, of probe which has hybridized, by contas:ting the Dlixture with hydroxyapatite u~der pred~t~nined conditions and ther~
processing the re-culting solution in a 3pecific manner.

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DISCLOSURE OF THE INVENT~O~

29 The pr~sent in~ention provide~ a method and mean~
for detec in8 3 identifying, ant quantitatin3 organis~a~
1~ blological and oeher sampl2R, and ~ore particularly to a m~thod for ~peclfically and ~en~itively de~ecting and quanti~cs1:ing any organi~m containing ehe ribo~oma~-RN~, ~hereinafter R-RNA) ~ ~ran~fer RNA (hereinafter t-RNA~ or other RN~; any me~ber~ vf large, intermed~ate, or ~11 sized categories or taxonomic group~ of such organl~c; and previously unkno~ organ~ms containing R-RNA or t-}WA. The ~ethod i3 eapable of detecting ehe :
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~L~7~{3~7 presence of ~ven one organis~, containing R-RNA or t-RN~.
The invention al.qo provide~ a method for using specifically produced nucleic acids com~lementary to specific sequences or pop~lation~ of different sequences of the RNA cla ~ mRNA, or hnRNA, or nRNA, or the class of RNA sequence~ (hereinafter precur~or specific RNA
sequences or p~RNA) which are present only in the pre-cursor mRNA, R-~NA, ~-RNA, hnRNA or snRNA molec~le~, and not in matUrQ m~NA, t-RNA, hnRNA or snRNA molecuie ~o deeect, ldentify, and quantitate speclfic organisms, ~roups of organi-Rm~, groups or eukaryotlc cells or viru~e~ in cells.
~he ~m~tion also pro~ a me ~ d 2nd.~ h~n~ ~ ~t~-~z~n~ itie~: (a~ ~.abilit~ ~o ~cifically dbtecc ~ Presen~:e of an~ ane of a læge ~er of diffsr~t or~ wil:h a ~ingle assa~r procedure which al o works regardless of the l?attern : ~ of geneeic e2pres~iorl o~ any particular organiQm; ~b) ~: 20 the ability to ~odify the test to detect only specific cat~gories of organi~m~ 9 even in the pre~enee of organisms noe in ~he group of intere~t, tc~ extremely hi&h sensitivlty of deteetion9 ant ability to detect the presence of one organ~ or cell; (d~ the ability to quan~itate the numb~r of organis~ or cell~ present; and (e) doe~ not require a gro~th s c~p .
My invention proYide~ means for detecting the anti-mi~robial agent en~i~ivity or antivlral agent sens~ ti~i~y of a particular group of or~an~sms or viru~es; for assaying 30 the pre~nc~ o~ an~ crobila or antiviral $ubQtsrlees in ,~ .

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blood, urine, o~ ~r fluids or tissues c~ other sa~les; for determining the state of growth of cellR; for deteceing microorganlsm or viru~ infections; and for rapidly assaying for the presence ln a Xy~ridiza~cion mixture 5 of probe which ha3 hybridized.
As described hereinbefore, R-RNA base sequence~ are partially similar in widely different organisms. The more clo9ely related two organis~ are, the larger the fraction of the total R-RNA which is ~imilar in the two 10 specie~. Tha ~-RNA sequence of any par~icular pecie~
or orgarll~m can be regarded a3 a serie~ of ~hort R-RNA
subse~uenceY, of which ~ne sub~quence 1~ 3im~1ar in vir~u~Llly all life r'or~. Therefore, the R-RNA of almo~t all life for~ t contain thi~ ~ub3equence.
15 A different subqequence i~ q~milar only in the R-RNA
~f the members of the Species ~o whi ch that organi~m belongs. Other subsequences are present in ~he Order or orga~ism~ thac the Specie~ belongs ~o, and so on.
Becau~e th~ R-RNA sequences of widely t$fferent 20 organis~ are a~ lea~t partially . imilar, the ~nethod of my inv~ntion, u3ing a proba which d~tect3 the R-RNA
sequence~ which are imilar in widel~r tif~erent crganis~as, can tet~c~c the pre3e~ce or ab~ence of an~ one or mor~ of those organism~ in a sample. ~ ~ar}ced nucleic acid 25 sequence, or 3equences oomplemen~cary to ~he R-~IA
~equence3 ~imilar in widely di~,rergent organism~, can be uced as such a probe in nucleic acid hybridization assay .
Because of the R-RNA sequences of closely related 30 organis~n~ are more si~ilar than t~o e of d$~caIltly related organism~, the ulethod of my in~ention, which includes u~:Lng 5 probe whlch detect9 only the R-RNA sequeLces . ..

~7~87 -2~-which are si~ilar in a part~cular narrow group or organi~m~, can detçct the pre~ence or ab ence of any one or more of those particular organis~ in a sample9 even in the pre~ence of many non-relatet organisms.
These group specific probes can be ~pecific for a variety of different ~ized cat~gories. One probe might be speclfic for a particular taxonomic Genu~, while ano~her is specific for a particular Family or a~other Genu~.
Group specif~c probes ha~e the ability to hybridize to the R-RNA of one group or organism~ but not hybridize to the R-RNA of any oth~r group of organ~sms. Such a group ~pecific eomplementary ~equence will deeect the pre~ence ~f R-RNA from any me~ber of that spe~ific group of organi~ms even in the presence of a large amoune of R~RNA from many organlsm~ not belonging to that specifl~
group.
The total number of R-RNA molecule~ in a 3ample i~
~easured by u~ing a marked ~equence or sAquences co~ple~en~ary to R-RNA and ctandard exce~s probe or exce3s sample ~NA nucleic acid hybridization methodology.
Th~ R-R~A con ent of cells fro~ a wide variety of organi3m~ i~ known in the ~rt. In a broad group of ~imilar organi~, fo~ example bacteria, the amount of R-RNA per cell ~arie~ roughly 2-5 fold. Therefore, if ~ th~ number of R- ~ A ~oleculeQ in a ~ampley and the broad - : . class iden~ty of the source of the R-RNA i~ known, ehen a good e~timate of the number of cell~ pre ene in the ~am~le can be calculated. If th~ broad cla~
lten~ty ~9 not known it can be de~er~ined by hybrid~zing the ~ample eo a serie3 of seleotet probeQ com~le~en~ary ~o R-RNA, each of which lx 3pecific for a par~ieular ':
:

' ., -29~ 789~7 broad category of organisms.
At the presen~ ti~e, the operation~l detectlon and quantitation rang~ of a single a~ay procedure i3 fro~ 104 R-RNA ~olecule~ (1 bacterium or 10 2 mammailan cell.~) to abou~ 1012 R-~NA ~olecule~ (108 bacteria or 106 mammalian cellR) a ~pan of about 108 in cell numbers.
A single te~t could also be don~ in such a way a~ to operaeionall~ quantiea~e from 103 bacteria to 1~1 bacteria. The t~t is quite flexible ~n thi~ w2y.
Because the test for R-RNA i~ specif~c an'd has the ability to detec~ the presenee of ~ery few organlsms there is no need to ampliPy t~e numbers of organisms through a growth step.
The practice of ~hat orm of my invention which i4 directed to determining the presence o~ ~n organi~
whlch contain~ R-RNA, in a sample which ~ight contain uch organis~, comprises basically:
a) bringing together the sa~ple, or isolated nucleic ac~dq contained in that ~ampl~, wlth a probe ~0 wh~eh co~p~i~e~ mark~d nucleie aeid ~olecule which .; ar~ comple~entary to he R-RNA of all organism~;
b) incu~atin~ ~he se~ul~ing ~ixtur~ under pre-deter~ined hybr~dizaelon conditions for a pr~teter~i~ed e, a~d th~n;
c) a~aying the resulting mixture for hybridization of ~he probe.
~: When ~ in~entio~ i~ directed to determlning the ~ . presence of any me~ber o a speciie catego~y of organisms .~ ~hlch contain R-RNX in a ~a~ple which migh~ con~ain cUch ~ 30 organls~ ~ ehe m~ hod compri~es:

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, ' a) contacting the sample, or the nucleic acids therein, with a probe compri~ing marked nuclele acid molecule.~ which sre complementary only to the R-RNA
of meDlbers of the specific category or organis~s, but 5 no.t complemen~ary to R-RNA from non-related organism~;
b) incubatinE~ the probe and the sampl2, or the i301ated nu~ leic acid~ therein; ant c~ as~aying the incubated mixture for hybri~ization of said probe.
My inven~ion can lso be used to deterr~ine the nuDlber of organism~ pre~eat in th~ ~ampl~ under investi-gation, bg adding to the as~ying in the second above da~cribed method in the event probe hy~ridization has occurred, ~he s~ep of comparing the quantity of R-RNA
15 present in the ~ample with the numb~r of R-RNA molecules nonnally present in individual orgarli~s belonging eo ehe said ~peci~ic group.
And, of course, included in the Yariations ~ within the Ccope of D:~ invention ~ which can be u~ed, i~ that 20 which co~pri~es, in lieu of the single pro~e o~ step ~a) in the 3econd ~f the above methot~, a multipl~ city or ba~tery, of di.fferent probe~. In such case, each ~eparaP~ prob~ co~pri~es marked nucleic aeid molecules which are co~pleme~ta~y only to the R-RNA of a ~pecif i~
25 group of organl^~n~s and each probe i9 ~pecific for a different g~oup of organism3; ~tep (a) i~ followed by i~cubating e~ch probe-~a~rple ~ixtur~ under predete~mlned hybridizatioTI eondit~on~ for a pre~d~ter~rled time, and then assaying each ~ixture for hybridization of ~he probe.

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~ 9~7 As descrlbed hereinbefore t-RN~ base ~equences are partially ~imilar in widely different organis~.
~he more closely related two organix~s are the larger the fraction o~ t-RNA ~equence~ whlch are related.
Each t-RNA gene ~equence can be regard~d as a ~eries of short t-RNA subsequence~. One ~ub~equence is similar in a large related group of organisms. Another sub-sequence is si~ilar ln an in~ermediate sized related group of organism~ while a third qubsequence i3 similar 10 in a small related group of organi~ms and ~o on.
Since, alqo, ~ t-RNA sequence~ of widely different organi~s are at least pareially similar, the method of my invention, u ing a probe which det~ct~ the t-~NA
sequences which are simllar in widely dif~erent organis~, lS can detect the presenee or absence of any one or ~ore of tho3e organis~ in a sample. Thu3, a marked nu leic acit sequence, or se~uence~ comple~entary to ~he t-RNA
sequence~ similar in widely divergent organism~, can be u~ed as ~urh a probe in a nucleic acid h~bridization a~ay.
And 3ince the e-RNA sequences o clo~ely rela~ed organi-~s ar~ ~or~ -~imilar than tho~e of di~anely related organi~m~, the ~æ~hod of my invention, whic~ includes u3ing a probe whieh de~ec~ only the t-RNA sequences 25 whlch are ~i~$1ar in a particular narrow group of organi~ can d@tec~c the pre~ence or abRance of any one or ~nore of those particular organlsms in a sa~ple, even in th~ pre~erlce of many non-rela~ed organisms.
Such group ~peci~ probrs can be ~p~riflc for a varlet~
30 of differene ~ized categorie~ . For example p one probe :, `:

mlght be speclfic for a particular ~a~onomic Genus, while ano~her ~3 ~pecific for a par~icular Fa~ily or another Genus.
Group specific probe have the ability to hybridize S to the t-RNA of one group of organi~s but not hybridize to the t-~tA of any other group of organi~ms. Such a group specif ic complementary ~equence w~ll detect the pre ence of t~ A fro~a any ~ember of ehat specifi~ group of organi~ms even ~n the presence of a large amount of t-PJM from rrlany org~ni~m~ not belong~ng to rhat specific group .
In the pract~Ye of that forln of the inveneion which iq direc~ced to deeennining the pre~enee of any me~ber of a specific category of organi~ which contaln e-RNA In a ~ample which might contain ~uch organi~aG, the method compr i.~e ~:
a) contact~n~ the saDIple, or he mlcleic acid~
the~rein, with a probe compris~ng marked nucleic acid molecule~ which are co~pleDlentary only ~o the e-RNA
of meD~ber~ of the speci~ic category of organism~, bu not co~ple~ntary to t-R~A fro~ non-related or~anisms.
b ) incubating th~ probe and th~ le, or the i~olaeed r~iucleic acid~ therein; anid C) a388yi~ig the incubated mixture for hybridizationi :~ 25 of -qaid prob~.
~r in~ention c~i also be used to det~rsaine the numiber . of organi~m~ pr~ene in the ~a~plg under in~7estigation~ by id~lng to the as~ing ~n the secont abo~re d2scribed methodi in the e~en~c probe ~y~ridizatiorli ha~ occurred, the step of c0~2iring t}~ie quanitity of t-RNA pre~ent in the sample ':

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~1 2789~37 with the number of t-RNA ~olecule~ normally present in indi~ridual organi9Dl8 belonging to the said speoific group.
And, of courqe, included ln the variations, withi~
the scope of my invention, which can be used, is that which comprlses, in lieu o the ~ingle probe of qtep (a) in the second of ' the above methods~ a multipllcity or battery~ of diferent probe~. In ~uch caqe, each ~eparate probe comprise marlced nuele~c acid moleculeq whlch are comple~en~ary only to the t-RNA of a specific group o organl~ms and each probe i~ ~pecif i c fo~ a different 3roup of organi~; 3tep ~a) is followed by ~ncubatlng eaeh probe-~ample mixture under predetermined hybridization condie~ on~ for a pre-determined time, and then a3~ayln3 each mixture for hybridlza~ion cf th~ prob~.
The method and measn of ~y invention are Dlor~ fully illustrated in the following descrlp~ion of characterizing features of te3e method~ ~n aceordance with the in~entionO
Nuclei~ Acid Hybr~dization Test Procedures A desirable de~ectlo~ te~ ~houldo a) be rapidi b) be easr to U9~!i C) be highly se~a~itlve; d) be able to detect and quantl~ate in ~u~ one lab a~ay.
The exiqtent of a nucleic acid probe wh~ch will ybridize 'co R-RNA from any me~ber of the Genu~ Legionella, but d~es not h~bridize to R~RNA rom any other ource, make~ posslble a rap~d, ea~y ec, u~e, sensitive, in solu~ n deteetion test which can bo~h de~ect and quantita~ce, for exampl2, ~ bacseria with th~ performa~ee of just orae laboratory a~ay and do~-Q not require the purification of Aucl~iG ac~d8 fro~ the ~a~le.

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~1 ~7~3987 A description of the ba~ic aspect~ of thi~ in ~olution hybrid~zation te~t procedure ollow~. While the procedur~ te.~cr~bed i~ designed for detecting members of the Ge~au~ Le~ionella, it iq ob~r~ ou~ that this same te~t procedure can be used with the appropria~ce probe to detect many other group~ or organism~ or viruses.
Step 1. Preparine the S~ e M~x the ample wi~h a solution containing a detergent and a protealy~ic enzyme. The detergent ly es rhe baceeria and help~ solubilize cellular com~onent~ whil~ ehe enzyme destroys ~he cellular prote~ns, includ~ng ~hose enzymes which d~8rade RNA a~d DNAo The com~o~ition of the detergent-enzyme ~lx depends upon the type of detergent : and proteolytic enzyme u~d ant the amount and type of sa~Gple to be check~d. Detergent~ used include ~odium lauryl sulfate, -~arkosyl and Zwit~ergent, while the enzym~s u~et lIlclude Proteinase K and Prona~e. A wide variety of en:zy~e~, -qolubilizing agents such as chao~cropic : agent~, can be u~ed. The probe can al o be pr~3ent in the detergent-e~zyme ~ix added to the sample.
The enzy~-~detergent act~ v~ry quickly on any Lep~ion~lla bacteria in th~ ple. Irl lao~t case~ it i~ not nece~ar~r to incuba~e the mix ure in order to make ehe R-RNA available ~or in solu~lon hybrid~zation wi~h ehe probe. In eertain ca~e~ a short incubation perind i9 neet~d.
In other ~ituations it 1~ noc neee~ary to include the proteolgtic enz~e, and de~cergent alone w~ll make ~che ~- R-RNA a~ailable for in ~olution hybridization with the ~`~ probe ~

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., ,, 789~7 This approsc~ pro~tides a very rapid and easy method for gett~ng the sa~le R-RNA into a ~tate where it can hybridize with the probe is~ an in Qolutlon a~ay. In addition, it allow~ ~he hybritization to occur in olution 5 w~thout purifying the qample R-RNA. A key to this method that the probe detect~ Le~ionella R-RNA. R~
~ingle ~trand~d in the cell and reaty to hybridlze with the probe onc~ the ribosomal protein3 are remove~ fro~
the R-RNA. In con~cra t, ~o directly detec~ the ribosomal 10 R-RNA DNA (l.e., the gene for R-RNA) or any other DNA
sequence it would be nece~sary to add a procedure which cau~ed the double ~tra~ded R RNA gene to ~parate ln~o two ~lngl~ strsnds befor~ the probe could hybridize to ~t .
To the best of my knowledge, there i9 no prior art conc~rning the u~e of ~ enzyrn~-detergene-~ample method for making R~RNA, transfer R-RNA, RNA in general or DNA
available for in solue~on hybridization ~ith a probe for ehe purpc: ~e of detec~ing and quantita~ing the presence or 20 ab~ence of organ1-qm~ in general or a ~pecific group of organi~m~ .
Step 2. Pre~srin~ the HYbridizatlon Incubation Mixture To the sa~le-en~y~e-detergent miac add the probe and sufflcient 3al~ to enable hybrid1zation to occur 25 and incubat~ 'ch~ resultant mixture at a~ appropriate ~emperature. The ~alt ccnce~tration and the temperatu~e of hybridizatlon incubatisn combine tc determine the cri~e2 ion. The crlterion of the incubation contition mu~t be equal to that used to ~elect tha probe or the 30 ~pecifiri~y o~ the probe may change.

., ~ 7~ 9 -3~-~ e incubation mi~ture mu~t be incubated for a long enough tlme fpr hybridlzation to oecur. The salt typ~ and concen~ra lon determine~ eh~ rate of hybridlzation which can be attained. Thu~ certain salts will promote ~ery rapid hybrid~zation when u3et at the proper concen-eratiOn. An example of ~uch a salt i3 sodium pho~phate.
Legionella ~pecific probe mixed with purlfied Le~ionella R-RNA in 3.0 M ~odium pho~phate buffer tpH - 6.8~
(hereinafter ter~ed PB) and incubated at 76 C hybridizes la over 100 time-~ more rapldly ehan the ~am~ amounts of Le~ionella prob~ and R-RNA incubated u~der ~tantard cont~tlon~ of 0.72 M NaCL, 76 C (~he~e two condltiona are equal in crieerlon). Other sal~ ean al50 be uset to effec~ ~his hybridization rate acceleration. The~e include ~ost sodiu~, a~mo~ium, ru~idium, pota~ium, ceslum, ant lithlum salt~.
. In 3 M PB at 76~ C the hybr~dization r~te of ehe spec~fic probe wlth Le~ionella R-RNA present in the PB-enzy~e-detergent-~ample probe mixture ~s al~o sccelerated by o~er 100 time~ o~er the hybridization rate~ seen for the standard incubation conditions.
:~ Hybridization al30 occurs between the proba a~d R-RNA in an enzyme-detergen~-~ample mixture under sea~dard ~alt concentration condltion~.
: 25 One o~ the feature~ of the invention, a.~ previously poi~ted out, iq th~ ab~lity to detect very ~mall nu~bers o~ organi3~s by detecting their R-RNA. Thi5 i~ po~sible because of ~he large number~ of R-R~A molecule~ in each ; cell. Xn ~g~ like organisms 5,000 to 10,000 R-RNA
~olecule are present in each ~ndividual bacterial cell.
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.' _37~ 78 98 7 One of the major determlnants of the ~en~tivlty of detection which can be achieved with nucleic acid : hybrid~zatlon i3 the rate of hybridlzatlon which can be atealned. The co~bination of tetection of R-RNA
and the u~ of the rate accelerating incubation conditions described abo~e ~ake ~ e pos~ible to attain extremely high senqltivity of detection of baceeria and other organi~m~ in a very short period of time with the use of ~ery ~mall amounts o sa~ple and probe. An illustrative example of thl~ is de~cribed laeer.
To the bes~ of ~y knowledge there i3 no pr~or art concerning the use of rate-accelerat~ng ~y~tems with in olut~on hybridiza~ion ~est~ for deter~lning the presence or abRence o a~ organi~ or group of organisms by detecting th~ R-RN~, transfer ~NA, other RNA or DNA of the organism~ of intere~t. Ther~ i~ also no prior art of which I a~ aware concern~ng th~ u~e of a com~ination o a rate-accelerating ~yqtem and the enzy~e-detergent-sa~lR-probe ~ixtures to determine the pre~e~ce or absence of a ~pecif~c organis~ or ~iru~ or group of ; 2n organism~ or vlru~ by detectlng th~ R-RNA, tran3fer RMA, or other RNA or DNA of the specific organism or ~ group of organi8~ of iatere~t.
- 5t~p 3. A-~s~ying th~ Incubatior~ M~xture for The signal that the 3a~p1~ contain~ the target R-RNA
lecules ~and thcre~ore th~ target organi~m)`i~ the : pre~ence of hyb~d~zed prob~ in th~ i~cubatlon mixtur~.
Thus the incubatlor~ ~ixtur~ r~t be asa~yed for th~ presence , :

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of hybridizPd pr~be at the end of the incubatlon period.
It is desirable tha~ such an assay be ea y to perform and rapid. For this a~say the lncubatlon ~ix i~ pro-cessed by utilizing hydroxyapatlt~ (HA). Under the 5 proper condition~ HA binds R-RNA rapidLy and oompletely but doe~ not bind ~he non-hybridized probe molecule~.
If a probe molecule is hybridized to a target R-RNA
molecule the probe also bind~ to the HA because tt is phyqically attached to the R-RNA.
Detection of organi ms by detecting their R~
ls a feature of the invention. The abllity of the HA
to bind R~ or R~A ln general, in econd~, whlle no~
binding ~ehe probe at all, ha~ allo~7ed the developDIent of a hybrldization as~ay method whieh take~ nute~ to perfor~a, has grea~ flexib~lity and whieh adapt~ well for handling multlple ~amples. I~ addition the sample-detergent-enzgme-probe incubation mlxture, can be diluted ~n~o the appropr~ate bufer and directlg proce~sed to as~ay or the pre~enc:e of hybridized probe.
HA i~ known in th~ art a~ a ~ubstance used for as~aying hybridiza~ion of probe The as~ay T~lethod d~scribed h~e" whlch ha~ great advantag~ oYer ehe prior art use~ of EA (Brenner et al., Analytical 3iochm (1969 ( 28 p . 477), can be carried out at roo~ temperature a~nd w~ll work o~rer a temperature rang~ of about 15a C
to abou~ 90~ C. It ha~ fewer ~tep~ as~d doe~ no~ require heating at each s::enerifugatior~ ~tep; it casl be carried ou~ ln th~ pre~enc:e or ab~ence of de~erg~n~c~ such a3 Zwltterger~t tCalb~;oche~, Dan Diego, Cali. ) ant sodiu lausyl 3ulfa~ce. I~ i~ 3 - 5 tim~3 fa~er, aIld a ~ingl~

,, ~ , , 1 ~ 78 9~7 assay can be done in 3 - 5- minuce~. It requires about 5 time~ leqs HA, Deter8~nt concentration can range from O to lOZ, wh~le ~he pho~phate concen~rationC can range from 0.1 M to O.2 M depending on the type of assay. The method can also be readily adapted for handling multiple sample3.
Methods o~her than HA are avail ble to assay for hybridization of the probe. The3e include enzyme assays such a~ the Sl enzyme method, ~ize separation methodq, and a variety of sample immobilization method3. The probe.~ discus~ed here can be used effectively with these ant any oeher method of conducing hybridization ant hybridization a~ ay~.

Procedures for th~ Product~on ~
~: Different approsche~ can be used to produce group spec~fic prob~s. All of these approaches but one, rely on differen~ial nucleic acid hybridization methot~ to ide~tify and purlfy the group speciic probe sequence~
2G Procedure A:
The ~08t u~ful procedure for producing group speclfic R-R~A probss uses recombinan~ DNA methodology.
~ ~ ~he ~ep~ in~olved in thi~ procedure follow: (The ::~ pecific detail~ of ~tandard DNA recomblnant techniqu~s ; 25 ar~ de~cr~bed i~ ehe book, Manual, ~. Maniati~ e~ al., Cold Spri~g Harbor Publication (1982)) olat~ n~cleic acid fro~ a ~peclfic organis~
:: of in~er~st. Standard i301ation ~ethod~ are ; ~ 30 u~ed.

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~l~'789 2. IJsing t~iQ i~olated DNA, clone the R~ A
~enes of thi~. organi m and then produce arge a~o~nts of the r~bosomal gerle DN~, u.~ing ~tar~dard DNA recomblnant tec}~ology, a3 qhow~ in Man~ ati-~ et_al ., ~upra .

3. ~educe the ~-RNA gene DNA to shor~ pieces with re~tr~ ction enzyme~ and make a library of tl~ese ~hor DNA pieces, u~ing ~tandard DNA recombinant methods, as shown in ManlatiY et al., supra.
4. Screen the library and ideneify a clon~
whieh contain-~ a short R-RNA gene sequence which hybritlzes only to R-RNA fro~ other Dlember~ of ~he taxonomic SPec~e3 of the organl~ of lnt~res~. Isolate e~hi~ clone.
It con~s~n3 a Specie3 9peclfic DNA ~2quence whlch i~ eomple~ntary only to ehe R RNA of the speolfic Specleq to wh~ch the organism~
of interest belongs.
Screen the library further a~d identify and i~olate th@ following clones: a) a clone which contain~ a DN~ ~equence co~ple~sentar~r to R-RN~ which will only hyb~id~ze to R-RNA
froDl ~emb2rs of the taxono~ic Genus to which S ~he organi~D~ of in~e~e~t belon~i) a clone which contain~ a DN~ ~equenc~ complementary to R-RN~ which will only hybridlz~ to R-RNA
fro~ ~e~be:c~ of ~he taxono~ic Ort~r eo which : ~he org~is~ of lntere~t beIong~; c~ a clone : whlch coatain~ a i)NA sequence co~plementary eo R-}UaA w~ich will hybridize only to R-~tA
: ~ fro~ ~eD~b~r~ of th2 taxono~ic F~ily l o which the organi~ of~ in~ere8~ belon~ a clone whic~ con~ain~ a !)N~ ~equenc~: complementary to R-RNA wh~ch will hybridize only to R~ from me~ber8 of the taxonomic: Cla~8 to :which ~e organi3~ of intereslt belong~~p and e) a clone whic~ conta~nY a DNA sequence co~plesl~entary:
~o ~-}IPaA'whlch w~ll hybridize eo R-RNA ~ro~
: 40 ~ : a~ man~jr diferent 1~fe forTIl~ a~ po~ibl~.

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f ~ ' 1.~7~387 The foregoing clone qelectlon ~che~e i~ only one of a nu~ber of pos~ible ones.
Standard method~ of cloning and 3creening are to be utilized, a~ discussed in ~aniati~ et al., supra .
5 . a) Produce larg~ amount~ of each clone ' s DNA.
From the DNA of each individual clon~ isolate asld purify only the DNA sequence which is complementary ~o R-RNA, uqing one of the many method-~ existing ~co accomplish thi~ , e O g., as in Maniatl3 et al. ~ .~upra.
b) In cer~ain instances the total DNA present in ~ clone is useful as a probe ~ ~n which ca~e the total DN~ i~olated from ~he clonlng vector ~s uced.
c) In certain other in eance~, the DNA single strant of the cloning vec~cor which contain~ the DNl~ ~equ~nee comple~nentary to R-RNA i~ used as a probe. In such case thi~ ~trand muse be isolated and puxifled, using on~ o:E ehe various method3 which ex~ ~t to aceo~plish thi~, a~
de~cr~bllad by Maniatis et al.
6. The probe DNA obtai~ed in 5a, 51:~, and 5c must . be rnar~d in ~ome way ~o tha~ ie can be ` ~ identifi~d irL the a.~say mixrur~. Many different ki.nts of T~arker~ can be uq~d, the ~o~t frequently u~ed ~rkder bein8 radioactivity. C)thers include fluoress:ence, enzy~es, and biotln. Standard : methoâ~ are uqe~ for marking the DNA, a~ set oue in Mania~i8 et alO, ~upra.
7. The ~roup 3pecific R-R~A gene ~equenc~ in the clon~ng v~ctor exi~ts in a double strand ~at~.
On~ of th~e 3trand~ 1~ complementary to R-RNA
and w~11 hybr~dize wlth it. The o~her strand wiIl not hybridize to R-RNA but can ~e used to :: ~ produc~. ~arked group specif~c ~quence~ com~
mentar~ R-RNA. Thi3 i don~ b~r u~cilizing a D~A or RNA pol~era~e and nucleic acid precursor . ~ :
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~.~ 7~38 molecules whlch are marked~ The enzyme will utilize t~e marked precursor~ for ~ynthesizin~
DNA or RN~ u~ing the DNA ~trand as a tem~late.
Th~ newly ~ynthes~zed mærked molecule will be comple~entary to R-RN~ and can be used as a group 3pecific probe. The template DNA can be re~oved by various established means leaving only single ~trand ~arked nucleic acid, as described in Mania~i~, et al., ~upra, and the artlcle by Taylor et al., in 810chemica and Biophy3. Acta (197~ 2. p. 324.

Procedure B~
: Several enzy~es can utilize R-RN~ from any ~ource as a te~plate for ~h~ ~ynthesizing of marked DNA complementary to the entire R-RNA sequence. Group qpeci~ic sequences co~ple~entaxy only to the R-RNA of a particular c~as3 of -~ organis~ can b~ i~olated by a hybridization ~election proc~s~. The fraction of the synthe~iz~d marked DNA
~ : which hybridize3 onl~ to the R~RNA from member~ of a :~ 20 specific class of organi3m~ ca~ be i~olated by standard ;~ hybridization proceduse~. An example of ~his proce~ i3 pre~ented her~inafter. Such a probe can be produced in uficiene qua~title~ to clone ~ i5 de-~cribed in A.
The~ base sequence of ~hi~ clone can be determined by tandard met~od~ and the ~eque~ce u~e~ to direct the produc~ion of the probe by chem~cal synth~is using ea~dard method~.

: Procedur~ C
30 : Th~nucl~otide Qe~uences o~ R-RNA fro~ wid~ly di~ferent orga~is~.have been~d~teræined. Çroup specific e~uences ~ilar to a specific:group of or~anis~ can ., ~ :

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~1 ~ 789~37 be identified by comparing the~e known sequences. A
sequence complementary to thi~ group speclflc R-RNA
sequence can then be chemicall~ synthe~ized ant marked, using standard methodology.

Psotuctior~ of Specific Probes Complementary to t-RNA
While differenc approaches can be used to p,roduce specific t-'RNA probe3, the same ba~ic approache3 de3cribed for producing R-RNA probe~ can b~ used to produce t-RNA probe~. Standard ~ethod~ are available to i~olate indi~ridudl t-RNA species and gene~ and ~hese are well known in the ar~. The for~ of ~he probe may be DNA or RNA, and the l~ngth of the probe may be 12 to :~ thousands of bases long. The probe need no~ be perfectly cosnplemerltary to the nucleic acid it i~ ~pec:ific for >
i.e., the targe~c nuole~c acid, and the whole length of the probe need not be comple~entary to the tar~et molecule.
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`; Productlon of Spec~f~c. Probe~ Compîemen~a~y The sa~e ba ic approache3 used to produce ~pecific probe~ co~Dplem~ ary to R-RNA can be used to produce ; i ~ 3p0Cii~iC probe~ for ~pecific elasses or populations ~f mRNA, hn~A, ~nRNA, or psRNA. The me~hod~ for isolating each C:12~!~ of RNA and ur~her frac~c~ onating it are well ~S ~ known in the art . Again the form: of the probe ulay be - DN~ or~RNA, and the leng~ch of the probe ~ay ~a~y from about 12 to thou~ands of bas~s long.. The co~sple~entary region of the probè need not be perfectly complem~ntary eo the targe~c nucleic~ acid and the whole lengt~ of the 30 probe n2ed not be compl2mentary to the target moleeule.

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789~7 Isolatin~ Sample Nuclelc Acid Standard prior art me~hod~ can be uqet tO i301ate nucleic ac~d from the ~ample~ to be assayed. One standard method of nucleic acid i~olation and purification is pre~ented in the example section and iQ alss discu.~sed.
in Maniata~ et al., supra.
A new ~echnique for making nucleic acid3 available for in ~olution hybrldization without performing purificat~ on step i~ described hereinafter.

Perfonnin~ the Nucleic Acid Hybridization An approprlate amount of marked probe ~9 mixet wi~h the sample nuc:leic acid. This mixture i~ then ad~ustet to a specific ~ concentra~ion (NaCl is u~ually used) ~ and ths entir~ mix incubated at a ~pecifi~ temperature :~ 15 for a -~pecific ti~e period. At ~he ~nd of the ti~e per~ od the mix~ure i~ analyzed by perfonaing a hybridi-zation as3ay. Many different combination~ of salt, solven~, ~ucelic ac~d conceneration~, volum~ , and temperatures ; exi~t which all~w nucle~c acit hybr~dization. The pre-; 20 ferred c~binatio~ depenting on the circum~ta~Pes of ~ the a~say. It i~ important, however, ~ha~ the criterion -~ (s~e "Defin~tion~) of ~he hybridization ~tep~ be idsntical ~o criterla used to iden~ify and select th~ group probe.
If the criteria of the hybridization step is different, the probe speciiclty ~ay change. See: "Repeated Seque~e~ in DNA",.by Britten ant Ro~n~, Sc~ence (1968~
161 p. 529; "Kinetic~ of Renatura~ion of DNA", by Wetmur and Da~it~on, J. M~: Biol. (1968) 31 p. 349; "Hydro-x~apatit~ Technique~ for Nucleic Acid Reas~ociaeion", by Rohne and Britten~ Proced~r~ in Nucleic Acit Research . :

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Two different approaches are used with regard to the amount of probe and ~ le nucleic acid presene in the hybridization mixture. In one, the exces probe m~thod, there is ~ore probe present ~han sample nucleic acid, in thi~ ca~e RNA. With the other, the excess RNA
method, there is more R-RNA present than probe. The excess probe method i3 ehe method of choice for detectin~ the presence of RNA ln unknown samples. I~ ha3 se~r~ral advantageq which are discuc~ed below. See Table~ 1 and 2 for further discusslon of theYe ewo approaches.
Using the exces3 probe method9 the d~tect~on and quantita~lon can b~ done with just one lab a~say point, if the proper RNA prone i8 available. If the hybrit~-za~iorl has gone to completion the a~nount of probe which ha~ hybrldized i~ a direct ~ea~ur~ of the amount of R~A preYent in th~ sample. The fact that the probe hybr~dize~ at all indicateQ ~hat RNA 1~ presen~, and -~ 20 the amount ~of probe which hybridiz~-~ indicates . he a~nount o~ RNA p~esent ln the sample;
Making 311r~ eh~t the hybridization ha~ gone to completlorl in a known l:lme i~ i~nportant in order to quaneitate thg RNA. l~ read~ly dorle by adding 2S enough probe o ~nsure that tha hybri~izatiorl goe~ eo co~pletion in a ~elec~ced ~ime period. The more probe : addedt the ~a~ter eompletion is rearhed. Thlls the excesq probe me~chod provite~ a mearls to en3ure ~ha~ the hybrid~-: za~on ha~ gon@ t8~comple~10n and to know whesl thi3 ha~
occurred.

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-4~-In contra t, the deteotion and quantitatlon of RNA
can'~: be done wlth,one lab as~ay point when using the exce~s R-RNA ma~hod. In addition, the ~ime when the test poin~ ~hould be taken cannot be predicced in the excess RNA me~hod. Unknown samples with small amount-~
of RNA w~ll hybrldize muoh more slowly than sample~
w~th large amo-mt~ of RNA.
The As~ay for H~rLdizat~on The ~ignal that RNA of the specific group ~S in the .~ample 1~ the preqence of double str~nd market prob~.
Man~ different method~, well document~d iFI the literature, ax~ available for a~sayislg the hybrid~zation mix~ure for the presence of marked probe in the double 3trand for~.
The choice of me~hot depend~ upon th~ me~hod chosen or th~ hyb~idization ~tep, the eompo~ition of the hybrlti-zation mixtur~, th~ type of marker on th~ probe and other factors. One co~monly u-ced method i~ descrlbed hereinafter.
Se~ also Wetmue and D~vit~or~, Kohn~ and Britte~, and l~ et al., ~upra. Al~t) the ar~icle by Fla~ll et al., Eur. J. BiochR~. (1974) 47 p. 535. And al~a, the article by Maxw~ll c~ al., ~ leic Acid~ Re~a~ch ~1978) 5 p. 2033.
I21 all ca~ owe~ex ~ protant to either as ay at or abo~7e the 8am~ criterion u~d for ~he hybrldi-za~ion reactio~ or at a criterlon at which hybridlzation canno~c occur.
Quan~itatlon o~ Nuc:leic Acid Sequence~
by ~ucleic Acit HYbr~d~:zation Th@ ~uanti~cy ~f nucleic acid pre~en~ in a ~ample can ~` be det~rmlnQd in ~everal ~;3y~ by nucleic acid hybritl-30 zation, u~ing method~ w211 known to . hQ art. The two .

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78~87 me~hod~ are di~closed hereinafter u~ing the example of quant i ~ at ing R-.RNA .
It will be unterstood that th~ pre~n~ ;nethod i~
generally appllcable in any ca~e where ~ t is neoessary 5 to deter~ne the presenc~ or absence of organism~
which contairl RNA or DNA and that ~uch inelude~ bio-logical sarnple~ such a~ sputum, seru~ tl~ue swabs, and other ani~al fludis ant tissues as well as industrial and pharmaceutleal samples and w~er. Specific tetail~
10 of the approach will charge depending orl wh~th~r R~IA
or DNA is being quantitaeed but the general approach i~ th~ -~ame for both DNA and RNA.

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~ 7~9 -EXCESS SELECTED PROBE METHOD

PROBE: The probe is a specific, selected, marked sequence from a member of bacteria group B, which represen~s 10 percent of ehe bas~
sequence of the R-RNA, and hybridizes completely with R-RNA from group B bacteria, but doe~ not hybridize with R-RNA from other : organism The probe cannoe hybridize with itself.

A. Positive Hybridize to a) One percent o Homologou~ completion the probe wlll Control and assay for~ touble . for double strand mole-- ~ 0.1 mlcro^ s~rand pro~e cule~.
- 15 gram Probe b) Thi~ is a direct measure of ~he ~0 3 R-RNA sample.
m~crogra~$ The n~mber of : 20 Sam~le probe molecules ~:: group B hybridized equals R-RNA the number of :~: . R-R~A ~olecule : present.

:: 25 ~ B. H~tero- Hybridize to: The~ probe does l~gous completion not hybridize Control: and assay with any R-RNA
for double but R-RNA from û.~l mlc~o- strand probe group B bac~ria gram~ Probe 30 ~
10 3 micro-gram~ Sa~ple human R-RNA
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~L~789~37 TABLE; 1 ( Cont ' d) C. Unknown Hybritize to a) If no group B
Sample completion R-RNA is pre-n 1 m~ c~o and assay sen~, no probe grams Probe f~r ~Ubleb will hybridize.
b ) If group B
+ R-RNA is pre-Unknown will hybridize Sample and form double s tr and mo 1 e -cul~s.
c ~ The number of probe molecules hybrldized equals the number of group B R-RNA mole-cules present in the sample.
d) If one pereent of the probe hybridi~e-~ ~
group B R- RNA
is pre sent ~:: since ehe probe . wa~ selected so : that it would hybridize only ~; wi~ch R~RNA fro~
~ ~ ~ a group B
-~ ~ 30 : bac~eria. Since the probe will only hybridize to group B
R-E~NA, ~he pr~sence of : other R-RNAs : : will not inter-.~; ~i ~h ~ne ~ction or the quan~citation of.
any bacterial R-RNA present .
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TABLE 1 (Cont'd) e) Usin~ a selected probe nake~ it easier ~o ensure ~hat the hybridization is complete. A
selected probe repre-sen~ing 10 percent of the R-RNA se~uence will hybridize 10 times faster ~han a probe which is representa-eive of the ~otal R-RNA
sequence.
f) The detection of R-RNA
in ~ner 1 is not poscible ~ince the probe hyb~idizes only with group B R-RNA.
The ~en~itivity of de~ection o group B
R-R~A i~:exeremely : ~ high.
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D.
T~e exces probe me~hod need~
jus~ one as~ay point~ orter 25: t o de~ect and quantiea~e group B organi~m~.

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~789~17 TAB~E 2 EXCES5 R RNA METHOD: THE USE OF A SELECTED PROBE

PROBE: The probe is specific, selected, marked sequence from group B baceeria, which represeneq one-centh of the R-RNA base sequence of one member o group B. Th~
probe hybridize co~pleeely with R-RNA
from group B, but does not hybridize to R-RNA from other organism~. The prob~
canno~ hybridize with it elf.

A . Posit~ ve Hybrid~ze to a) The fraction Ho~ologou~ completion of probe which atld a3~aY a direct Sa~ stra~d prob~ mea~ure of ~he gra~ ~roup R-RNA a Id ~his + ca~@ 10û per-cent of the 10 3 ~nicro- probe can gram~ Probe hybridize.
b3 Thls percene-`~: age is r~oe a `~ ~ : measuse of the a~olmt of R-RNA
;~ ~ ` 25 pre~ent. In order to deter-mine chis ehe kinetic~ of th~ reaction . ~ t be deter-~ined.
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~78~387 8. Hetero- Hybridize to The probe does logous completion not hybridize Con~rol and assay to non-bacterial for touble R-RNAR.
0 .1 m;cro- s trand probe .
grams human R- RNA
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Probe micro-gr ams C. Unknown Samole Hybridi~e to a~ If no group B
completion R-RNA is present Samole and a~ay i~ ch~ ~ample for double ehere will be serand no hybridized probz. probe.
P ~ obe ;: . b) If group B
10 3 R-R~IA i present micro- the probe will ~ grams be hybridized.
:: 25 c) The amoun~ of R-RNA can ' t be determi~et from ehe perceneage : hybridization at 30 . the comple~ion o~
~he reaction . In order to de termine this the kinetics of the hybr idiza-: 35 tion ~ust be deter-minet. Since the pro~e will hybri-dize with only one type o~ R-RNA, the : 35 kin~ic determina-tion i~ simple.

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d) If 100 percent of th~
probe has hybridized with the sample, this meaII~ that group B
S R-RNA is presen~ in the sample. It does not indicate that only this R-RNA is prPsen~.
Other R-RNAs which do not hybridize with the probe may also be pre-;: sen~c in ~he samp le .
e~ ~ 100 percent of the probe hybridizes with lS the sa~pIe, it is pos~ble to :~pecifi cally quantieate the group B R-RNA in the present of human R-RNA
by de termining the l~in~tics of hybridiza-tion :o~E the probe with the sampLe R-RNAv Sinc~
he prob~ will: hybridi~e o~ly ' w~th group B: R-R~
uch a kineti~ reaction:
will have only one coE~Ipon~nt, the qne from reacting with group B
; 25 : ~ R-RNA.
f) There are ~ituations :: : : wh2~e eh~ hy~ridiza~
tlon Gan ~ t 8 to com-pl~tion. In thi~ me~h~d 30- ~ . the~sample R-~NA musc drive: he hybridization to :cc~ eion, since only a very smalL~ :a~unt :of probe i~ pre~es e.
: 35~ If :th~re ~ no~ su~fi-:
cie~at~ R-RNA in the sa3~ple:, the hybr~diza- -t~on ~ill xlo~ be :: : : : compleeod. The inte 3S: : ~ pretation of such a:
ltuation i~ d~3cus~ed : bslow.

~8~3~37 : If hybridization of unknown s~mple results in 20 percent hybridization of the probe - at the usual assay time, S it is not possible to tell if th2 reaetlon is complete with only one time poine.
It is neceqsary eo take another poine at double ~h~ original ti~e to deter~ine if ehe hybridiza-tion value increases. If ic does not iner2ase then : the hybridization is complete.
: 15 In thi~ ca~ the R~RNA is at such low concentration in the ga~pl~ that the proba is in exee~, and the nu~bex of R-RNA molecules present in : 20 the sample i~ equal to the :~ ~umber o~probe ~olecule.q ~ hybridize~.
: If the hybridizaeion value i5 ~:~ : increa3ed, the hybridizatIon :
`:: 25 wa~ not over a~ ~he first ime-point. A third time-: poin~ mu9~ ehen be done to . : tetermine whether the r~action wa~ over at the ~ second time point.

: The ~xce~ a~ple R-RNA method need~ T~ultiple as~ay point~ :
in ord~ o deeec~ and ~ :
:~ 35 : quan~i tate~ and i~ much more ~isne-con~u~ing :~ ~hat : th~ exce~- probe method. ~ :

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USE OF SELECTED PROBE5 COMPLF-ME:NIARY TO ONLY A
PARTICULAR E~CTION OF THE R-RNA SEQUI:NCE FR0~2 A PARTICUI,AR SOIJROE TO DETECT R-RNA VERSUS USE OF
IJNSE~GTED PROBES Cû~?l~:NTARY TO THE ENTIRE R-RNA
SEQUENCE FROM A PARTICULAR SOURCE TO DETECT R-RNA

On~ aspec~ of my invent~on, which comprise~ using specifically selected probe~ comple~entary to only a particular fractior~ of the R-RNA sequence~ to d~tecc, quantita~e, and identify R-RNA ha~ ~mpor~cant capabilities 10 and advantages over anoth~r a~pec~ oi~ the invention, that of u~lng unseleceet probe~ or 3equences co~plementary to the entire R~ equence to d~tect R-R~A. The advantage~ of u~inE5 a 3elected probe in b~Eh exce~
R-RP7A and exc~s probe hybridization methodologie3 are ~ 15 sst orth below. The proble~s with uslng a co~pletel~
repre~en~cative probe are al~o pre~nted.
Th~ advan~czg2~ of using a selected probe ov~r u~ing a cor~plQt~ly repre~neative R-RNA prob~, with exce~s probe hybridization, a~ well as wi~h exce~ R-RNA hybrldization, 20 l~ set out below:

Proble~ wit~h CaDpl~ely A~ of Us~ng 1, R~ ected in a ~e selec~ed probe ~ be used sa~le ~rith ~he ~:ess pr*~ ecr~ d specifically detPr:~ ~ ~ of l~ ence of a pa~iculæ R-RNA, prese~lt. T~3 ~ probe ~n't ~n an ~ ~a~ple wh~ used 'be u~ed to spec~ y detect i~ an exce~s p~robe }ybridizati~n ~nd qu~ntitaee 1~ p~e of D~t}d. Tnls ~ be dc~e with . ~lie., w~ hs exce~ pro~a prese~e of R~R~ frasl o~r ~g8~.

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~,7~3~3~7 2. As s~ated abave, t~ exces~ The use of a selected probe Dro~e ~thod carrlot be used with n~ke~ it pocsible tl~ use the ~s probe to detect or quanti- excess probe m~t~od for tate the presence of a particular detect~n8 and qu~ticating R-~ in a s~ple. For this pur- the presenf e o~ a pæ~i-pose the probe ~st be used ~n cular R-RNA in art ~awn the excess R-RNA m.ethod. sa~le. This greatly s~li fies the task.
The excess R-R~ nE!thod i~ ~ch more t~ne cons~i~ng, re~es nuch more ~ric, and is m~ch D~re colT~licated ~ the excess probe n~thod.

Advanta~es_o tlle Exce~s R-RNA Hybridization Method Probl~ with ~pletely Adv~tage~ of Us~
lle~re~ Rde Seleceed Probe 1. R-RNA cal ~ deeected in The seiected pro~e c~ be used al ur~awn sample with this to spec;fically tetect and : probe, but in m~r cases there quantitate the presence of i no way of ~etermining thE~ a pæticular R-~NA in æ
~:ype or quanei~y of R-~ ur~ow~l sa~le ~n all which is presen~. l~s in si~ation~. This c~n be m~ instances the pro~e . dar2e even in the presence nnot be.used to specific- of l~g~ a~unt~ of R-RNA
ally detect and qu~titat~ from other organisms.
ehe presence of a p~ti~ulæ
R-~ i~ an ur~ sa~l~.
2. In mar~ ca~e~ ~ sensi- With the selected probe the ~: ti~ r of detec~o~ of a pre~nce of R-R~ fra~ oth~r ~: 30 spec~ mLtet organism~ d~es not la~ar the by the pres~æe o E~-~A sen~itivi~y of detection of frc~ oth~r o~ga~s~. a particular R-~A.
3. In m~ case~ ~ere it is The dbtection e~ld quantitation . possible to det~:t and ~ti- of the presence of a p~ticular ~te the presence of p~rticul~ R-RNA i~ u~xh easier when a R-RNA9 lt require8 a lo~ of selected probe is utilized.
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Il lu~ trat ive Embodlment _ My invention,. illu3tra~vely, m~y be u~ed to determine whether a ~ample contain~ any member of a partlcular gs-oup of li~ring organi9m~. Th~ m~thod, 5 de~cribed ln the following example~ a te~t which may be u~ed eo detect and quantiaee th~ presence of a~y member or member~ of a partlcular group of baceeria in a sample, even in the pre~enc~ of large n~ber o~.
organ~sm~ which are not mellDber~ of thae par~ cular grsup .
A~ se~ forth in the examples, applicant's method irnolve~ f~r~t producing a group 3peciflc R-RNA probe wh~ ch, at a pecific criter~o~, hybridlze~ eo R-RNA
fro~ any me~ber o the ~peclfic group o~ interest, but toe no~ hybridize to R-RN~ fro~ a¢ly ot~er organims.
15 The u~e of ~uch a probe in a nuclelc acid hybridiza~ion te~e allows che de~ction of any n~ember oi~ that qpeoifiG
group, ~ven in the pre~ence of large number~ o other orgarlism~ . .
ExaD~ple~ of the practic:e of the in~ntio~ are lis;ed 20 la~cer. Each exa~ple involve~ the product~on o~ a marked nucleic: ac~d pro~e .which will hy~rid~z~ only ~ieh R-RNA
roE~ ~e~er~ of a partlcular group of organi~Ds.
The ba~lc ou~l:llne of the ~ethod used ~o produce each pro~¢ ~ ollow~:
2S 1. Producc ~a~ d nucleic acld co~ entary ~o the R~ of a member of th~ group o~ inl:erese.

. Hybrit~ze thi~ D~ to R~RNA fro~ a ~e~ber o the group o~ ~roups of :3rgan~3~ evolu~ionarily ~o~t clo~el~ related to thls group o organisms fo= ~-h1ch th~ p~obe i~ to be sp-ci~ic, Selecc ~' :

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'1;~78~387 the fraction of t~ marlced nuclelc acld which, at a ~pec,ific criterion, to~ ot hybridize to R-RN~ froD~ a ~mber of this clo~eat related group o~ organi m~. This frac~lon i~ ~peci~ic for the R-RNA of the organi~m gro~p of lnterest and doe~ not hybr~dize wi~h R-RNA ~rom the most closely related group or group~ or arly other organis~.
Exampl~ l: Produc~ion of Probe Whlch Will Hybridize ~In ~ ~ypical ~tuation, abou~ lO~ -107 ~ammalia~
cells are grown in~a ei~3u2 cul~ure pla~ce at one tim~.
Bacterial ~pecie~, expecially member~ oi~ the Eaxonomic Cla~ Mollîcute3, are known to conta~rlat~ ti~3ue culture cell~. Memb~rs of the t:las~ ~ollLcue-J, ~llke 1~03t ~: ~ other bact~ra, are noe r~adily eli~ina~ed b~ arlt~biotic~, and aré trouble~o~ conta~inant~ o~ c~ll culture~. Many - diffe~ent ~olllcute~ specie~ have been deeeeted in ti~lSUe c~lture cells:. If ~u3t one o the~org~l~m~ i~ pre~ent : 20 in the cul~ur:~ pl~te9 it ha3 the poten~al~ ev~n in the pre~en~e o~ ant~biotic~, ~o ~ul~iply a~d produce hundreds o~ organls~ p@~ cell. Such organis~ are capable o~
erin~ th- act~Yl~ o~ cells, there~ ~ff~c~g the :
result~ of ~ariou~ s~udle~ and:the marke ablli~y of cell 25 ~ culture~product~.
Prio~ art~ethod~ for detecting eh~se or~ani~
involve:baa:ically:~ualitatlve te~ts, the E~t commonly~
u~ed~being growth ~e~t~, differe~ial ~aiain8 te~t~ and i~Naolog~ a~ay~. The grow~h te~t~, while quiee : ~:

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1.~789~7 o59 sen~itive, ~ake 3 - 6 week~ O They have . he adtitional disadvantage that many organis~ns are difficult or i~pos ~ ible ~o grow.
While the actual detec~ion senqiti~rity of che ~taining meehod i not: known, it i~ known that more than qeveral organisms per cell ha~e to be pre3ent.
Immunologic tes~ are quali~ative test~ ant in~rolve using ane~body toward a particular specie While ehey can be car~ied out rapidly, they are nc~t very sen~itive;
furthermore~ many different antibod~e~ would be required to tetect all typ~ of Mollicutes.
The embodimen~ o~ applicant ' ~ me~ho~ described below, i3 a test which may be u~ed ~o detec~ arld quan~itate ehe presQnce of a~y. member of the group Qf jqlll bacteria, includlng ~he taxonomlc Class Moll~cue~l, to detec~ the presence of ~ollieutes in tis3ue culture, to detect the presence of bact~ria ~n ti-~su~ which i~ nonnally free of baeteria, a~d to detect ehe pre~ence o the bacteria evea~ i~ the pr~ence o~ larg~ nber~ of ma2nrllal~ an ce11~ .
A~ ~et foreh in th~ example, applican~ ' ~ ~ethod - in~olve~ fir3t maki~s~ a pecif~c R-R~A prob~ ~hich i~
complen~en~cary to R~ ro~ any bacteria but is not eo~Dplem~ntary to ~alian cell R-RNA. The U~@ of such a probe 1~ a rluclelc acid hybridization t~t allows th~
d~tection of any bacterla type, e~en in th~ pre3Pnce of large numb~s of ~2am~lian cell~.
A detai}~d de~crlptio~ of thi~ e~bodi~en~ of the in~ention follow8 ~: :
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~ ~ 78 9 Prepa~ation of R-RNA from Mammalian and Bacterial Cell~ .
Mammalian cells are resu~pended i~ 0.3 M Nacl, 0.02 M Tri~, pH - 7.4. Sarko3yl ~ B adted to a final concentrae~on of 1 percent to ly~e th~ cell3. Immediat~lv up~n lysis an equal volume of a 1/l mLXtUre of phenol/
chloroform iq added and the re~ulting mixture shaken vigorously for 2 minute~. The mixture i3 then centri-fuged (8000 x g for 10 minute3) to ~eparate the aqueous and organic phases. The aqueou~ pha~e i~ recovered, and to thi~ is added another ~rolu~ of phenol/chloroform.
After shaking and cerltrifugaeion a~ abo~ve 9 the aqueou3 phase ~ g again recovered. To thi i~ added 2 volumec of 95Z ethanol and thi~ ure i~ placed at -20 C for 2 hours to facilieate precipitatian of the ~ucle~c acid~.
Then the ~ixtur~ i~ centrifuged ~8000 x g, lO minutes) in order to sed~ent the pr~cipitate to the bottom of the tube. The liquid i3 ~hen removed. The p~lleted nucle~c acid i9 redi~301~d in water. Th~e solution is then mate to 0.2 ~ NaCl, 5 x 10 3 ~ MgCl2, 5 x 10 3 M CaC12. 0.02 M Tris (pH ~ 7.4), 50 ~icrograms per ml of deoxyrib~nuclea~e I and incubated at 37 C for 1 hour.
Then add an equal volu~ of phenol/chloroform and shake as abo~e. Cenerifuge a~ abo~e and recover th~ aqueouY
~5 pha~e. Ethanol preclpat the RNA a~ above. Centrifu~e the pre~pitate a~ abo~e and red~_~olve the pelleted RNA
;~ in water~ Make ~hi~ solut$on to 2 M LlCl and place ie at 4 C for 10 - 20 hou~3 in order to facilieate ehe precipitatio~ of t~-h~gh molecular wel~ht RN~. Then ~: 3~ cen~riguge thls ~olueion to collect the precipaee and ~;
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789~7 ret~-~solve the precipitate in water. This preparation of RNA coneains9 gr~ater than 95% R-RNA.
Bacterial R-~NA i~ lsolated in a s~ilar ~anner wlth ehe following exceptions. In ~hose cases wh~re deter-gent alone doex not lyse th~ bacteria, other meanq are employed. Thi~ usually involves pretreating the bacteria with an enzyme (lysozyme3 to make them susceptible to lysis by sarko~yl. After lysis of the bacteria t~e isolation procedure i~ as described above.
Purifled R-RM~ is stored at -70 C.

Produ~eion of Radioacti~e DNA Complementary (3H-cDNA) to Mollicutes R RNA
R-RNA from the specie~ MYC~e1aSma ho~ini (M. hominis), a member of the taxono~c class ~ , i9 used as a te~plate to ~ynthe3ize radioac~e cDNA oomplementary to M. homini R-RNA.
Thl~ cDN~ i~ produced by utllizing the abili~y of an enzyme, reverse transcriptase, to utilize R-RNA a~ a ~emplat~ and produce 3H-cDNA com~lementary (cDN~) eo R-RN~. Th~ r~erse tran ~ a~e reacelo~ ~i~tu~e contains ehe foll~wln ~ 59 ~ Tri~ CL (pH - 8~3) 9 8 ~M MgC12, 0.4 ~M d~thiothreitol, 50 ~ KCL, 0.1 ~M
~I-deO:~y~chy~idln~tr~phO~pha~e (50 C~rie~ Per Dlilli~Ole~, O . 2 ~M deOXYadenO8inetriPhO-~Pha~e, 0 . 2 DM deOXYOYtidi-netr1PhO3Phate~ 0 . 2 m~I deOXY8UarlO~inetriPhOSPhate, 200 m~cro8ram~ per ~1 o~ oligodeoxyribonucleo~ide primer ~ate from E. coli DNA, 50 mlcrogra~ per ml of M. hominl~
R-RNA and 50 unlt~ per ml of AMN reverse transciptase.

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This m~eure i~ incubated at 40 C for 30 mizlut~s .
Then ethylene dia~ine tetraacetic acit ~EDTA) (pH D 7 . 3), ~odiur~ dodecyl ~ulfa~ tSDS), NaCl and glycoge~ are added to final concentration3 of lû 2M, 1 percent, 0 . 3 5 M, and 100 microg~ per ml re~peGtively. ~he solueion i~ then ~aixed with 1 volume o~ phens~l~chlcrofon~ tl~l) and shakesl vigorou-~ly for 2 minute~, ehen een~crifuged (8000 x g for 10 minut~) and the aqueous pha~e recovered.
The nueleir acid~ a~e precipitatet by th~ addition of 2 . 5 volume of 952 e~hanol . Th~ precipica~ce i~ re~overed by centrifugatiorl and redi~olved in ~ 901ution ontain~ the te~plat~ R-RN~ and eh~ newly synthe.Qized H- cD~A .
Thi~ solutlon is th~n"nakQ to 0.3 M NaOH and incubat~d a~ 50 C for 45 r~l~nuEs~, and cool~d in ice and neu~rali~ed with 0.3 ~f Ht:l. Two and on~-half ~olumes o~ 95Z EtOH ara ~n added to precipi~at~ eh~ r~maining nucl~ic acid an~ th~ re~ulti~g pr~cip~:at~ redissolYed ~n water. Thi~ ~olu~ion is ~he~ pa~-d over a S~phadex G-10~ colum~ ~quili~tet to 0.3 M NaCl, 0.1 percent carko~yl and th~ ~xclu~d volum~ r~covored. Thi~ ~nlu~ion hanol pr2cip~t~t~d a~d th~ r~uleinE~ p~cip~ tate red~.3~01~r~d i~ a $mall volu~ of ~at~ Th~ p~oc~
descri~ed in eh~ p~æagraph re~o~re3 eh~ ée~plaee R-RNA
ant an~ re~ainisl~ pr~c~or ~ater~al fross eh~ 3H~cDNA
pr~p~atioT~.
3~cDNA 1~ eh~a hyb~ z~d to IS. ho~lnl~ R-RNA
co ensura that le i~ lndeed co~ple~entary to ehl~ R-R~A.
Th~ hybridlzation DliX~ oo~ t~ o~, 0.0S mic~ograms of ~ingle strand 3H-~DNA, 20 mic~o~r~ of 1( ho-lniJ

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This mixture is incubated for O . 2 hours at 65 C
and is then diluted to O.14 M PB and pa~sed over a hydroxyapatite (HA) column equilibraeed to 0.14 M PB
and 65~ C. 3H-cDNA.hybridized to R-RNA absorbs to the hydroxyapatite (HA) column while non-hybridized 3H-cDNA passes through the column. The hybridized 3H-cDNA is then recovered by elution of the HA column with 0.3 M PB. This frac~ion is then dialysed to remove the PB, ethanol precipitated to concentrate the nucleic acid, centrifuged and the nucleic acid redissolved in water. This solution is then treated with NaOH as described above in order to remove the R-RNA. After neutraliza~ion, addition of glycogen carrier and csn-centration by ethanol precipitation, the 3H-cDNA is redissolved in a small volume o~ water. This solution contains only H-cDNA which is complementary to M. hominis R-RNA.

Selection of 3H-cDNA Which is Complemen~ary ~o ~; 20 M. hom nis RNA but is not Complementary to Human R-RNA
The purified H-cDNA is further frac~ionated by ~ hybridizing it with a great excess of human R-~NA. The ; ~ hybridiza~ion mixture consists of 0.05 micrograms o ;~ H-cDNAj and 40 micrograms of human R-RNA in one ml of Q.48 M PB. This is incubated at 68 C for 1 hour and ~ the mixture is then diluted to 0.14 M P8 and passed over : ~ HA equilibrated to 55 C and 0.14 M P8. The fraction (about 50% of the ~otal) which does not adso~b to the ~;~ : HA (i.e., 3H-cDNA not hybritized to human R-RNA) is colleceed. This fraction is repassed o~er a new HA
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fraction i~ collected. This fraction i~ dialysed to remove the PB, etha~ol precipitated to concentrate the nucelic acid and redissolved in wat r. This solution is treated wi~h NaOH, a3 described earlier, in order to remove the human R-RNA. After neutralizatio^., addition o~ glycogen carrier, and concentration by ethanol precipitat~on, the 3H-cDNA is redissolved in a mall volume of wAter. Th~ 3H-cDNA preparation iQ c.omplemen-: tary to M homini~ R-RNA but is not com~lemeneary to human R RNA.

Hybridization of Selected 4H-cDNA with R-RNA from Di~ferent So~rce The production of the select d 'H-cDNA probe allows the d~tectio~ of bacteria, including member of t~e Class ~: 15 Mollicuee~ in mammal~an tisque culture cell~ and ma~mal-ian tissues b~ detecting the pre3ence of bactPrial ~-RNA
: by nucleic acid hybridizatlon. A nece-~ary requir2ment oX such a tes~ hat the seleceed probe must no~
hybridize to R-RNA fro~ mammalian cell~ whlch do no~
contain bacteria. That thi~ requirement i8 ~et is shown in Table 3V.
Tabl~ 3, parts II and III shown that the probe will detect al~ ~embers of the class Mollicuee~ and should detec~ all type~ of bac~ria. For example, Le~ionella_p 25 and E. coli and B8CillUJ sub~ilis are repr~sentatives : of very differen~ bacterial type~ ~nd the probe hybridizes ~with R RNA ~rom each. of ehe~e ~ype Evolu~lonasy con-s~derations indica~e that.thi~ probe will hybridize to . ~ ~
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39~7 R-RNA from vlrtually any known or unknown bac~eria.
Th~q i~- due ~o the extrerlle coneer~ation of the R-RNA
nucleotide .~equence during evolution.
This ~elected probe is u3eful for detecting the presenoe of a specific Clas3 of bacteria, Mollicute~, in tissue culture rell~. In most tissue culture cells antibiotlc~ are present in the growth ~ed~um and this prevents the growth of virtually all bacteria bu~
member~ of ehe Class Mollicutes. Thu~ any cont~mination of a tissue culture preparatlon i~ almo~t certain to be due to a member of the (::lae~ Mollicutes.
An ~poratnad aspect i3 the ability to determine the number of organ:l.sms pre~ent. In mo~t oases, cell l~ne3 and ~heir product~ are di~carded when cell~ are shown, by prior are ~ethod3, to b~ contaminated. The ability to quantita~e the~e organism~ makes it possible ~co ~ake judgements as to the ~everity of any effectc tue eo contaminaelc~n. The degree of a contaminatlon may be very ligh'c, ant only one organis~ per 1000 cell3 present.
~i~ level of conta~lnation would have very litele effeo~
~: 20 on the cell~ and i~ IQany cituations the cell prQducts neet not be tiscarded. l~he deci~io~ rQi8ht be ~de to s,~ta~n val~able cell lines until they beco~e more heavily cont~inaeet. Quantatiti~re considerations are :- impo~tane for 3utging the importance of any kind of a :~: 30 bacterial conta2alnation.

.

'~ ' 7~39 Hybridizat~on of Selected Mollicutes 3H-cDNA
with R-~A from Widelv Differen~ Sources Percent ~ybridizatic~
So~ce of R-RNA

I. Con~:rolA. N~ R RNA added, ~ 1 Ex~er~tRSelf Reacticm of 3H~
B. ~ck R-RNA i~ol2tia~
C. E~an c~ll R~ 1~ to be 97%
coneaminatet with M. h~inis R~

ybridizatialA. M~-Q of the Order of of 3~ cDNA with ~f~rc~ ales of t:axo~ ( ecr-~ ) 97%
` ~ cute9 - -~ 2~ ~plasma salivarius -- ~ ect~ 93Z
3. ~e~ ~W'W'W
( ~CtS pl88) 84Z
:: :
4- ' w~W~ i' W'W
ir~ect~ ~ce) 82 B. ~ers of the Order wh_ulcew o e ect~ c ows, bi~ , dogs, ~ : ; hcn~;e cat R, m~e, 3heep, pig8j ~)t pr~a~es) 52~
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~7813~7 Percenc ~ybridizati~
of H-cD~A
with S~ce of R-RNA R-RNA

II .Cl~nt ' d 2 . AcholePlasma laidIawii ( isolate ~2) 53 %

C. 2~ers of ~he Order .~ SDirOP1aS
. ~ ta eae i 1. $~ (infect~
insects and .~ 15 nic~) 69 Z
2. E~ney bee (isolated fram honey bee) 68 Z
~: 3. Cac~s (isolaced : 20 frw~ cactu~) ~ 71 4. Corn Stunt (isola~ed from ~: CorTI) 69 %
. ~ :
5. Corn Sh~nt (isolaeed ~ran ~sect) 65 ~ %
. ~
ybri~tion of A. ~5err~er of the ~amily 3H cDN~ w:Lth R RNA Enterobacteraceae fr~ o~hçr :type5 0~ 1. Escherischia coli :30 : :b æ teria (t~ ~infect5 m~mmals) 52 ; ClaJ~ ~ ) B. ~emb6r of the F~mi}y 1. Legionella:
: : pneumoph~la (infec~ man) > 28 , ~

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~78C387 Percent ~ybridizatior of H-cD~
with ~ce of R-RNA R-RNA

III. C~n~'d C.~rr~er of the Fami ly crococcaceae 1. Micrococcus luteus 50-60 %
2. Sea~lococcu~
auretls 50 Z
D. M~er of t~`te Family La~tobacillaccae 1. streptococ~Ls ~:~ ~ 50 %
E. Y~er of ~he Family ; ~ Bacillaceae : 1. Bacillus ~: 2~ su~tilis 40 n. ~ybrid~ Atian of cD~ wi~h R~ 2 Yeast ~ %

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:1~7~39~7 TABLE 3 ~Con~'d~
;

Percent ~ybridization of H-cDNA
So~Drce of R-RMA ~ith R-~A

V. Hybridization ~ (pr;~ee) 1 Z
R-RNA from ~ls and ~*use (rodent) 1 %
a bird Rat (rod~t) 1 %
~ ~amster (rod~t) 1 %
;~ Rabbit (lagw7Drph3 1 %
ick~n ~avian) 1 %

Excess R-RNA hybridization~ are don:e at 68 C, 0 . 48 M
15 PB. Hybridization a~say~ are don~ with hydroxyapatite ae 67 C in 0.14 ~ P13, 0.005Z sodium dodecyl sulfatP.
The hybridiza~ion exposure is sufficient to ensure c:omplete reaction of he ~H-cDNA with nuclear R-RNA
~: or or mitochondrial R-RNA. Nor~ bacterial R~RNA Cot ' S
;~ ~;: 20 ~ of ~t : least 2 x 103 are reached in the ca~e of the ~: mammals and bird. A non-speciL'ic sig~al of 1~2 percent has~ been substracted from the hybridization values prese~tet abov~

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Quanti~cation of R-RNA by Nucle~ c Acid HybrLdization __ _ The amount of bacterial R-RNA present in a sample can be deeermined by measuring the kinetics of hybridi-5 zation of the selected 3H-c-DNA probe with the RNA
i301ated fro~ a tissue and comparing these kinetics to tho.~e of a known ~andard mixture. This can be done even in the pre~ence of a large exce~s of mamma~ian cell R-RNA since the probe does not hybridize with thi~ R-RNA (see Table 3, V).
For measuring the kinetics, the hybridization mixture~ contain, 10 5 to 10 4 micrograms of 3H-cDNA
and 1 to 103 microgram of purified sample RNA in 0.01 to 0.1 ~l of ~.48 M PB . This mixture i~ incubated at 68 C and aliquot~ are removed, dilutet to 0.14 M
PB and assayed for hybridization at various times after the initiation of the reaction. Hybridization assays are performed using hydroxyapatite as de~cribet earlier.
The re3ult~ obtained are compared to the hybridization of the probe reacted with ~tandard RNAs con~aining known amounts of bacterial R-RNA. These standards are mixtures of mammall~n c~ll RNA and known amount~ of a ~pecific bacterial R-RN~.
:
Detecsion and Quantitation of MemberY of the Class Mollicute~ in Tissue Culture Cell~
Table ~ pre~ent3 daea obtained by hybridizing the selected probe with RNA i~olated (a-~ described earlier~
from three differe~t ti~sue culture cell sampels. Only cell line number 3 i detectably contaminated and the kinetic~ of the reaction indicated that aboue 5 x 10 bacterial cell~ are presen~ in ~he ti~ue culture cell~

. ~

1.~78~387 TABL~ 4 Detection and_~antitation of Mollicute3 in Tissue Culture C lls Hybridl- Hybridization Time of ~-cDNA Num~er of Bacteria 5Cell ll~e (hours) with RNA DYtected .. . ~ .... _ . _ 1. 44-2C (rat) 17 1 None detected 1 Ncne detected 2. P388 D~M
(mouse) 1.1 1 Nbne deteceed 22.5 . 1 None detected 3. P388 DlC 7 (mouse) 0.025 20 5 x 10 16.2 78 (ab~ue 1 M~llicute per mammaLian Ln cell) .
,'' Excess R-RNA Hybridizations are done at 6B C in 0 . 48 15: M P33 in a vo}ume of 0.01 to 0.04 ml. Each mixture contains 2 x 105 micrograms of 3~-cDNA probe and : 50 200 micrograsl-s of sampl~ RNA.

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~ 78~8 ~he foll~wing example i9 another embodiment of the method of ~y inYention, used for detecting very small numbers, even one trypanosome, ln ~he pre~ence of a large number of blood cell~.
The detection of trypanoso~es is important since certain members of the protozoan group TrYPanosoma are pathogenic for humans, causing d~seases that include East African c1eeping sickness, We~t African sleeping sickne~s, and South Amerlcan trypanosomiasis. These organisms are large and have varying characteristic shape , depending on the stage of the life cycle. Prior art methods rely mainly on serologic, differential taining coupled w~th microscopic examination and animal inoculation prooedureY for detecting these organisms in hu~ans. The serod~agnostic method~ ~ary in ensiti~ity and specificity and may be difficult to interpret. The microseopic method~ are most used, however small number3 of the trypanosome3 are cten difficult to detect in the presence of larg~ nt.~bers of blood cells. Animal inoculation 1~ a long and co tly procedure.
. The embodl~ent of ehe in~en~ion set forth in the following ~xa~ple 1~ a method which make-R it relarively ea~y to detect the presence of one trypanoso~e even when co-pre~en~ w~h a large number of ~lood cell~.
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Radioactive DNA complementary (3H-cDNAo to try~an-osoma bruee~ R-RNA iq produced in the ~ame w y a~

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~ ~:7~ 7 M. homini.~ 3H-cDNA, which is de3cribed above in detail, . .
execpt that ~r~2~no~ma b R-RNA iq used a~ a template.

Selectlon of Trypano30me 3H-cDNA Which is Co~plementary to Trypanoqome R-RNA but is not Com~lementary to ~UE~ R-Thi~ i~ done in the same way as described earlier for M. hominis except that TrypanoQoma b. H-cDNA is hybridized to the human R-RNA.

USQ of Selected Trypanosome 3H-cDNA ~o Detect and Ouantitate n~anos ~ s ~ ~En Tis ~ or ~uid The production of the selected 3H-cDNA probe allows the detection and quantitation of trypanoso~es in human samples by detecting the presence of trypano~ome R-RNA.
A necassary requirement of such a test is that the selected probe mu t noe hybridize to R RNA from human cells which do no~ contain trypanosomes. Tabl~ 5 shown that this requirem~nt i~ met.

.~

_ 7 4 ~ 7~39~7 Eiybridization of Selected Trypanosoma brucei ,, 'H-cDNA with R-RMA from Different Source3 _ _ _ _ Percent Hybridization R-RNA Source ofH-cDNA with R-RNA_ .~ 5 No R~IA added 1 %
Trypanosom_ brucei R-RNA 98 %
Bacterial ~Mycoplasma hominis ) Human R-RNA 1 Human R-RNA known to be coneaminated with Trypanosome 98 %
, Exce.~s R-RNA hybridizations are done at 65 C in 0.48 M PB~ Reactions are run fo~ 24 hour~ ant the hybridi-`~ : 15 zat~on exposure is ~ufficient to ensure complete reaction of the human nuclear or ~itochondrial R-RNAs and the ~: ~ bacterial R-RNA. Hybridiza~ion assays are done wieh :~ hydroxyapa~ite at 72 C in 0 .14 M PB, 0 . 005~ sodium dodecyl sulfate.

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-75~ 7 8 987 One illus~rative probe which I have prepared i~
specific only for member~ of the Genu~ Le~ionella.
The probe hybridizes to greater than fi~ty percent with nuclelc acid~ from diverse members of the Genus Le~ionella, and does not hybridize signif-ioantly wlth nucleic acids from mammals, yea3t and a variety of widely diverse baeterial strains (Table 8). The probe hybridize~ well even with Legianella species such as L. pneumo~hila and L. icdadei which show little or no bulk DNA relatedne~s. Other known ~ species can be detected by thi~
probe u~ed in Table 6; as listed ~n Table ~ All of the known Legionella specie~ (ehus far 23 differen~
~pecies) ha~e been examin~d and all can be speclfi-cally d~tected withthe probe used in Table 6.
: The ~pecif~city of th~s probe make i~ pos~ible to detect and quantitate the presence of ~egionella ~pecies, eYen in ~he pre~enee of large number~ of non-related bacterial or mammaliam cell~. Thus, liver cell from a L. ~ oph~la infected hamster was assayed for the pre~ence and number of Legi nella organis~s by u~ing the specific probe and well established ~: n~cleic acid hybrld~zation procedure-~. The liver had preYiou~ly bee~ a~sayed by a ~icrobiological growth te~t .: 25 whlch inticatet tha~ abou~ 107 ~ organis~s per : gram were prasent in the infeceed livex. Nucleic acid hybridization analy~i3 indicated about 1 - 2 x 108 Legionella organi-qms per gra~ of liver. The~e results ~ug~e3t9 t~at the~a~ing ef~iciency ~n the growth test i8 about 5 ^ 10 percent.
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-76~ 7~

The specific probe allows the highly sensitive and rapid detection of Le~ionella org ~ ~ even in the presence of large numberc of mammalian cells.
In an assay which took les~ than 1 day the probe easily detected the presence of about 400 Le~ionella organ sms which were mixed with 0.4 mg of liver (about 2 x 10 cells).
`: :

Hybridizat~on of Legionella Speclfic Probe With Nucl~ic Acits from Widely Different Sources Normalized Nucle~c Acid Percent Probe ' 50urce Hybridized I. Con~ol~ 1~ No n~cleic ~id ;~ 15 2) Mbck nuc~ ~ acid Isolat 3) L ~neum. ~ ected issue 100 (Ac~
percent ~ 81) II. Le~a~l~-:~ . ~ ~ 1) z~Enii ~GA) ~ 59 2) L. ~ff~ (TEX-K~j ?50 ___ ~: :
3) L. ~æ~E~i (LS-13) ?50 4) L. jor~ (~ 40) ~50 S~ 50 :~ 6j L. m:lcdadai (H~) ~ 50 7) L. M~ ~ 50 8) L. oao~ J (~i ~ 10) > 50 , ~ ~

' ~77 ~7~3~38 ~E 6 (Cont'd) No~malized ~clel~ id Percent Probe Source ~ 5 ~) L. pneu DphilA (P~A 1) 100 ; 10) L. ~ans~ 2 > 50

11) L. SC-32C-CB > 50 III . Other 1) Aec r~as c~r~Dhila Bac~erial Specie 2~ B s~b~ilis 1 3) !~
; 43 ~5~ ~!a~ 1 .: 5) 1:. ~oli 1 6~ Fl~bacteriull bre~
, ~ ~
7) ~ " ~ 1 8) " ~iDgosept~
9~ " ~ltivar~

:18) Ps~ ~ al~es 1 ",`, '~ ~i; ~

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, ' '~ ' ' ' 'i` ' `:
,.','' ' ' ' ' 7~187 Nor~ 1 ized Source of Percen~ Probe Nucleic Acid HYbr dized III. Other 19) Vlbrio El Tor Bacterial Specieq 20) Mycoplasma homini~
( cont ' d) 21) " ~
22) " salivarium 23~ Acholeplas~a Laidlawii, 2 4 ) Sp iro~ 1 a~ma SMCA
25) " corn qtunt 26) " honey bee 27) " cactu~ 1 15 .IV. Yeast S. cer~r 1 .
V. Mam~als Human Hams ter Mou3e ~:: Excess R-~NA hybridi2ation~ are done a~ 76 ~, 0.48 M PB.
20 Hybridiza ion a~say~ are done with ~ydroxyapatite at 72 C
in 0.14 MPB, O.OOS% sodlum dod2cyl ~ulfate. The hybriti-zat~or. e~Cpocure i~ qufficient ~co ensure compl~ee reaction q of th~ JH-cDNA with nuclear R-RNA or for mi~ochondrial :~ ~ . R~RNA. Non-bacterlal R-RNA Cot ' ~ of at lea~t 2 x 10' ~ 25 ar~ reached in the ca~e of ~he mammals and bird-~.
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1~78{3B7 TA:13LE 7 Oth~r Legioneila ~pecie~ Which Can Be Detected B~ Specific Nucleic Acid Probe of Table 7 SDecies _ WA-316 L. WO-44-31:: (L. feeleii) L_ Phoenix- l_ _ L. PF-209C-C
L. SC 65C3 (ORW) L. Jamestown 26Gl-E2 L . MSH- 4 L. ans~
: ` L. SC-I8-C9 :: , L. SC-63~C7 : I, 81-7l6 :

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1~78~7 Example 3: Production of a Probe Which Will Hybridize 0~ly to R-RNA cm ~rs of ~ ~u~ Le~ionel~

Pro~t~ of Ra~ac~ive DNA ~x~l~mentary to Le~ella R-RNA _ _ _ R RNA from the ~pecies Legionella pneumo~hila is u~ed as a templ~te to synthesize marked (radioactive) cDNA (eomplementary DNA) complementary to Legionella : pneumo~hila R-RNA. This cDNA is produces by utili~ing the abili~y of an enzyme, reverse transc ptase, t~
utilize R-RNA a a template and produce H-cDNA.comyle-mentary to R-RNA. T~is is done in the s~me way a~
described for producing M. hominls 3H-cDNA excep~ that R-RNA fro~ Legionella ~_eumophila i~ u ed as ~ template.

lS Selection of Radioactive Probe which Hybritize ~: only to R- ~ ember~ of the Genu~_LeRionella The purified H-cDNA ~-~ fract~onatet by hybridizing it with a 8reat excess of R-RNA rom E. coli, Acheola~lasma 2Q laidLswaii and Prsviden~ia stuar~ii. The hybrid~zation ~xit~re consi3t~ of 0.o5 - 1 micrograms of 3H^cDNA and 20 microgr2~s of each bac~erial R-RNA in 1 ml of 0.48 M PB.
~ Thi~ mixture 1~ ~ncubatet a~ 76 C for 1 hour and ~he : ~ixture i~ then diluted to 0.14 M P8 and pa ~ed over HA
equilDrated eo 72 C, 0.14 M PB. The fractio~ of 3H-cDNA
: wh~ch doe~ ~ot adsorb to the HA (~.e., the H-cDNA no~
hybridiz~d to the R-RNA) i~ collected. Thi8 raction is ` then passed o~er HA under the ~ame condi~ions as aboYe :~: . and again the non-ad~orbed fraction is collected. Thi 3H cDNA is ~hen concenerated and aga$n hybridized with ~ ~ bae~erial R-RNA ~ describet above. The non-adsorbed .~ fract~on i3 collected and co~centrated and ~h~n hybridized .~ ..

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with bacterial R-RNA for a third time as described above and fractionated on HA a~ above. The non-adsorbed fraction is collected, base treated to remove any R-RNA
present and concentrated into water. This 3H-cDNA pre-paratlon will hybridize to any me~ber of the Legionella genu~ and will not hybridize to R-RNAs from other sources.

Hybridization of Leg~onella specific 3H~cDNA Probe Wi R-RNA and R-RNA Genes from Different Source The selected probe allow3 the d~tection o~ any me~ber of the genu-R ~ in a ~a~ple by detocting the presence of ~ R-RNA by nucleic acid hybridizatlon. A neces3ary require~ent of 3U h a ~es~
i~ that ~he ~ pec~fic probe ~U3t not hybridize to R-RNA from o~her ource~.

Q~aneitation of Le~ionella R-RNA by Nucle~c_ id Hybrid~7ation The amount of bacteria~ R-RNA present in a sample can be tetermi~ed b~ ~easuring the k~net~c~ of hybridi-za~ion of the sel~cted 3~-cDNA probe wlth the RNA
isolated fro~ a ti-~ue ~ample and comFaring these kinetics to tho e o~ a k~own ~tandard ~ixture. Thi~ can be done even in ehe pre ence of a large exces~ of m G alian ce~l R- ~ ince the p~obe doe~ not hybrit~z~ with this R-RNA.
For mea~uring ~he kinetic~, the hybsidization : mix ure may conta~, for exam~le, 10 5 to 10 4 micrograms :~ of H-cDNA ant 0.01 to 10 microgram~ of purifi~d sæmple RNA in 0.01 to 0.1 ml of 0.48 M PB. Thi~ ~ixture i~
in~bated at 76 C and aliquot~ are removed, dilutet to ~ .
: ' ~ ~ .

~ ~ ~8 ~7 0.14 M PB and assayed for hybridization at various time~ after the initiation of the reaction. Hybridi-zation assay are perfromed using hydroxyapatite as described earlier. The results obtained are compared to the hybridization kinetics ~: of the probe reacted with standard RNAs containing known amounts of bacterial R-RNAo The~e standards are mixtures of mammalian cell RNA and known amc~unts of a specific bacterial R-RNA.
Table 8 present~ data on the quantitation of Legionella pn umophila present in water samples and in an infeceed ham~ter liver sample. Th~ water samples and the liver sample-Y were titered for the presence of L. pneum by standard quant~tative growth assays at the Cen~er for Di~eaqe Control in Atlanta, Georgia.

: M~d by ~sured by E~CPSS R-RN~
a Grow~h Assay Nucleic A~id HYbridization L. hila bacteria 107 bacteria 1 - 2 x 108 bacter~a per ~ ected gra~ liYer ~ lr~er . ~ hamSt:OE live~
:: n o L. ~ bæterl~ 1.5 x 10 bacter~a 2;1 x 10U bacteria ~ ~er o water ~le --~ ml i'~ .
Excc~ Prob~ M-Cbod The amoun~ o~'bacterial R-R~A pre.~ent ~n a sample can al50 be measured by doing hybridization under con-, :; . :'~ ..
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~ ,78 ~87 -~3-dition~ where there $~ an exce~ of the Le~io~ella speciic 3H-cDNA probe, relative to the amount of Le~ionella R-RNA prese~t. Thi~ ~ixture iq hybridized to completion. At this point each Le~io~ella R-RNA
molecule pre~ent in the sample i~ saturated with probe molecules, By compar~ng the amount of probe hybridized to the R-RNA to an appropriately con tructed standard calibration curve, the amount of R-RNA in a samp~e can be deter~nined. A goot e~ti&ate of the total num.ber 10 of ~ Pneumoph-ila bacteria present: in the sample can ~hen be calculated by knowing the a~verage number of R-RNA molecules per L. pn~u~nophlla bacterium.
Table 9 present~ data on ehe quantitation of L.
pneum~hila presen~ in water sample~ as determined by 15 the exces3 prob~ accelerated hybridization rate - enzyme-detergent-sample method described in deta~l in a la~er section. The water samples were ti~er~d for ehe presence of L pneumophila by ~tantard quaneitati~e growth assays.
The~e assayq take day~ to complete while the hybridization as~ay take~ about 1 hour.

'~' ~ Mea~ured byMea~ured by the Exces ~' ' L ~ 1.5 x 108 bactcri1 2.2 x 10~ bæter~a 25 bacterial per ~ 3r-~
water sample .
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' :~' 7~38 obe S~ecific On1Y for R-RNA from M~ers of t~ ~ Le~ionella A. wlwi1 cf ~a~er a~: Accelerated ~ybridization Rate ~thod 1. Preparatian o 5a~1e and ~bridizati~ Inc~bation Mixture:
Mix in the following order æ quiclcly a~ pos3ible.
S a) 9 mlcroliters of sa~le b) 2 ;rrolit~rs of enz~-detergent solution coT~tainin~:
5 milligrams/~nl Prote~se K, 0.5 M Tris ~pH ~ 8.1), 8% sodium dodec~l sulfate (SDS), 4% sodiu~ sarcosinate, 0 . 25 M Na~l, 0 ~ 016 M EI~A, 0 . 016 EOEA
c) 1 ~ierolit~ of probe d~ssalv~d in water d) 20 micxolit~ of 4.8 1!~ PB

~: 2. ~a~e the mi~e at 76 for an ~ppropria~e ti~ so that *le hybrldizatior reaction i~ c~lete.

3. The h~b~idiza~ assay is perfonE~d as follows:
a) A~d the ina~bation mixn~re to one ~1 o~ a r~
tes~ature solution containing: 0 . 05 gram~
h~apa ~te (HA), O.054 M PB, O.02Z Zwitte~-gOElt 14 (~i~chem) (hereinafter referred to a~ Z-14) b~ Sh~ae ~ mlci~e ~or 30 sect~nds at ro~ te¢~3erature, add 5 ~1 0.14 M PB, 0.022 Z-14, ~nd inn~ate ~ Dix~re at 72 C ~ar 2 m~sute~.
G) Centrifilg~ the soluticJn to pellet *~ ~. All centri~i~ æe d~ne at r~ te~era~re.
~S and ~e the liquid fracti~ wash tl.

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_ ~ 5 ~ 7 d) Add 6 ml of 0.14 M PB, 0.02Z Z-14 ~olution to the pellet. Re.~uspend th~ ~ pellet by vortexlng it.
Centrifuge to pellet the HA and decant S and Rave the liquid fraction. Thi~ is wa~h ~ 2.
e) Repeat step d. Thia re~ults in wash ~3.
f) Add 6 ml 0 . 03 ant resuspend the HA pe~llet by vortexing. Centrifuge the suspes~sion io to pelle~ the HA and decan~ the liquid and a~say :i.t for the pre~ence of the probe.
Thl~ fract~ on ontain3 the hybrldized probe, if any is pre sent .

~: It i~ not necessary to elute the hybrid~zet probe fron~
. 15 the HA under certa~n conditions. Thus, if the probe ismarked with a marker which can be detectèd in the presence of HA, the pellet from 4tep e can be assayed tirec~ly for ~he probe. In the case of a marlcer suoh a-~ Iodine-125 ~ ~ ~ the tube containing the HA pellet can be placed direc~ly : :~ 20 in~o a gam~a detec~ion machine.
:. ~
Other modifica ionQ can al~o be made to make the :~; ; te~c fa~t~r and ea~ier. Thu~, the volume and asnount of ~ u~et caR be ~calet down, arld the nu~b~r of waqhe~
can also be ~ r~duced, tepending on ~he ~ituation. In ~: 30: ~ oeher in3tance~ ~ t ~ay be de~irable ~co increase the Yolu~e ~of ~ :or slu~beI of wa~he~ variety of salts other: ~han ~odiu~ pho~pha~e, and other detergentQ can alJo be u~ed~ he assay.

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~1 ~7~ 37 B. Arlalysi~ of a Liquid Sa~: Standar~ion Rate ~thod 1. Preparatiorl of Sa~le ~nd ~ridization Irn~batior~ mix.
Mix in the ~ollowing order and as qu$ckly as possible.
a~ 14 mlcroliters of sa~le b3 2 microliters o~ e~-detergent solution described in A.
c) 1 microliter of probe d) 3 microliterq of 3.2 M PB, 0.03 M EDIA, O.û3 M EOE~

2. In~ubate the n~ure ae 76 C for an ~ropriate tise so that ~ybridization will ccuple~e.

3. l~ br~dizati~n assa~ i3 perfor~d as follows:
a) A~d ~he in~atian m~ to 1 ~il of a solutia~ cc~ntaining 0.14 M PB, 0.02Z Z-14, Q.Q5 ~am~ of HA.
b) Fra~ point on the protocol is identical to that : ~ 15 desc~ ed in A.

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~; A 10 percene liver hc~glate in water is used as ~e tissue sa~le.
1. Prep~atian o~ Sa:~}e and ~nQibation Mix.
~y a~ possible ~n *~e follawing order.
~ a~ 8 microl~ of sa~le ~10~ 7er ~genate) b) 3 ~ll~lt~s o~ enzy~-detergent ~c cocltaining:
8%~ i.~odiun sa:rco4inate9 0.15 M~NaCl, .016 M E~rAg 0.016 M EOEA, 0.38 M Tri8 (pH a 8.~) ~ 195 millig~fisd of Prana~e.
25 ~ : c) l ~oli~ of probe spe~fic only f~ ~ R-R~.
d) 20 D~s:roli~rs of 4.8 M PB.
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~ 78~7 2. Incubate the mixture t 76 for an appropriaee time to ensure that the hybridization reaction is com~lete.

: 3. The hybridization assay is performed as described in the section on analysls of a water`sample;
: Accelerated Rate Method.

D. ~h~: Standard ~oridi2ation ~ate ~thod ~ ..
A lO percent liver ha~ge~ate in water used as the s ~ le.
1. Preparation o ehe ~le ~nd ~ ation M~x.
Mix as qui ~ y ~s po~sible in ~ foll ~ order.
~; a) 12 m~ollter~ of s ~ le.
b) 4 ~dsroliter~ of ~ detergent solution descr ~ d : in B~
; : c) 1 md~roli ~ of probe ~ec~fic only for : R-~M~.
.d) 3 ~ liter~ of 3.2 M PB, 0.03 M EDrA, Q.03 M EGEA.

: :15 2. ~ ate ~ ~ixe or an ~ ropriate t ~ at 76~ C.

3. The hyJrs~zat~on a~say is perf~æd a~ foll ~ .
a) adt ~ ~ ation m~x to 1 ~1 of a ~olutian c~nCi~ng 0.14 M PB, 0.02~ Z-14, 0.05 gr ~ HA.
: b) ~ron~ 3 point ~ as~ay ~ ~t~cal to ~ t ~cr ~ d in A.
A ~ e de~led ~escription of n~cleis acid ~ ion te~t Oo :: detect ~Ek~ E~ bact~ n water and live~ 5 ~ les pre~ d below.

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~1 ~78987 Examp le 4 Rap~ ~iti~re Detect~on of Le~e~la B~teria in Water Sanrle: Aceelerated }~te ~ethod 1~ Ihe ~ollow~ng componenss were mixed as quiekly as pos s ibl e and in the fo llowing crder ~
a) 4 . 5 microliterq of a water sample con~aining about 105 ~ ~ baec.erial per ml . The number o bac ceria in t~e water wa determ~ned at the Center of Disease Control in Atlanta by a grow h tes~.
b) 1 microliter of the enzyme-detergerlt solutio~
de~ribed in A.
c~ O . 5 ~icroliters of ~ spec~ :fic probe .
The amount of probe equalled ~ 5 x 10 6 micrograsn3.
d) 10 microliters of 4 . 8 M P~ .
~: Asse~bling the hybridization mixture about 2 minutes.
' 2. Incubat~ the hyrbidization m~x'cure for 36 minutes at 76 C .
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3. Perfo2~ the hy~idization a3say a~ de~cribed in A.
This t~ok about 5 ~inu~ce-4.
4. As~ay ~ch2 fractiolls for ~he p~e~ence of the probe.
Tlli9 took ~ou~ 10 minute~. ~
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The n~ber o~ l;egionella bac~er~a presen in the hybridizae~on mixture wa~ abou~c 500. Thi~ number of ,.
organisms was ~det~ct~d and quan~itated in a~out one ..

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~1 ~78~38'7 hours, from ~eart to finiqh, with the use of less than 10 5 microgram~ of probe. Twenty-three percent of the -probe hy~rid~d with ~e Le~ione~a R-RNA in thi~ test which wa.q de3igned to be an exces~ probe hybridization test. Control te~e~ were done under the ~ame conditions, one with no bacteria added, and one with about 105 E.
coli bacteria present in the hybridization mix. In both cases only 1-2 percent of the probe behaved~as if it were hybridized.
The above te~t can be modified to acsay larger volumes for the presence of Le~ionella bacteria. Thus, one ~1 of a water sample containing 104 e~ionella bacteria per ml wa~ centrifuged for 3Q minute~ ~o pellet the bacteria. A small amount of enzy~e-detergent was added to thepellet and a hybridization test was performed on this mixture u ing the accelerated rate method and the LeRionella bacteria were readily detected. Much larger volumes of .qa~ple can be c~ntrifugcd and other methods of concentrat~ng the bacteria, includ~ng membrane filtra-tion, can al~o be u~et. These modification make itpossible ~o detect a s~all number of bacteria in a lar~e : sa~le volume. Air Qample can also b~ concen~rated by ~: methods, including ~embrane filtration methods, ant small number9 of bacteria can be detected in large volumes of air sample.

Example 5 =le }. The follawlng cc~ts ~ere mixed a~ quiclcly a~ possible ;~ ~n the fo~l~ ord~.

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~ -~.~ 7~987 a) 4 microliters of a 10 percent liver homogenate of a hamster liver infected with Le~ionella pneumcphila. This i9 equivalent to 400 micrograms of liver or about 6 x 104 liver cells. About 750 Le~ionelLa E~
were present in this sample.
b) 4 microlieers of an enzyme-detergent solueion : composed of: 45 milligrams/ml Proteinase K, 8~ SDS, 4% sodium sarcosinate, 0.5 M Tris (pH = 8.2), 0.008 M EDTA, 0.~08 M EGTA, 0.25 ~ Nacl.
c) 4 microliters of Le~ionella specific probe.
The quan~ity of probe was about 10 micro- -grams.
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: ~ 15 2. Incubate ~he hybridization mixture at 76 C for 3 hours.

3. Perform the hybritization assay as described in A.
,. ' :~ 4. Assay ehe re~ult~ng fractions or the presenca of ~ probe hy~ridized ~o ~E~ R-RNA.
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: 2~0 The nu~b~ o~ ~ bacteria present in the hybrldization m~xture was about 7~0 and the amount of : : R-RNA presene ~n this:n~ber o~ Le~__nelLa cells is about 1.1 x 10 5 microgram~. The number of liver ceLls : present wa~about 6 x 104 and ehe amount of liver R-RNA
:pre5ent:wa3 about one microgram. Ten percent of the sp~cific probe hybridi~ed. Control tests were ,, ~

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done wi~h unifected liver in the same manner and less than one percent of t~e probe behaved as if hybridized.
Examples 4 and 5 illustrat_ only two of the ~any possible conf~guration~ for such a te~t. Test3 utilizing different volumes, salt~, detergents, probes, sample types, prote-olyti~ enzyme~, amounts of ~A, incubation per~ods, organism types, amounts of probe, temperatures of incubation, ant hybridization rate accelerating sy~tems can be succes fully utilized within the general ~o~tex~
of the test3 described here. Any of the R-RNA probes can be used in a sy3te~ comparable to tho~e dcscribed above. Non R-RNA probes can also be u~ed to good effect in the~e sy3tem~ with so~e obvious ~odification~.
: For example, a teqt -~pecific for a particular DNA ~equence ; 15 in a specific organism or group of organi m~ can be done exactly as described above if a step i.~ included which converts the double strand DNA ~o the singel strand for~.
In other case~ different modification~ of the method mu.~t be used. Bacteria such as Mycobacteria and Bacilli are difficult to break open. A step which breaks open - these bacteria mu~t then bc used in con3unction with the method deccribed abov_. A single incubation, in : the absence of detergents, with ~h~ enzy~e ly-~ozyme, ~:: will ~ake mo t ~aclllus bacteria su~cept~ble to lysis -~ 25 by detergent~, or example. On the other hand, Myco-~ baceeris ar~ very difficult to lys~ and may haYe to be ; . phy~Icall broken open before they can be tosted for.
;~ A modification of th~ above method ~an also be ~ : uset in con~unctl~n^with any ~ran3fer ~NA probe or a :~ 30 probe specific for any other RNA present in an organism.
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~78~7 ~92-A qtep designed to concentrace small numbers of bacteria or other cells out of large volumes of sample~ ~uch as alr or liquid can al~o be used in con~unction with the hybridization te~t to detect mo~t other bacterial organi~ or other types of organisms .
While I ha~e described above, in detail, the produc~ion and ~se of a nucleie acid probe which~
hybridizes only to nucleic acids fro~ me~ber~ of.
the Genu3 Legionella, it wlll be readily apparent to thoqe skilled ~n the art fro~ that exa~ple and the others, that o~her probes can be produced, based on ~he procedures illustrated above. Thu~ th~ method uset to produce quch other probes would be a~ follow~:
1. Produce market nucleic ac~d complementary to the R~RNA of a member of the gro~ of interest.
2. Hybridize thi~ DNA to the R-RNA from a member of ~h~ group of orga~is~s evolutionarily most closely related to the group of organisms for which the probe i pecific. Select the fraction : of the mar~et nucleci acid which, at a specific criterion doe3 no~ hybridize to R-RNA fro~ a : me~ber of ~hi~ closest related group of organ~
i~3. Th~3 fractio~ is speclfic for the organ-is~ group o~ interest.
Example~ ~f the~e are:
a. The produc~ion of a market probe which hybridizes : only w~t~ R-RNA fro~ a member of the bacterial : : Genu~ and toes not hybridize with ~ 30 R-RNA other ource~.
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, : ~;, ' ~ 7 8 ~87 b. The production of a marked probe which hybridizes only with R-~NA from a member of the bacteri~l Genu~ M~coplasma and does not hybridize with R-RNA from other sources.
c. The production of a marked probe which hybridizes only with R-RNA from a member of the bacterial Family Enterbacteriaceae and doe~ not hybridize wi~h R-RNA from other source~.
d. The produceion of a marked probe which.hybridizes only with R-RNA from a member of the anaerobic group of bacteria and does not hybridize wi~h R-RNA from other sourres.
. The production of a marked probe which hybridizes only with R-~NA from a me~ber of ~he group Fungi and doe~ not hybritize with R-RNA from other ~ources.
f. The productlon of a marked probe which hybridizes only wi~h R-RNA from any member of th~ Chlamydia : group and does not hybridize with R-RNA from other 30urce~.
g. The productlon of a marked probe which hybridizes only with R-RNA from any member of the bact~rial ; famlly MYcobacteriaceae and doe~ not hybridize : with:R-RNA fro~ other sources.
25 h. The production of a marked probe which hybridizes R-RNA fro~ any living organ~sm.
The production of marked probe which hybr~ dizes ~: only w~th R-RNA from any mammal and toes no~
hybridiz~ w~th R-RN~ from other sources.

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7~39~3'7 Illustrative l~mbo~t of the Use of Pro~es for t-RNA to Detect, ~rntitate and Identi~ 2nis~

t-RNA probes may be used in the same mann~r as the R-~NA. probes to detect, identify and quantitate organisms S ant in some case~ viruses. For example, a t-RNA probe specific for Le~ionella can be produced and used in a manner similar to that descrlbed for the R-RNA probe specific only for e~ionella. The ilLustrative embodi-ment described for the Le~ionella specific R-RNA probe thus also er~res a~ an illu tration of a t-RNA specif ic probe.
The genes present in ;~any DNA and RNA v~ru~es include t-RNA gene~ which are ~pecif~c for the virus.

Illustrative ~t of t}~e Use of Probe~ Speci:Eic far ~A, knRNA, lS snR~ or~sR~ to: Deeect! Qu~titate ~d Id~ntif~ isms, Cells ~nd VL~
ProbeQ specific fos mRNA, hnRNA, snRNA or psRNA may be usat in a manner alalogou~ to those for R-RNA arld t-RNA to detec~ dentify and quantita~e a specific ` ~ 20 large or small group of organisms, cell~ or vlruses in ce3.ls. S~nc~ the evolutionary con~er~Jation of the individual gen~ which produce tho~e various RNAs varies grea~ly it is pos~ible to produce probes wh~ch will detect ~; m~mb2r~ of very large classe~ of organ~ s~ and probes which 25 d~eect member3 of relati~ely s~all classes of organism~, cells or viru~eq in cel}Q.
One example of highly conser~ed gene . equences are the hiseone gene~, a fa~nily of genes pre~ent in eukaryotic . .

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~.~ 78 cells. Histones are nuclear structural proteins which are present ln all eukaryotes. The histone DNA sequences show greae similarity even in widely diverged organisms.
Thus, the histone genes of sea urchin and man are similar enough. to hybridize together. A probe which is specific for a particular histone mRNA or for a population of histone mRNA~ can detect, identify, the presence or absence of any member of a large group of widely diverse organisms. The sensitivity of detection of cells or 10 organisms is enhanced by the abundance of the histone mRNAs. In ord~r to grow, a cell or organism must synthesize histone mRNA in large amounts.
Another example invovles certain gene sequences which code for psRNA and are not conserved during evolution.
Such gene sequences fro~ one type of organism do not hybridize to DNA from distantly related species. A
; probe specific for one partlcular psRNA sequence or a population of different ps~NA sequences from one organ-is~ type or viru~ type, can be used t~o detect, quanti~ate, and identify member~ of a small group of closely related organis~s or a small group of closely related viruses ~: which are i~ cells.
: Another example i~ the developme~t of a prob~ specific - for a sequence or sequence-~ o mRNA, hnRNA, snRNA or psRNA
which can be u~ed to examine body fluids for evidence of specific cell damage and des~ructi~n. In certain dlseases cell~ are destr~yed and their content~ including cell nucleic acid~ ar2 ~pilled in~o the circulating blood.
Liver damage due~to hepatitis is one such situation and :- ~ 30 i~ is known that both DNA and ~NA from liver eP.lls enters ,' ::~

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789~37 the circulating blood as a result of c211 damage. A
probe can be produced which is specific fo~ an RNA
sequence or sequences which are characteristic only of liver cells and are not present in other nor~al cell types. The exis~ence of such RNAs is well known. This ~robe can then be used to detect, idencify, and quan~itate liver speci~ic mR~A, hnRNA, snRNA or psRNA sequences in blood samples by nucleic acid hybridization m~thodology as described herein. The presence of liver damage and 10 its extent can be determined since the amount of RNA
present in the blood will reflect the extent of cell damage.
P~obes specific for a particular ~RNA, hnRNA, snRNA
or ~sRNA sequence or sequences present only in a specific 15 - cype of liver cell can also be produced and used to de~ect and quaneitate the presence in the blood of the RNA
sequences resulting from the damage of a specific liver ;~ cell type. Obviously the morc abundant the specific RNA
sequence in a liver cell the higher the sensitivity of detection of ehe RNA.
Damage to any body tissue or organ (including heart, kidney, brain, muscle, pancreas, spleen, etc.) may be detected and quantitated in this manner. Other body fluids including spi~al fluid and urine can also be assayed for the presence of these specific RNAs.
A useful initial screening test for tissue damage from any source can be done by utilizing a probe speciric for R-RNA and examining blood or other body fluids for the presence of R-RNA or t-RNA sequences. Quantitation of R-RNA or t-RNA present will provide an indication as to the extent of tissue damage without identifying the ~- source.

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~;~78~87 Another sample of the use of the nucleic acid hybridization tests and approaches described herein is the detection and quan~itation o E. coLi cells containing the plasmid genes which code for the E.
c_ entertoxin pro~ei~. Such a test involves the use of a marked nucleic acid probe complemen~ary to the enterotoxin protein mRNA in order ~o detect and quantita~e the presence of E. coli bac~eria containing the enterotoxin protein mRNA. This can be accomplished by utilizing the in solution hybridization methods described herein.
As discussed herein bcfore the use o a probe complementary to E. coli enterotoxin mRNA as a means to detect and quantitate the presence of E. coli bacterial which are producing E coli eneerotoxin and therefore contai~ enterotoxin mRNA, by using nucleic acid hybridization methods, has signifioant advantages over methods such a3 de~cribed in the Falkow et al.
patene discussed earlier.
; 20 The sa~e approach as described above can be utilized ; to detect the specific gene product of a particular microorganisal which confers resistance to a particular antibiotic or other anti~icrobial agent. Th2 genes which confer re~i tance to mv~t antibiotic-~ are almost al~7ays pre~ene on pla~mids in the cell. In order for an organism to produoe the faetor which confers resistance, the gene for the fac~or and the mRNA for ~he factor must be present in the cell. Thus a probe sp~cific for the facto~ mRNA can be used to det~ct, identify, and quantitate ~; ~ 30 the organism~ whi~h are producing the factor by utilizing nucleic acid hybridiza~ion methods.

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~7~9~7 -9~3-The above examples of the use of nucleic acid probes specific for ~articular ~equences or populations of sequences of ~RNA, hnRNA, snRNA or psRNA for the purpose of detecting, identifying, and q~antitating 5 pa2ticular group~ of organisms, cells, or viruses in cells containing such mRNA, hnRNA, snRNA or psRMA
sequences, by nueleic a~id hybridization methods, are illustrative only, and not limiting.

The Determination of the Sensi_ivity of Microor~anisms to Antimicroor~anism A~ents A large number of differen~ clinical situations require the determination of antimicroblal agent suscep-tib~lity for a variety of differen~ bacter~ a and anti-~: 15 biotics ( see "Antibiotics in Labora~ory Med~ eine" by V. Lorian, Editor, Publi~er, Williams; and Wil~ens Bal~imore, 1980) All of these situation~ utilize a method for detecting and quantitating specific classes 2û of microorganism~. In many of these situations useof ~he nucleic acid hybridization tests described ~; ~ earlier would greatly speed up the de~ermination of antimi~robial agene susceptibility.
As the organ~ sm in a sample grow and divide, the 25 : a~ount of RNA in ehe culture increases . A doubling of organlsm~ r~qult~ in a two fold increase ln the qu~nti~y of R~A~ of differen~c type~ which is pre ent in the cul~re.
~; Thu~ organism growth c n be monitored by determining the : quantity of RNA present in the cul~ure at different times 30 after the -qta~c of growth incuba~ion. An increase in the amoun~c of RNA presen~ w~th time ind~cates organism .

7~387 99_ growth. The magnitude of the increase indicates the extent of growth. The rate of growth i~ then the extent of growth per time period. Probes specific for, R-RNA, t-RNA, psR-RNA, pst-RNA, certain mRNAs or psm~As, certain snRNAs or pssnRNAs~ or hnRNAs or pshnRNAs can be used indivldually or in combination to measure the ~rowth of organisms since the quantity of each of these RNAs in a culture will increase as the organism~ grow.
A culture of specific category of organisms grown in the presence of an agent or agents which completely inh~bit growth will not shown an increa~e in RNA with time, whil~ cultures which are partially inhibited by such age~t will show a lower ra~e of RNA accu~ulation.
A cu~ture whi~h i~ no~ inhibieed will show ~he sæ~e rate `~ 15 of RNA increase as ~he control culture which does noe ~:~ contain the agent.
~ One example of thi~ is in de~ermining the suscep~i-: bility of Mycobacteria tuberculocipresen~ in a clinical ;~ sputum sample. The first step in diagnosing such a sample is to prepare a direc~ s~ear of the spu~um for staining ~n order to detec~ acid-f2-~t bac~lli.
~:~ It i.~ esti~atet that it r~quire~ at least 104 - 105 M. tuberculosls organism~ per ml of sputum to yield a positive direct smear. However, onIy 10 to 100 of the~e organisms are recoverabl~ by growth culture methods.
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9~7 If ~he sputum specimen shows a positive smear, the specimen is then treated to kill all bacterial except Mycobacteria, and a dilution of the treated specimen i~ plated on aBar medium containing anti-microbial agent and on control agar which does not contain the agent. Viable individual bacteria will from colonies on the control agar while growth will be inhibited on ehe agar with the appropriate an~i-microbial agent. The ration of the number~ on the control agar to those on the agent treated agar is then a maeasure of the e~fectiven-s~ of the antimicrobial agent.
A small colony will contain at lea-qt Io6 bacteria.
This meanR that at least 20 div~sion are needed to form a colony from one bacteria and each division will take at least lX hour~, for a total of 240 hour-~ or 10 day , at a minimum. In ~os~ cases ~t ta~e~ 2 - 4 times thi~ long (3 to 6 week~) for colonie eo appear.
A methot describ d earlier for ~ , would greatly decrease the ~im~ needed for deter~ining an~i-microbial agent su~ceptibiliey. A probe ~peeific only for R-RNA fro~ ~ember~ of the genus M~cobacterium could - be used in such a te~t. Such a probe woult allow quanti-tation and a detection sensitivity equal to that described ~5 earlier for L~ionella. A nucleic acid hybridization : test u~ing ~he aecelera~ed hybritization rate conditions and the ex~e s probe mode of hybridization would easily allow ~he detec~ion of abou~ 200 Mycobac~eria cells. A
: step woult be atded ~o en~ure the di~ruption of ehe ~` 30 Mycob:aceeria celIs so tha~ ~he R~RNA would be free to hybridize. ~ycobac~eria do not readily lyse in the :

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As mentioned above, a minim~m po~itive sputum specimen ( as determined by acid staining) contains about 10 to 10 ~y~ cells per ml and these 10 to 102 cells can ~e detected as colony forming units.
For drug susceptibility studies on agar, enough ~yco-bacteria are added to the control and experimental agar surfaces to ensure that 40 to 50 colonies will appear : on the control agar where no antimicrobial age~ is present. If such a practice is followed when usi-lg a nucleic acid hybridization assay thi~ means that the culture is star~ed with about 50 MYcobacteria and it will the~ take about 3 - 4 cell d~visions or about 2 - 3 tay~ in order to obtain a detectable level of cells.
If any ~ignificant inhibition of growth by t~e agent ha occurred the control will be posiei~e and the culture : ~ containing agent will be negative. It is clear ~hat the use of ~he highly sensitive nucleic acid hybridization ~ ~ me~hod can greatly reduee the tim~ needed to determine ; ~ 2a susceptibility by 5 to 10 fold.
The abo~e is ~ust one example of the use~ of nucleie ac~d hybridizat~on te~ts such a~ those deseribed for E~ for de~erming antimicrobial agent sensitivities.
The sensltivity of any microorganlsm can be determined by utilizing a co~bination o~ ~he s~a~dard growth method-: :ology an~ an assay for microorganim~ based on nuclei~
. acid hybritization. In addition, in many case~ the spe~ificlty and sen~tivity of the nucleic acit hybridi-~: : zation test~o~ ~ croorganisms allow the determination ~ ~ 30 of antibiotic sen~iel~ity of specific organis~ even in .'.:,:~ ~ :

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~.~78~3~7 the pre~ence of a large excess of other microorganisms or eukaryotic cells.
It i obvlouY th~t the ~ame approach can be used to determine the presence o~ antimieroarganism activity in blood, urine, other body fluids and ~ssues and other samples. In this case my nucle~ c acid hybridizat~ on procedure can be used to monitor and quantitate the effect of the bLood, ur~n~ 9 or other sample on the grow~h of a specific group of microorganismS which are put ipeo contact with said blood, uring or other samples under conditions where growth oc~ur~ if anti~icrobial activity is not present.

The overall rate of pro~ein synthe~i in a c211 : 15 ic determined by the number of ribo~ome~ per cell. The rate of t-RNA synthe~is is aLso related to the ~umber of ribosomes per cell. Inhibition of protein synthesis : in a cell results ~n the cessation of R-RNA synthesis by : the cells. Indeed, stopplng cell growth by any means results ~n th~ ce~at~on of R-RNA synthe is and slowing cell growth re~ult~ in a slowing down of R-RNA synthesis.
~; The newly s~n~hesized R-RNA molecule is larger ~chan the suss of ~che mature R-RNA ~ubunits prese~t in the ribosome.
25 For example the R-RMA of E. coli is -~yntheslzed as a pre^
curqor molecule 6000 baseY long. The precus~or molecule then proces~ed to yield the R-RNA ~ubuslits (totaling about 4500 b~ses3 w~ieh are then incorporated into .~ r~bo o~e and "extra" or pr~cursor speci fic R-RNA

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R-RNA i~ not ~ynt~esized in non-growing cell~ and therefore no precursor specific R-RNA sequences are present in ~hese cell~. In ~his case, large numbers of R-RNA molecule3 are present ln the cell but no pq R-RNA sequence~ are pre.~ent.
In a ~lowly growing cell a s~all amount of R-R~A
precur~or i synthesized ant a small amount of p~R-RNA
is present.
In a rapidly growing oell a large amount of R-RNA
precur~or i~ -~ynthesized and several thousand psR-RNA
sequenees are presen~.
The ab ence of psR-RNA in a cell signal that the cell is not growing. The ratio of R-RNA to psR-RNA in a eell is an indication of the growth rate of a ~ell.
Antimicrob~l agents inhibit cell growth. Cells which are not growth inhib~ted by the agene will contain large amou~t~ of psR-RNA. In cells whioh are only :: 20 partlally growt~ inhibited the psR-RN~ will be present ~;~ in a loer a~ount. The ratio of R-RNA to psR-RNA will give a ~ea~ure of the degree of inh~b~tion.
A nucleic acid probe specific for the psR-RNA
~5 sequences o~ a par~icular group of microorganisms can be used in a nucleic acid hybridization eest to determlne and quantitate the pr~sen~e or absenca of : psR-RNA in tho3e microorganis~ when the organisms are growrn in the Rre ence and absence of a particular 3~ anti~icroorganis~'i~ent or a group of such agents. This ca~ be done even in the presence of large number3 of '~

~ 7~3~7 -10~
or~anisms which are not related ~o the microorgani m group of intere~t.
It is obviou3 that this nucelci acid hybridization method can also be used to determine the presence of substance~ with antimicroorganism aceivi~y in blood, urine, other body ~luids and tissue~, and other samples.
This method of determining growth of cell.~ can be used tO determine the ~tate of growth of any cell which synthesizes ~-RMA. The abo~e exa~ple ~ only one of many us~d for such a method. A method based on u~ing a ~: probe specific for the p~t-RNA sequence~ of a particular group of organisms or cellq can al~o be used to deter~ine - the state o~ growth o~ those organ~sm~ or cells.
A method baset on u~ilizng probe specific for cer~cain mRNAs, psmRNAs, hnRNA~, pshnRNA, snRNAs, or pssnRNAs, which are abundanc in rapidlv growing organisms or cells but absent, or pre~ent in low amount, in non-growing or slow-growing cells can also be used to determine the statP of growth o~ the~e organis~s or cells. For example, the mRNA for a pEotein, ~NA
polyerase, i pre~ent in abundance, several hundred copies per cell, in rapidly growing cell~. In non growing ~ cells cery little ~NA iq ~ynthe~ zed and little ~RNA
: i~ pres~nt.
: 25 A method baset o~ u~ilizlng probe speciflc ~or er~ain ~iru~ m~NA~ or psmRNAs which are abundant when ~aid viru3 i~ rapidly gowing in a cell and ab~en~ when the viru~ i pr~3ent in a eell bu not growing, eab also - :~ be ~et ~o deter~ina~he ~tate of growth of viru~es in cell :
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~ ~ 7 ~ 9 Thu~ in qituation9 where member~ of a particular category of organi~m~ are known to be present in a sample it iq po~sible to use a ~lngle probe to determine the growth state of said organis~s. For example if no 5 p~ R-RNA can be detectet in the organisms, ~hey are in a non-growing state. If psR-RNA is detected in ~he organisms but in low amount relative to the number of organisms preRent, the organisxnc ar@ growinE~ 910uly.
If large a~nounts of psRNA are detected, relati~re tO
10 th~ number of organisms present, the organismx are grow~ng rapidly.
Another approach to determinlng the s~a~e of growth of a particular ~rganis~n or class of orgar~isms relies in utilizing two probeq, each of which ~ill hybridiæe only to RNA from a particular category ~f organis~s one probe is ~peciflc for a ~table RNA (R~RNA or t-RNA) which ~NA
~: is present in said organi~ms in roughly ehe same amount Ln non-growing organisms or cells and rap~dly growlng : organism~ or cells; the other probe is specific for a 20 particular mRNA, psmRNA, pst-RNA, pssnRNA, hnRNA, p~hnRNA or psR-RNA ceqllence or sequences which RN~ is p~eseslt in abundance in rap~dly growing cells or organis~ns, absent or present ln low a~ount in non-growing organism~ or cells. Thes0 probe~ are ueilized to detect, 25 iden~i:Ey, and quan ~ta~:e the amount~ present in ~he sample of the ~ each i ~pecific for . The rat~ o of the amounts : of the3e R~A~ i~ an indication of the growth s~ate of the organi3m~ or cell3.
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~ .~78987 A specific example of this ~nvolvec the u~e oftwo probes, one speclfic for the R-RNA of members of & spec~fic category of organisms or cell~, and the other specific for the p~R-RNA of the ame category of organisms or cells, in order to detect, identify, and quantitate the R-RNA and p~R-RNA present in a s~mple. The ratio of the amount o p~RNA to R-RNA present in the sample is an indicator of the state of grow~h of the organism or cells. In rapidly gorwing cQll~ there are s~veral thousand copies of psR-RNA and the psR-RNA/R~RNA ratio is at a maximum. In slowly growing cell~ a relatively small amount of psR-RNA i9 presenc and the psR-RNA/
R-RNA ratio $s m~ h lower. In non-grow~ng c~lls psR-RNA
shouid be absent and the p~R-RNA/R-RNA ratio is at a 15 minirrrum.
This same two probe ~ethod can bs u~ed with a variety of different combina~ions o the probes mentioned abov2 and can be done in the presence of orgarlis~s or cell~
which are not members of the said specific category 20 de~ec~cet by the probe.
An obviou~ application of the methods described here to deter~ine th~ state of grow~ of specific categorîes : of organism~ is the u~e of these m~thod~ to: deeer~i~e : the presence of antimicrobial agen~Y in blood, urine, 25 other body fluld~ or tissues or other ~amples; determine the sen~itivity of specific categorY es of organisms to specific sntimicrobial agent~ or groupq of ~uch agents.
For example bacteria which are completely growth inhibited : ~ by a particular a~n~ will have a minimu~ psR^RNA/R-RNA
~ ~ 30 raclo, '~

~, -107- ~ ~ 7~

Detectin~, Identifyin~, and Quantitatin~ Viruses It is often important to be able to quickly determine whether a particular virus or group of viruses is present in a sample. This can be done by utilizing nucleic acid hybridization tests described herein.
The rapid nucleic acid hybridization test which combines: a) the me~hod for rapidly making nucleic : acid available for in solution hybridization; b) the ~ethod for greatly accelerating ~he rate of nuc~eic acid hybridization; c) and the rapid method for aqs~ying for the presence of hybridized probe; is dirsctly appli-caSle to the detertion, identification and quanti~a~ion of any group of DNA or RNA viruse~ present in a sample by ~he use of a nucleic acid probe which is c~mplementary to the virus group of interest.
In addition, ~uch a viru3 assay mcthod could be ,u~ed to determine t~e effectiveneQs of particular anti-viral agent and to determine the presence of antiviral activity in blood, urine and other s~mples.
, .
Meehod for Detecting Microorganism Infections by Exa~inin~ o~ Or~anism's Pha~cy~ic Cells ~ The extremely high sensitivi~y an~ specificity of : detect~on characterizing the nucleic acid hybridization ~ tes~t.~ speci~lc for R-RNA which ha~e been described above, .~ 25 permits of a simple solution to the problem of obtaining . .
an appropriate clinical specimen for microorganism : : : diagnoci.~. A simple blood test sample which eontain~
the white blood ~ell (hereinafter referred to as WBC) fraction will suffice in a large number of cases.

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1~ 7~ 9~7 .-108-One manner of using this WBC approach i~ to first hybridize the '~BC sample with a marked probe which will hybridize to R-~NA from any member of the group of all bacterial but doe~ not hybridize tQ R-RNA fro~ any other source. Such a probe ser~e~ a~ a general screening device for any ba-teria. Sample~ which are positlve for bacterial R-RNA are then aqsa~ed with a hierarchy of other probes in order to further identify ~he bacteria which s present. For example, a probe whi~h h~bridizes to R-RNA from any member of the Famlle Enterbacter but not to R-RNA from any other source can be used to detect or rule out. Eneerbac~cr bacteria while a probe specific only for anaerobic R-RNA woult be used to detect anaerobes.
The above illu~tration is just one of may possible way~ of using the WBC~ a~ the prim3ry cl$nical sample for the quick diagnosis of microorganis~ infections by nucleic acid hybridization. For example, d~pending on the clinical symptom~ of t~e patelnt, different co~bination~ of probes would be used in order to ob~ain a diagnosi~.

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~ 789~7 ~ he publicat~ons listed below are of interest in connection with ~arious aspects of the invention and are incorporated herein as part of the disclosure.

1. Repeated Sequences in DNA
R. J. Britten and D.E. Kohne, Science (196~) 161 p 529 2. Kinetics of Renaturation of DNA
J. G. We~mur and N. Davidson, J. Mol. Biol.
(1968) 31 p. 349 3. Hydroxyapatite Techni ~ s for Nuclelc A~ld Rea~K~iati~n D.E. Kohne and R.J. Britten, in Procedures in Nucleic Acid Research (1971), eds Cantoni and Davies, Harper and Row Yol 2, p 500 4. Hybrid$zaeion of Denatured RNA and S~all Fragme~ts Transferred eo Nitrocellulo~e : P.s. Thoma~, Proe. Natl. Acad. Sci. USA ~1980) 77 p 5201 5. DNA-DNA IHybridization on Nitrocellulose Filter~:
General Corl~ideration~ and Non-Ideal Xinetics R. Flav~ll et al., Wur. J. Bioche~. (1974) 6. Assay of DNA~A Hyrbits by S Nuclease Digestion and Ad~orption to DEAE-Cellul~;e Filtcrs I. Maxwell et al., Nucleic Ac~ds Research (1978) 5 p 2033 7. Molecular Cloni.ng: A ~aboratory Manual ~: T. Mania~i~ et al., Colt Spring Harbor Publication ~1982) : ~ 8. Efficien~ Tran~cr~ptio~ of RNA ln o DNA by Avian Sarooma virus Poly~erase J. Taylor et al.~ B~ochemica et Biophy-~. Acta (1976) 442 p 32~: :
~ 9. U~e of Specif~c Radioactive Probes to Stu~y Trans-`~ ~ cription and ~epLication of the I~fluenze Virus Genome J. Taylor~e~ al., J. Virology (1977) 2} t2. p 530 :::: `:

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~1 ~7~39~7 10. Virus Detection by Nucleic Acid Hybridization:
Examinat~on of Normal and ALs Tissue for the Presence of Poliovirus D Kohne ee al., Journal of Gene~al Virolo~y (1981) 11. Leukemogensis by Bovine Leu~emia Virus R. Kettm~nn et al., Proc. Natl. Acad. Sci. USA
(1982) 79 ~8~ 5-2469

12. Prenatal Diagnoqi of a Thalassemia: Clinical Application of Molecular Hybridization Y. Kan et al., New England Journal of Mediclne (1976~ l p 1165-1167 .13. Gene Deletions in a Thalassemia Pro~e that the 5 ~ ~OCU3 i~ Funtional L. Pressl~y et al. 9 Proc. Natl. Acad, Sci. USA
(1980) 77 ~6 p 3586-3589 14. U3e of Synthetic Oligonucleotide as Hybridiza~ion P~obes. S.V. Suggs et al., Proc. Natl. Acad. Sci.
USA (1981) 78 p 6613 -:~ 15. .Identifica~ion of Enterotoxigenie E. coli by Colony Hybridization Using 3 Enterotoxin ~~ne Probes S.~. Mosely el atl., J. of Infect. Diseases (1982) : 145 ~6 p 863 : 16. DNA Reas~ociation _n thé Taxonomy o' Enteric Bacteria. ~ D. Brenner, Int. J. Systema~ic Bacteriology (1973) 23 ~4 p 29~-307 ~ 1?. Compara~ive ~tudy of Ribosomal RNA Cis~rons in I Enterobacteria and Myxoba ter~a ~: R. Moore et al., J. Bacteriology (1967) 9~ p 106~ 7~
~ 18. R~bosc~al RNA Si~ilarieie~ in the Classification :: ~ of Rhodococcu~ and Related Taxa . M. ~ordarsk~ et al., J. General Microb~ology tl980) : 118 p. 313-31~r ".
19. Re~ention o'~on Nucleotide Sequeno~s in the ~: Ribo~omal R~A DNA of Eukaryote~ and Some of their Physleal Characteristic~
J. Sinclair et al., Biochemistry (1971) 10 p 2761 . ~

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~ ~ 7~387 20. Homologies A~ong Ribosomal RNA and Messenger RNA ~ene3 in Chloroplasts, Mitochondria and E. coli ~ert et al;, Molecular and General Genetic~
(1980 179 p ~ 545 21. Heterogeneity of the Conserved Ribosomal RNA
Sequenceq of Bacillus subtili~
R Doe e al., J. BacterioTogy (1966) 22. Isolation and Characteriza~ion of Bacteria~
Ribosomal RNA Cistrons D. Kohne, 3iophy~ical Journal (1968) 8 ~1~ p 1104-1118 23. Taxonomic Relations Between Archaebacteria Including 6 Novel Genera Examined by CroRs Hybridization of DNAs and 16S R-RNAs J. Tu et al., J. Mol. Evol. (1982) 18 p 109 24. R-RNA Cistron Hbmologie Among ~YDoh~robiu~ 2nd varicuS O~r Bacter~, R. M~ore; C~an J. ~ g77) ~3 p 478 r ~ ~"25. Conservation of Transfer RNA and 5S RNA Cistrons in Enterobacteriaceae D.J. Brenner e~ al., J. Bactcrlology Yol 129 ~3 (Mar 1977) p I~35 26. Seqeunce Homology of Mitochondrial Leucul-tNA
C~tron in Different Organi~
S, Jako~cic ~t al., Biochemistry Vol. 14 tlO
(May 20, 19753, p. 2037 ~: 27. Synthe~ic Deoxyoligonucleotide~ a Çeneral Probeq : for Chloropla-ct t-RNA Gene.
J.A. Nic~oloff and R.B. Hallick, Nucleci Acids ~ Research, Vol. lQ ~24 (1982) p 8l9l-82lo :~ 28. Antibiotic~ in Laboratory Medicine ~: V. Lorian ed, Willia~s and Wilken~ (Baltimore/London) : 1980 29. Diagnostic Microbiology Finegold and Martin, Edi~or~, C.V. Mosby Co.
(St. Louis) 1982 :: ~

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~ ~789~7 30. spotbloe: A Hybridization Assay or Specific DNA
Sequence~ ln Multiple Sample~
M. Cunningham, Analytical Bio~hemistry Vol. 128 (1983) p. 415 31. (29) Analy~is of Repeating DNA Sequences by Reaqsoc~ation R. Britten et al., in: Method~ in Enzumology XXIX, page 3~, Eds. Grossman and Moldave, Academic Pre~s, New Yor~ (19743 32. Studiec on Nucleic Acid Reassociation Kine~lcs Retarded Rate of Hybridiation of RNA with Excess DNA
G. Galau et al., P-oc. Natl. Acad. Sci. USA
Vol. 74 ~ T4) p 2306 33. Acceleration of DNA Renaturation Ra~e~
3. Wet~ur, Biopolymer~ VoL. 14 (1975) p 2517 34. Room Temperature Methot for Increasing ehe Rate of DNA Reassociation by Many Thou.~andfold: The Phenol Em~lsion Reassociation Teckniqu~
D. Kohne et al., Biochemistry Vol. 16 ~24 (1977) 35. Molecular Biology D. Freifelder, Scienc~ Book International (Boston) ~ Van Nostrand Reinhold Co. (New York) 1983 : 36. Gene Expre~3ion 2 . ~ B. Lewin, J. Wiley ~ Sons, Wiley-In~erscience Publlcatlon (1980) New York :: ~
37. Gene Expres~i~n 1 :~ ~ B. Lewin, J. Wiley ~ Son~, Wiley-Interscience Publicat~on ~1974) New York .
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~ ~ 7 8 ~ ~ 7 As used in the specification and claims the following terms are definited as follows:
DEFINITION OF TER~lS

base (see n~cleo~ide) base pair mis~atches (see i~Derfectly ccmDlementary base sequence) base seqeunee, (nucleotide A DNA or RNA lecule ccnsisting 10 sequence or ge~e sequence of mLl~iple bases.
or ~olynucleotide sequence or single strand nuclei~ asid sequence) .
15 complementary base pa~rs Certain of the base~ have a chemical affinity for each other and pa;r toge~her, or are comDle~gneary to : one another. The coFplementary : ~ase pairs are A:T nd G:C in O DNA and A:U in RNA.

` complemEntary strand~ or Perfectly co~plYmen ~ nucleic `- ccmplementary ba~e acid lecules are nucIeic acid sequences moIecule ~ in which each:base in one m~lecule is paired:with its c ~ lementary base in ehe other scrand, to form a stable helical dbuble strand mDlecule. The indiYidhal strands are termed : complementary strands.:

30: criterion Mbs~ preeiesely defined as ehe :~ difference between the te~perature of nelting of the double strand : nu~leic æ id and the temperature at which hybridi~ation is done.
35~ The melting temperature of a . .. ~ . .

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' ~.~7~98 DEFINITION OF T~FUIS (Cont'd) crieericn, conei~ued. double strant n~cleic acid is determnr.ed primarily by the sal~
concentra~icn of the solution.
The criterion deter¢ines the deg~ee of co~plementæ ity ~eeded for two single stranls to form a seable double strand molecules. The criteri~n can ~e described as highly stringent, or no~ very stringent. A highly stringent criterion requlres that ~wo interacting com~lem~n~ary sequences be highly complementary in sequence lS in order to fon~ a stable double strand molecule. A poorly strLngent criterion is one which all~w~ rela-tively disslmil3r compl:mentary strands to interacr znd form a double strand molecul~. Hign stringency allow~ the p~esence of only a small ~raction of b~se pair ` -~ mQsmstches m a doubIe str2~d ~ole-cule. A poorly stringent criterion ` ~ 25 allows a much Iarger ~raction of base pair mismatches in the hybridi-zation pr~duet.
e bont betw~ the paired bases in denæ~ed or d~ssoclated a do~ble g~and rlucleic acid molecule 30 ~ ~lelc æld : ~ : can be broken, resultinz ~n ~o single s~r~d m~lecul~s, ~which then dif~use ~way fran each oth.er d~le 8traSlt; ~n~cle~c acid As it is fa~t in eh.e cell, ~ost DNA
is in the d~le st~d staee. The :~
35 ~ :DNA is :made up o two:~DNA ~leculesor ~ strands ~und~ h~lically OEo~d eac~ other. : The: bases fa~e i~ard ::
:i and each bas~ is~specifically bondet eo a canç~lementary base in~
4:0: ~ the ot~ir strand. For exa~le, a A in or~!e s~rant is always paired h :a T~ ~ ~he other strar.d, ~ile ., . .:
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39~317 DEFINITION OF TE ~ S (Cont'd) do~ble strand nucleic a G in one s~Iand is paired with a acid, con~nued. C in the other strand. In a bacterial cell th~ double strand molecule is abcu~ 5 x 106 base pairs long. Each of the bases in one strand of this ~olecule is paired with its base ccmDle~.ent in the other strand. The base se~uences of the individual double st~and molecules are termed comple~entary strands.

hybri~;~ation ~see nucleic and hybridization) ; i~perfectly cc~plementary Stable double strand ~Dlecules can 15 base sequences (base pair ~e form.ed between rwo strands where mismatches) a fraction of the bases in the one strand are paired with a nan-complement æ y base in the other strand.

20 marke~ probe or ~arked Single strand nucleic aoid ~Dlecules s ~ ce which are used to deeect the presence of o~her nucleic acids by the process of nucleic acid hybridization. The probe molecules are ~ark~d so that they can be specifically detected.
This is done by incorporating a ; specific marker ~olecule into ~he nNcleic acid or by attaching a ;~ specifie marker to ~he nucleic acid. The st effec~ive probes ~ ~ are ~arked, single s~r~nd sequences,-~ which cannot self hybridize but canhybrid~ze only if the nucleic acid to be~detected is present. A large ; ~ 35 number of differEnt ~ar~ers are available. These include radio-active and fluorescent ~olecules.

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~7~987 DEFINITION OF TERMS (Cont'd) nucleic acid hybridizatiqn The bond ~etween the cwo strands cr hybridization (reassociation, of a double strar.d molecule c~n or renarurati~n) be br~ken and the tw~ single . strands c~n ~e comDletely sep~ated frcm each ot~er. ~nder ehe proper cond ticns the corDle-meneary single st~nts c3n collide, ~ecogni7 each other and refo~3 the double str~nd helical m~lecule.
This process of formaticn of double strand molecules ~r~m comple~entary single strand molecules is called nucleic acid hybridization.
Nucleie acid hybridizaticn also occurs benween partially c~mple-~entary single s,rands of R~ and D~A.

nucleotide, nucleotide M~st DNA consists o~ sequences of base or base only four nitrogeneous bases:
adenine (A), thynrL~e (T), guanir~e (G~, 2nd cytosir~ (C). Together these bases fo~ the genetic alphabet, and lGng ordered sequences of ~he~ coneain, in coded fon~, m~h of t~e i~far~aticn present in genes.
Mbs~ RNA also c~nsists of sequences : of only four bases. However, in R~A,~ ~: 30 ~ th~e is replaoed by ulidine (U).

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~ 7 8 9~7 DEFINIIION OF TEgMS (Ccnt'd) reassociaticn (see ~cleic acid hybridizaticn) rena~uration (see nu~leic acld hybr~di7ation) ribos al R~A or R-RNA The R~A which is presenc Ln ribosomes.
Virtually all ~ibosomes c taLn 3 s ~ le strand RNA suDunits: one large, one medium-sized, and one small.

riboscme A cellular par~icle (containing RNA
: 10 and protein) necessary for protein synthesis. All life forms except viruses contain riboscmes.

R-RNA DNA or The base sequence ~n the DNA which codes for ribosomal RNA. Each R-~NA
R-RNA ge~e subunit is co~ed for by a separate .
R-gM~ probe A m æ ~et nucleic acid sequence which ls cc~pleIent~ry to R-R~A ~nd there-: fore will hybridi2e with R-RNA to form a stable double strand molecule.

mgNA Each individual niU~A is a direct gene : product containing ~he information necess~ry to specify a p rticular prote~n. The ma~hinery o the cell tr2nslate~ ~he sequence of the ~RNA
. into a specific pro~ein. Many tifferent ~RNAs exi~t ~n each cell.
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~7~g87 DEFI~ITION OF IERMS (Cont'd) hnRNA A c ~ lex class of ~NA sequences presen~ in the nucleu~ of eukaryotie cells which includes precursor ~RNA
~olecule~. Most h~ R sequ~nc~s never lea~e the nucleus. The func~ion af mos~ of chese ~Dleclles in ~awn.

snRNA A c13ss of relaeively stable s~all nuclear RNA lecules which æ e present pr ~ ily in the nucleus of ~: eukaryotic cells in large n ~ rs.

precursor ~ M~DIY RNA mD~eculec in both prokarvotes : ~nd eukaryote~ are synehesi7ed as part :~ I5 of a large gNA m~lecules which i5 ~hen processed to ~ eld ~ature RNA ~Dlecules - of v æ icus types 2nd o~her ~ ller sequences which are apparently tiscarded.
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: ~ 20 pre~ursor specific ~NA T~ RNA sequences present in ~re~rsor ` ~ ~p ~NA). ~NA, t-~NA, ~-RNA, snRNA, an~ h¢~YA
w~ich are t ?resent in the mat~;e : R~ RNAI n~, snRNAI and ~NA
: moleo~les.

; 25 eher=~l stabili~y of The thermal seability or melting : double strant nucleic population of d~uble strand mDlecules acid mDlecules ~ha~ been converted to the single scr~hd for~.

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119 ~ 3~ 7 DEF~NIrION OF T~MS (Cont'd) restric~ion ~ eq Compcnents of the restricticn-~odificatio~ cellul æ defense system agains~ foreign nucleic acids. These enzymes cue unmodified (e.g., methylated) dcuble-stranded DNA at specific sequences ~hich exhibit ~ofold sym~etry about a poine.

tr~nsfer RNA ~t-RNA~ During protein synthesis individual zminD acids are aligned ~n the proper order by v æ ious specific ad~ptor nolecules or e-KN~ ~ole ~ es. Each amino acid i osdered by a different t-RNA specie~.

le the invention has been described ~nd illustrated in detail, it will be apparent to those sXille~ in the art that various ch~nges, equivalents and alternat~ve~ may be resorted to without dep æ tir~ fro~ the spIrit of the i~vention, and all of sush 2~ ~ es, eou~valents 2nd alternative~ are cont~mplated as ~ay come within t~e scope of th~ appended claims ~nd equivalents ~reof.

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Claims (4)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A method for determining the presence of organisms which contain RNA in a sample which might contain such organisms, which comprises the steps of:
a) bringing together the nucleic acids in the sample with a probe comprising marked nucleic acid molecules which are complementary to a RNA subsequence which is conserved in all organisms;
b) incubating the mixture under hybridization condi-tions for a specified time; and c) assaying said incubated mixture for hybridization of said probe.

2. A method for determining the presence of any member of a specific category of organisms which contain RNA, in a sample which might contain such organisms, which comprises:
a) bringing together the nucleic acids of the sample with a probe comprising marked nucleic acid molecules which are complementary only to the RNA of members of said specific category of organisms;
b) incubating the mixture under hybridization conditions for a specific time; and c) assaying the incubating mixture for hybridization of said probe.

3. A method for determining the presence of any member of a specific category of non-viral organisms which contain rRNA, in a sample which might contain such organisms, which comprises:
a) bringing together the nucleic acids of the sample with a probe comprising marked nucleic acid molecules which are complementary only to the rRNA of members of said specific category of organisms;
b) incubating the mixture under hybridization conditions for a specific time; and c) assaying the incubating mixture for hybridization of said probe.

4. A method for determining the presence of any member of a specific category of organisms which contain nucleic acids, in a sample which might contain such organisms, which comprises:
a) bringing together the nucleic acids of the sample with a probe comprising marked nucleic acid molecules which are complementary only to the nucleic acids of members of said specific category of organisms;
b) incubating the mixture under hybridization conditions for a specific time; and c) assaying the incubating mixture for hybridization of said probe.