CA2157416A1 - Method for enhancing detection ability of nucleic acid assays employing nucleic acid sequence based amplification - Google Patents

Abstract

This invention relates to a new and novel method for significantly enhancing the sensitivity of assays aimed at detecting the presence of target deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sequences in a sample. Specifically, this method applies to techniques that employ nucleic acid sequence based amplications (NASBA?) or other nucleic amplification techniques to amplify copies of the target RNA or DNA (Sooknanan et al., Bio"Techniques, 17: 1077-1085, 1994) to allow for detection. The method of the present invention combines the following three steps for the first time: RNA amplification using NASBA; RNA:DNA hybrid formation; and immunochemical detection of heteroduplex nucleic acid sequences. This method extends the standard NASBA techniques to accommodate amplification of copies of the NASBA product, which, following binding to DNA sequences facilitates immunochemical detection of the RNA:DNA hybrids thereby formed. This combination of steps results in a new assay with increased simplicity, speed and sensitivity, including a greatly improved signal-to-noise ratio, over standard nucleic acid detection methods. This improved method will be extremely useful for performing analyses in the forensic, clinical, agricultural and biological fields.

Description

21~74~6 .

METHOD FOR ENEIANCING DETECTION ABILITY OF NUCLEIC ACID ASSAYS
EMPLOYl~G NUCLEIC ACID SEQUENCE BASED AMPLIFICATION
BACKGROUND OF TEIE INVENTION
The present invention relates to improved methods of detecting specific nucleic acid sequences. Examples of nucleic acid sequences are d~-y~ ..u,,l~.~, acid (DNA) and ribonucleic acid (RNA) sequences. The molecular subunits of both DNA and RNA are called 10 nucleotides which are linked together to form long poly ' ' chains. Each nucleotide subunit is made of a sugar moiety, a phosphate moiety and a bæe moiety. It is the sequential ordering of the bæe moieties [adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U)]
that contains DNA or RNA's genetic illrUl The ordering of these base moieties in a p~ly ' ' chain and the tendency of the bæes to attract and bond with other specific bæe 15 moieties, is exploited by this invention to locate, detect and isolate specific DNA or RNA
sequences.
DNA l-ormally contains two poly,.u.,l~,u~ , stMnds twisted about one another lengthwise in a helical manner resembling a ladder where the sides are made of identical sugar 2 0 (deu--y. il,~se) and phosphate molecules while the rungs are made up of bæes extending out from each stMnd, held together by weak attractive forces. In DNA, the base thymine on one stMnd always pairs with the bæe adenine on the opposing strand, and the base guanine always pairs with the bæe cytosine. This is called Culll~ lLaly bæe pairing.
RNA is also a p~ly ': '- strand. However, the sugar moiety is ribose (versus d~u~yl i~ose in DNA) and the bæes are adenine, guanine, cytosine and UMCil. In RNA, the bæe uracil on one strand can pair with the bæe adenine on the opposing stMnd, and the bæe guanine can pair with the bæe cytosine. Although RNA can pair with either a cc-~lr' y stMnd of RNA or DNA, it is normally single stMnded so does not form a helical structure.

2I~7~1 6 .

Techniques for detecting and/or isolating particular nucleic acid sequences of interest have increase~ in popularity during recent years especially in terms of application for detecting the presence of the DNA or RNA within pathogens such as viruses, bacteria, or other UU16~111;,~111~ and therefore the presence of these pathogens themselves. These techniques can 5 also be used for other purposes such as to screen bacteria for antibiotic resistance, to aid in the diagnosis of genetic disorders (for example in sickle cell anaemia and th~ ), and to detect cancerous cells. Several a~J~J' have been developed for the ~ ,lùbiu106i~,~1 analysis of clinical, food, Cl.viUI I, and forensic samples. A general review of a technique and its present and future v is provided in Bi~ nlngy (August 1983), pp. 471-478 0 which is i.,~ul,uul~L~d herein by reference.
One situation in which it is desired to detect the presence of target nucleic acids involves the detection of pathogens by means of diagnostic tesE which claims a large share of the health care market and the agri-food industry. The definitive ' ~ of microbial pathogens in i ~ ., ' (e.g., foods) and clinical specimens requires the observation of the infectious agents or their -~ ~lr , soch as specific antigens or nucleic acid (DNA or RNA) sequences. The traditional culture methods for the detection of pathogens are slow, expensive and labour-intensive.
2 o To overcome these di ,~lv ~ , simple and rapid methods exploiting new Ir~
have been developed for detecting the presence of pathogens of medical, c..~/iu~ ' and a6li ' ' importance which appear in sample matrices (e.g., foods) at very low Several ' O ' tests are now available which exploit the specificity of the antibody-antigen reaction, namely, ~' tests, nuul~...,e tests and ~., (e.g., Id.li~ ~, enzyme ~,.).
~ " y employs a radioactive isotope as the label. Because of the ;II~UA.~. , hazard and difficulty of handling radioactive materials, assay systems have been devised using labels other than ,~ ,.- as the label comronPnt, including enzymes, 3 o 1,~~ , metals and organo-metallic comple~es, co-enzymes, enzyme substrates, enzyme ~1~7416 activators and inhibitors, rh~mil ' ' reactants, fluorescent molecules, and others.
Enzyme ~O use enzyme-labelled ~Cllio (antibodies or antigens) for detection of antigens or antibodies captured on a solid phase. In this technique, enzymes (e.g., 5 peroxidase, alkaline 1' . ' ) are covalently linked to a detector ~g~ (e.g., an antigen-specific antibody) by using a cross-linking agent, such as, ' ' ' ' yJ~ or a procedure such as periodate oxidation. Adsorption onto an easily l~iCu..,l~le solid phase provides for a simple and rapid means of ~;li~iu.. of VU,~ I for the subsequent capture of antigens or antibodies from a test sample. Since antibodies and antigens contain l~yd~ ic 10 regions in their structures, they bind readily to l~yJIu~Jll~ic surfaces. Most commonly used enzyme y~ depend on the adsorption of ~Ab~ onto either a flat solid phase, a ~UIUUo surface, or a Il~ ,lUlJVIUUo surface such as polyester cloth. Solid phases, e.g., microtiter plates, tubes or beads, and plastics, e.g., pulyoIyl~ " polyvinyl chloride, nylon and poly,l,~ "y' have been commonly used.
Another approach commonly used for pathogen detection involves the use of a nucleic acid probe (DNA or RNA) labelled with a detector moiety (e.g., ~ , chemical label such as ~'iv v or biotin, enzymes, fluorescent markers, etc.), which can be hybridized with IIUE,~.. specific nucleic acid sequences ' ~ 1 on a solid phase (e.g., membrane or microtiter plate). The hybridized probe is then measured by detecting the presence of the detector moiety.
Yet another more recently developed approach for detecting pathogens involves the use of nucleic acid sequence based ~ lirl~ iull (NASBA), described in United States patent 5,409,818 for the a"",l ~ of unique nucleic acid sequences of bacteria and other t~rget cells. The advantages and limitations of this technique have been reviewed by van Gemen (J.
Virol. Metho~s, 43: 117-188, 1993, i,.~u,~ ' herein by reference). This powerfultechnique, which uses can achieve ~..,.,.~,..Ju . amplification of very low numbers of target nucleic acids to levels which can be easily visualized by vl~llU~lllUl~ analysis or by 30 llybli.li~dtiOIl with a DNA probe.

~ 2~7~16 The . ' of specific RNA sequences generated by the NASBA technique has been widely applied to the rapid and sensitive detection of bacterial pathogens. Although NASBA has great potential as a very sensitive and specific technique, it has not always been possible to ællieve this level of sensitivity in its application to the detection of pathogens directly 5 or in U.ll ' cultures of food and e.~ ' samples.
The sensitivity of the NASBA could be enhanced by detection of the product using DNA
probes tdrgeting the amplified sequences (such as the ' ' 'o,,y described in Holstrom 11..
1993, Anal. Biochem., 209:278-283). However, such methods usually involve labour-intensive 0 DNA-DNA l~ylJIiJ;~Liu procedures and require the use of probes labelled with detectable chemical or radioactive moieties, which can be costly to prepare and difficult to ~ Ldi~.
r, Lh~,,l.lUI~, DNA probes targeting the NASBA product sometimes only produce a moderate . U~ in the sensitivity of the test, which may still require more than ten to several hundred cells per reaction in order to give a detectable signal.
One way of enhancing signal amplification entails tdrgeting RNA. An example of target irl~liu., entails assaying for ribosomal RNA (rRNA) of a UUlo~lll; ..l rather than ' ul.lù ~VIlldl DNA. Since rRNA is present in any given cell at 104 times higher ~
than DNA, the number of possible target sequences increases, thereby increasing the probability 2 o of detecting the target organism. However, this approach suffers from the limitation of the type of RNA sequences being expressed and therefore not completely suitable.
Yet another i iL"iol~-based; .,lirl~Liu.l system utilising ~B-replicase can produce a 2-5 million-fold ~ rl~,iull of a given target after 4 cycles (Li~ardi et al., B- ' ' Oy 6, 1197-1202, 1988). However, this technique suffers from such problems as excessive noise, false positives, requires ~ ' ' ' technical expertise, and relatively expensive il..,.
and reaOents (Walcott et al. J. Food Prot. 54:387-401, 1991).
Accor~ingly, those concerned with the dl~v~ '(I and application of NASBA-based 3o diagnostic tests have recognized the need for simple, non-hazardous L~ for ~ v ~1~7416 .~

the sensitivity of NASBA tests. r. ih.,.lllvl~;, there is a great need for ~ methods for detecting pathogens, rather than the purely qualitative approaches currently being practised, since it is often necessary to estimate the degree of microbial hazard associated with a sample (e.g., foods) on the basis of the level of Therefore, there is a great need for simple and ill~.A~ methods to augment the sensitivity and reliability of NASBA tests, ~ ,ulally for the forensic sciences, the food industry, clinical d~licdLio.~ and other users who must routinely process large numbers of samples containing very few target cells. It is therefore an object of the present invention to 0 provide a method for the . ' of NASBA for specific pathogens without the need for ill~Vl~Ul~llil." hazardous " ~ "~ or expensive (and potentially umreliable) chemical labels in the nucleotide transcripts to permit their subsequent detection.
A furtller object of this invention is to provide a very simple and user-friendly detection 15procedure for the products of the NASBA.
Yet another object of this invention is to provide for the 4u~ liv~ assay of pathogens in a sample by using the combined target nucleotide; ' method and detection procedure described above.

~ , In accordance with the present invention and as used herein, the following terms are defined with the following meanings, unless explicitly stated otherwise.
The term "amplicon" refers to a fragment of DNA or RNA spanned within a pair of annealing primers; this fragment is amplified 1AIJV '' ~Iy by the enzyme DNA pol~-ll.,l~s~.
The term "i .: ' . ' " refers to a procedure for s ~ ~ 'y enhancing the number of copies of RNA sequences that are ~v~ y to DNA amplicon sequences ~1~741~
by ~ of those amplicon sequences into many copies of RNA by using an dl)lJlU~JI' ' RNA polymerase.
The term "lldiL~ i' I reaction mix" refers to RNase-free deionized distilled water containing 80 mM Tris-HCI (pH 7.5), 12 mM MgCIl, 4 mM ~. " , 20 mM NaCI, 20 mM
(1, 1 mM of each rNTP (Promega, No. P1221), 10 units of T7 RNA pOIylK~
(Promega, No. P2075) and 20 units of RNasin (Promega, Nû. N2511) per 25 ,ul).
The term "NASBA " refers to a process for amplifying a specific nucleic acid sequence, involving; ~y ' ~ single-stMnded RNA, single stranded DNA and double-stranded DNA;using the single-stranded RNA as a first template for a first primer, using the single-stranded DNA as a second template for a second primer, and using the double-stranded DNA
as a third template for synthesis of a pluMlity of copies of the first template; a sequence of the frst primer or the second primer is cu."~' y to the sequence of the target nucleic acid and a sequence of the first primer or the second primer is h~m~r~--c to a sequence of the target nucleic acid. NASBA is used to increase the quantity of the target nucleic acid sequence to allow detection or to increase the purity of such a target nucleic acid sequence.
The term "target nucleic acid sequence" refers to any DNA or RNA sequence that is desired to be detected.
SUMMARY OF TE~E INVENTION
This method applies to techniques that employ nucleic acid sequence based ~
(NASBA) to amplify copies of the target RNA or DNA to allow for detection. This method combines the following three steps for the first time: nucleic acid , ' ' using NASBA
of a target nucleic acid sequence; RNA:DNA hybrid formation; and ' ' detection of i,~,~,ludu~ ,A nucleic acid sequences. This metbod extends the standard NASBA techniques 2~S7~1 6 to r~ ---- ' ' the .~ r~ O~. of copies of the NASBA product, which, following binding to immobilized probe DNA sequences facilitates ' ' detection of the RNA:DNA
hybrids thereby formed.
FIGURES
Figure I is an Agarose gel Clc~,ll, . ' ~i~ of the NASBA reactions.
Figure 2 is graphic l~l~ of various COI~ " of NASBA products detected 0 by anti-HNA-antibody.
DETAILED DESCRIPTION OF THE INV~ON
The method described in this invention applies to techniques that employ nucleic acid 5 sequence based A~ lir~ . - (NASBA) to amplify copies of target RNA or DNA sequences to allow for their detection. This invention constitutes an exterlsion beyond the prior art rendering nucleic acid sequence detection more feasible and reliable while ~ improving thesignal-to-noise ratio of these techniques. This improvement is especially notable in situations where a target nucleic acid sequence is present as a minute component of a mixture of nucleic 2 o æid sequences or is present at low levels.
The method of the present invention combines the following three steps for the first time:
target nucleic acid ~ lirl~liol~ using NASBA; RNA:DNA hybrid formation; and - I detection of l~ `UdU~ A nucleic acid sequences. This method extends the 25 standard NASBA techniques to r ' ' the I .,lir~ ,.. of copies of NASBA RNA
product, which, following binding to DNA sequences facilitates ' ' detection of the RNA:DNA hybrids thereby formed.
In the first step of this invention, a nucleic æid sequence based r ~ l r ~' (NASBA) 30 process is used for amplifying a specific nucleic acid sequence, which involves: ~

2L ~7~ ~ 6 . ~

single-stranded RNA, single stranded DNA and double-stranded DNA;using the single-stranded RNA as a first template for a first primer, using the single-stranded DNA as a second template for a second primer, and using the double-stranded DNA as a third template for synthesis of a plurality of copies of the first template; a sequence of the first primer or the second primer is 5 : , l y to the sequence of the target nucleic acid and a sequence of the first primer or the second primer is l~n ~ ,JII!~ to a sequence of the target nucleic acid.
In the second step of this invention, RNA:DNA hybrid formation, advantage is taken of the fact that RNAis produced directly from NASBA, allowing the target nucleic acid sequences 0 to once again be selected out of b~h~,-uu--d nucleic acid sequences by ll,~/blil]i~illg the RNA
sequences to , ' y DNA probes attached to a solid support, to generate ' ili~d RNA:DNA hybrids. This allows for washing away of extraneous material.
The h~uduL~h,~ nature of the nucleic acids sequences allows for the use of antibûdies 15 that specifically recognize RNA:DNA hybrids and can be used to detect the hybrids in one of many ' ' methods of analysis. This antibody can possess a chemical group allowing for direct detection of the hybrids, or it can also be detected indirectly using another antibody to which a chemical group is attached, that recognizes the anti-RNA:DNA antibody.

t~; I r~r,~ - ,.1 Inll In one particular ~,...1,, ' ~, the sample to be tested is typically a piece of food, for example meat or cheese, or another source containing single or double stranded nucleic acids.
25 This includes ~ ,luu~ and/or other cellular material associated with these samples. The test sample is first treated to release the nucleic acids from the cells, followed by a step to denature the r~ucleic acids. This is typically accomplished by Iysing the cells in a Iysis buffer solution and the ~' of nucleic acids is preferably , ' ' ' by heating the resulting solution in boiling water or alkali treatment (e.g., 0.1 N sodium hydroxide). The denaturing step can often be used ' '~, with the Iysis method. The release of nucleic acids can, 21~ ~41 ~
.

also, be obtained through mechanical disruption such as free~ing/thawing, abrasion, sonication, physical/chemical disruption (eg. ~olyu~yc;al~lc.le ether detergents like Triton~, pulyu~.yal~ ulbiL~LIl detergents like Tween~, sodium dodecylsulfate, alkali treatment, osmotic shock, heat, or Iysing using enzymes such as proteinase K, Iysozyme, pepsin). The 5 resulting medium will contain nucleic acids in single stranded form which is then assayed according to present llyblidi~LIiu.. methods (Wang et al., Appl. Environ. Microbiol., 1992).
When the sample contains free single-stranded RNA sequences, the sample is in proper form for NASBA. If the sample contains a tuget sequence that is a free single-stranded DNA
10 sequence, then Lldl,.,~ iul. must first occur to generate the desired RNA sequence. When the assay is performed for detection of a Illi~,lU~ ,. , a bacterium for example, the cells must be Iysed and the nucleic acids have to be exposed in order to be available for llybl iJi~dtiu.. with the priming ~ . Methods of Iysis have been previously described and are well known to one skilled in the art.
RNA Amplifirotin~l ~ N~ R~
Target RNA is amplified by NASBA procedure (or the d~JlJlUlJI- ' RNA sequence must be generated from the target DNA). A standaud NASBA reaction mixture contains T7 RNA
20 pulyll.~.~se,RNaseH,AMVreverseLIdll,~ L~ nucleosideLlr~ ~' ,twoseparatespecific primers and dl~JIU~Jl buffer -ml One of the primers (Primer 1) is prepared with the T7 promotor sequence appended to its 5' end.. The reaction Lr ' has been previously described ( B. van Gemen et al, J. Vuol. Methods, 43: 177-188, 1993; European Patent No:
0329822).
In this process, single-stranded RNA is converted to single-stranded DNA which in turn is converted to a functional template for the synthesis of a plurality of copies of the original single-stranded RNA. A First primer and a second primer are used in the ~mrlifiroti~-~ process.
A sequence of the fust primer or the second primer is ,urrl~;~.lLly . y to a sequence 30 of the specific nucleic acid sequence and a sequence of the first or the second primer is 7 ~ 1 ~
sufficiently l~ ."~ to a sequence of the specific nucleic acid sequence. In some instances, both the first primer and second primer are rr ' '~ ' y and s~fr~
e.J--- to a sequence of the specific nucleic acid sequence, for example, if the specific nucleic acid sequence is double stranded DNA.

The RNA is converted to single-stranded DNA by l.yb- idi~ g an r~ ul ~rl~ primer(the first primer) to the RNA (the first template) and ~yllLh~i~illg a ~ . ' y strand of DNA from the first primer, (the first DNA sequence) by using a RNA-directed DNA
pol~ l~e. The resulting single-stranded DNA (the second template) is separated from the first template by, for example, hydrolysis of the first template and by using a l;l.. ". l;~.~ which is specific for 12NA-DNA hybrids (for e~ample, .i~ . H). The second template is converted to a form which is capable of RNA synthesis by l.~. idi~illg a syntbetic r~ ,J"", l~Ol ir~,S (the second primer), which contains at its 3' end a sequence which is rr- ~ Iy IL~Y to the 3' end of the second template and toward its 5' end a sequence containing 15 the antisense strand of a promoter and antisense sequence of a lldlr~ UliOI~ initiation site, and by Sy..a.~ i,.g a second DNA sequence covalently attached to the 3' end of the second primer using the second template as a template and ~ ih~i~illg a third DNA sequence covalently attached to the 3' end of the second template using the second primer as a templated, using DNA-directed DNA p~lylllci~. The resulting functional derivative of the second template, 20 which is a third template, is used for the synthesis of a plurality of copies of RNA, the first template, by using a RNA p~l~lll.,l~e which is specific for the promoter and LlL~ iUII
initiation site defined by the second primer. Each newly ~yllal~i~d first template can be converted to further copies of the second template and the third template by repeating the cycle.
In addition, repetition of the cycle does not require ~ ioll or . . I by the user.
RNA:DNA Hybri~l Forr~irn Once the NASBA method has generated an ~lUL" number of copies of the target nucleic acid sequences of interest, the NASBA products will be digested using an ~JIU~II' ' restriction; ' ' (eg. Hind lll) to cleave off the primer- l' y sequences, 215~41 6 leaving target-~ , ' y nucleic acid sequences for use as probes. These probes will be ~urr..,;~ ly long (typically greater than 16 nucleotides) and ~urr~ , ' y to thetarget nucleic acid sequences to allow for efficient binding of transcribed amplicons to generate T~NA:DNA hybrids.

The probes thereby created can be attached to solid supports such as microtiter wells, "dip sticks~, or III.I~,IUI,JvlU.I. polyester cloth. Modes of nucleic acid sequence attachment to various forms of solid supports are well known in the art. The primary objective of the mode of attachment chosen is to allow for secure attachment of the nucleic acid to the support while 10 allowing for future efficient binding of cu.ll~' y sequences.
The probe may be denatured by heating and then diluted in ice-cold coating buffer. The solid support Tnay be incubated with this coating buffer, air dried, and sequence cross-linked to the solid support, for example by exposure to ultraviolet light for an ~lu~,l amount to time 15 to allow for efficient cross-linking. The solid support may be washed with a~ lUlJI buffer and m,.~ ir~,, attachment sites blocked by incubation with l.ybli " solution, including protein blocking reagent, following by washing, air-drying, and storage under ~
conditions until use t see P. Tijssen, "Practice and Theory of Enzyme r y~ in R.H.
Burton and A.H. van ~nirpPnhPrg, eds., 15 Laboratory Techniques in Biul,}~ lly and Molecular Biology, (New York, Elsevier Publication 1985) at 549 illWI,U~ ' herein by reference].
T - ~ I ~PfPrfi~m of rT. . .~ rx Nucleic Ari~l SPqllpnrpe The RNA NASBA products are hybridized with a .' y DNA probe immobilized on a solid phase, and the resulting RNA:DNA hybrids are detected ~Iy using an antibody that recognizes the RNA:DNA hybrids. Such anti-RNA:DNA antibodies recognize and bind to the unique helix structure formed whenever RNA
3 o and DNA stral~ds base-pair with each other, regardless of the specific nucleotide sequence of the ~1~7416 .

h~ulidiLi.l~ strands. Thus, the anti-RNA:DNA hybrid antibody will be useful in the I assay of any NASBA product, provided that a suitable cu~ lelll.,ll~y DNA
probe is available for immobilization on the solid phase. The solid phase used for the ' ' of the DNA probe can be any suitable surface which will enable the binding of 5 DNA by either covalent or non-covalent bonds, including, but not limited to, plastic microtiter plates, Illi~ UI~ of nylon or nitrocellulose, llyJI~ c cloths, beads, etc. The immobilized DNA is any DNA segment in the single stranded form which is y to the amplicon ll~liL~ i' reaction product, but which does not contain sequences ~ y to the priming ~ .."~ used in the initial modified NASBA.
10 Thus, the ~iliL~ DNA can be created by isolation of a specific DNA fragment generated by restriction digestion, by the well know procedure of rol~.l,.,l~e Chain Reaction (as described in U.S. Patents 4,683,195 and 4,683,202), or by i~yll~ll~iLil.L~ an ~ ' using synthetic chemistry processes. DNA fragments originally in the double stranded form can be rendered single stranded by ~' ' (e.g. heating, alkali) prior to ' ~ iu.. on the solid phase.
15 In the i ' ' detection system, the RNA:DNA hybrids formed on the solid phase by reaction of the amplicon i i~ I product witb the ' ;1;~ DNA are detected by reaction with an anti-RNA:DNA hybrid antibody which is labelled with a detectable chemical moiety (e.g. enzyme, 11U~ c, etc.). Alternatively, an unlabelled anti-RNA:DNA hybrid antibody can be used, followed by detection of the antibody using a second anti-antibody labelled 20 with a detectable chemical moiety.
The present invention will now be illustrated, but is not intended to be limited, by the following examples.
25 ExamDle 1:
An example of a method of detecting the NASBA product is described where the RNAis detected using the anti-HNA antibody, in a microtiter plate assay.
30 The plasmid used as a template for l ' = was pEP20 (E. Emond, 1. Fliss and ~157~16 S. Pandian, ~ppl. Environ. Microbiol. 59: 2690-2697, 1993), which contains an insert of 784 bp, cloned into the vector pUC12. A 2 ,ul aliquot containing 0, 102, 104' 106 and 10~ molecules of P1- primed DNA as template was added directly to each 25 ,ul ( final volume) NASBA
reaction containing 40 mM Tris-CI pH 8.5, 12 mM MgCI2, SOmM KCI, 10 mM vliLllivll~
(DTT), 2mM of each rNTP, ImM of each dNTP, 0.2 fLM Pl primer, 0.2 ~m P2 primer, 15 %
(vol/vol) dimethyl sufoxide, 100 ,ug/ml bovine serum albumin, 8 units AMV reverse aDv, 0.1 unit E.coli RNase H 40 units T7 RNA ~vvl~ and 12.5 units RNasin The reaction was incubated at 40C for 90 min.
0 The Pl-primed DNA template was prepared by annealing Pl primer to linearized and heat-denatured plasmid DNA (pEP20), followed by DNA synthesis using 10 units of AMV
reverse l"" ~ . and NASBA reagents at 50C for 15 min. The P1 primed strand was separated by tllermal ~ ;.," and ' 'y chilled on ice to minimize ~ UldLivll of the DNA strands.
Analysis of the NASBA product r~c~. Fel 'vlvvhu~ . Two ~1 of eæh of the above NASBA reætions, vull~ r " g to varying amounts of the input P1 primed template was loaded on the agarose gel (0.7%). and the RNA reætion product was detected by ehidium bromide lluvlv~vv..vv. Figure 1 illustratvs the results. Lane 1: Molecular size markers (100 bp ladder), Lanes 2-6: NASBA reætion products, where the template DNA were 0, 102, 104' 106' and 10~ molecules respectively. It can be seen tbat tbe ~..I."lir has occurred in all the reactions (Lanes 3-6) exvept the control reaction (Lane 2).
25 2. HN~-El l.~. Into microtiter plate wells coated with capture probe DNA (: ,' y to the 784bp fragment of pEP20), 50 ~1 of h~ buffer was taken followed by the addition of ten f~l of eæh of the NASBA reætion products and 40 ~1 of RNase-free water. The plates were incubated for 9û min at 56C and then three times washed with TBST buffer. The following i ~ were carried out at room i ~ d~Ulv~ Anti-HNA antibody (100 ,ul; mouse 3 o monoclonal IgM) was added at d~)~)lU~I id~v dilution and incubated for 20 min. and then the plates 21~7416 were washed three times with TBST. Goat anti-mouse IgM - HRP conjugate (100 ,ul) was added and incubated for 20 min. and the unreacted conjugate was washed away as described above.
TMB microwell peroxidase substrate was added and the blue color was read at 540nm. The results are given in Table I and Figure 2. It can be seen that the NASBA reaction products can 5 be detected using the anti HNA-antibody or other reagents known in the art.
As disclosed in detail above, in one ,..ll.~.li,....,l of this invention the combined ~ ' and ~ ' assay procedure of the present invention can be applied to any instance where one wishes to employ the NASBA technique for the assay of a microbe 10 (bacteria, fungi, viruses, etc.) either directly or in an c... ' culture of a sample (e.g., food, clinical specimen, ~ t, etc.). This application will be ~.~u~il,ulally useful in the clinical (human medical and veterinary), agricultuMI (food safety, food industry) and ~,llVil~ ' ' (water systems, soils) fields, where it is often necessary to analyze samples for the presence of minute quantities of target cells (e.g. pathogens), and where the availability of an ul(~ ive and specific method would be pcui ' ly aJv In another ~ ~ ' t, the present system can be applied in the NASBA-based geneticanalysis of target cells (e.g., organs, tissues, cellular organelles, etc.) or specific genes. This application would enjoy many uses in the medical and biological fields, where it could be used 2 0 as a tool to aid in the diagnosis of genetic diseases or the ~ i. .., of genetic material in biological samples.
In yet another . ' ' t, the present invention can be applied in the NASBA-based analysis of target cells (e.g., microbes, eukaryotic cells, etc.) captured from a test sample on 25 a solid phase (e.g., ~ beads). This application would be of particular use in the ilubiologi~,dl, bi~,h~,l..;~l and medical fields, where it is often desirable to analyze for the presence of very low numbers of t~rget cells recovered from large volumes of sample (e.g., ~llViU~ l water samples, body fluids such as blood, etc.).
3 o Tbus, the principle of the present invention should be widely applicable not only to the 16 ~1~741~
detection of food pathogens such as L. I~u~o~,y tu~ s. but also in the analysis of a wide variety of other bacteria, fungi, viruses, etc., of clinical or economic interest.
From the foregoing d~ ir , one skilled in the art can easily ascertain the essential ~,ll~t~ ,s of this invention, and without departing from the spirit and scope thereof, can make various changes and 1..~ of the invention to adapt it to various usages andconditions. (~ ly~ such changes and -'~ are properly, equitably, and "intended" to be, within the full range of equivalence of the following claims.

Claims (3)

1. A method of nucleic acid sequence detection comprising the steps:
a) RNA amplification utilizing nucleic acid sequence based amplifications;
b) formation of RNA:DNA hybrids; and c) immunochemicals detection of RNA:DNA hybrids using procedures comprising antibodies directed to RNA:DNA hybrids.

2. A method as claimed in claims 1, wherein the RNA:DNA hybrid formation has one of the nucleic acid sequences attached to a solid support.

3. A method as claimed in claims 1 or 2, wherein the immunochemical detection isperformed by a monoclonal or polyclonal antibody that recognizes the target RNA:DNA hybrid.