CA2012982A1 - Process for rapid nucleic acid detection by incorporating a reporter moiety into amplified target nucleic acid - Google Patents

Abstract

TITLE

PROCESS FOR RAPID NUCLEIC ACID DETECTION
BY INCORPORATING A REPORTER MOIETY
INTO AMPLIFIED TARGET NUCLEIN ACID
ABSTRACT
A nucleic acid assay which incorporates a deoxyribonucleotide triphosphate containing a reporter moiety into a nucleic acid amplification process, followed by the detection of the reporter moiety, is provided.

Description

PROCESS FOR RAPID NUCLEIC ACID DETEC'rION
BY INCORPORATING A REPORTER MOITY IN~O

F I E I.D QF ~I NV~Q~
This invention relates to the detection of nucleic acid sequences and more specifically to a process of combinin~ amplification of target nucleic acid sequences with detection of a reporter group specifically incorporated into the target nucleic acid sequence.
ACKGRO~ND OF THE INVEN~Q~
The development of practlcal nucleic acid hybridization methods which can be used for detecting nucleic acid sequences of interest has been limited by several factors. These include lack of sensitivity, complexity of procedure, and the desire to convert from radiometric to nonradiometric detection methods.
A variety of methods have been investigated for the purpose of increasing the sensitivity nonradiometric procedures. In one general approach, improvements in the total assay procedure have been examined, with concomitant effec~s on the issues of complexity and nonradiometric de~ection. In another approach, methods wh~ch increase the amount of nucleie acid to be detected by such assays have been pursued.
U.S. Patent 4,358/535, issued to Falkow, describes a method of cul~urin~ cells to ~ncrease their number and ~hus the~amount of nucleic acid of ~he or~anism suspected to be present, depositing the sample onto fixed support, and then contacting the sample with a labeled;probe, followed by washing the support and detecting the label. One drawback ~o this method is that without culturing the organism first, ~: :

_, the assay does not have adequate sensitivity. Adding a culture step, however, is time consuming and not always successful. Maniatis et al.~ ~lec~la~
ClQnin~: A La4P~a~Qry-Manual~ Cold Spring Harbor Laboratory, pp.390-401 ~1982), descri~e a method in which a nucleic acid of interest is amp'Lified by cloning it into an appropriate host system. Then, when the host organism replicates in cu]Lture, the nucleic acid of interest is also xeplicated. This method also suffers from the requirement to perform a culture step and thus provides for a procedure that is time consuming and complicated.
An alternative approach to increasing the quantity of~nucleic acids of organisms has been described in U.S. patents 4,683,202 and 4,683,195.
These patents disclose "a process fox amplification and detection of any target nucleic acid sequence contained in a nucleic acid or mixture thereof". This process employs an in vitro cycling mechanism which doubles the nucleic acid sequence to be amplified after each cycle is complete. This is carried out by separating the complementary strands of the nucleic acid sequence to be amplified, contacting these strands with excess oligonucleotide primers and extending the primers by enzyma~ic treatment to form primer extension products that are complementary to the nucleic acid annealed with each primer~ The prQCeSs is then repeated as many ~imes as is necessary. ~n advantage of this method is that it can rapidly produce large quantities of a small portion of the sequence of the nucleic acid of an organism of interest. A disadvantage of this method is that the detection of the nucleic acids produced, using a direct assay method, is complicated in that the amplification process can produce nucleic acld 3 ~ 9~2 sequences which are not faithful copies of the original nucleic acid which was to be copied. These erroneous nucleic acid sequences can prov~de false positives in the assay which increase the back~round noise and thus decrease the sensitivity of the entire method.
Numerous DNA probe assays have been described in the past for the detection of nucleic acids of interest. Falkow's method ~above) first renders the target nucleic acid single-stranded and ~hen immobilizes it onto a solid support. A labeled probe which is complementary to the target nucleic acid is then brought into contact with the solid support. Any excess probe is washed away and the presence of the label in the resulting hybrid is determined~ A
disadvanta~e of this method is that it is time consuming and cumbersome. The assay steps, i.e., hybridiæation and washing steps are carried out in a sealçd pouch which contains the membrane ~solid support) as well as the buffer solution.
Hill e~ al., wO 86/05815, describe a variation of the above assay format employing nitrocellulose coated maynetic particles to which the target DNA is affixed, followed by direct hybridization with a biotinylated probe and detection using a streptavidin-conjugated reporter.
Dunn et al., ~11, Vol. 12, 23-36 (19773, describe a different hybridization format which employs a two-step sandwich assay method employing polynucleotide probes in which the target nucleic acld is mixed with a solution containing a first o_ capture probe which has been affixed to a solid support.
After a period ~f time, the support is washed and a second or reporter ilabeled) probej also complementary to the target nucleic acid but not to the capture "

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probe, is added and allowed to hybridize with the capture probe - target nucleic acid complexO After washing to remove any unhybridi~ed repor~er probe, the presence of the reporter probe, hybridi2ed to the target nucleic acid, is detected.
Ranki et al. U.S. patent 4,563,419, disclose EPA
0 159 505, w086/03782, and ERA 0 200 113. It is to be recognized that all of these employ an assay procedure in which the first or capture probe is :immobilized onto a solid support prior to hybridization.
A further variation has been described in German Preliminary Published Application 3,546,312 Al. This method, like that described by Ranki et al., employs a capture probe and a reporter probe which hybridize to distinct portions of the target nucleic acid. The target nucleic acid is contacted in solut~on by the two probes. The first, or capture probe, contains a binding component, such as biotin, that is capable of binding with a receptor component, such as streptavidin, which has been affixed to a solld support. After formation of the capture probe -target nucleic acid - reporter probe complex, a streptavidin-modified solid support is added. Any unhybridized reporter probe is washed away followed by the detection of the label incorporated lnto the complex bound to the solid support. An advantage of this technique over that disclosed by ~anki et al. is that the hybridization, which takes place in solution, is favored kinetically. Some disadvantages are that the length of the target nucleic acid affects the overall efficiency of the reaction which decreases with increasing target nucleic acid length. Also, sandwich nucleic acid probe assays, whether hetProgeneous two-step or one-step, or utilizing ~ ' .
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solution hybridization, are hot as sensitive as the direct assay method.
~ISCLOSUB~ QF_~E~ E~TIQM.
The nucleic acid assay of this inventio~ for the detection andfor measurement of a preselected nucleic acid sequence in a sample suspected of including a nucleic acid containing said preselected sequence comprises the steps of:
(A) rendering the target nucleic acid single~
stranded;
(B) amplifying at least one specific nucleic acid sequence contained within the preselected nucleic acid sequence in ~he presence of at least one deoxyribonucleotide triphosphate containing a reporter moiety in an amount up to the total replacement of the correspo~ding dNTP, by ~1) treating the strands with two oligonucleotide primers, for each different specific sequence being amplified, under conditions such that for each different sequence being amplified an extension product of each primer is synthesized which is complementary to each nucleic acid strand~ wherein said primers are selected so as to be sufficiently complementary to the different strands of each specific sequence to hybridize therewith such that the extension : product synthesized from one primar, when it is separated from its complement, can serve as a template f~or synthesis of the extension product of the other primer;

~2) separating the primer extension products from the templates on which they were synthesized to produce single-stranded molecules; and ~3) treating the single-stranded molecules generated from step (2) with the primers of step (1~ under conditions that a primer extension produc~ is synthesized using each of the single strands produced in step (2) as a template;
: (4) repeating steps ~2) and ~3) to produce sufficient primer extension produ~t for detection and/or measurement;
~C) rendering the product of step (B)~4) single~stranded;
(D) contacting the product of step (C) with an oligonucleotide which i.s attached to a solid support and which is complementary to a portion of a primer extension product not including the nucleic acid sequences defined by both primers;
(E) removing any unhybridized material; and ~; ~F) detecting and/or measuring the reporter moiety immobilized on the solid support.
DET~ILED ~SCRI~l35~L5U' ~ Y3~
The nucleic acid assay of this invention comprises the following overall process for the detection of target nucleic aci~s of a preselecte~
: 30 sequence:
a) Usi~g the polymerase chain react~on (PCR) nucleic acid amplification method described ln U.S.
4,683,202, incorporated herei~ by ~eference, specific nucleic ac~d sequences are amplified by anneal~ng the denatured target nucleic acid present ln ~he sample ~: :

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with primers specific for ~he target and forming extension prod~cts. In this process, a deoxyribonucleotide triphosphate containing a reporter group (moiety), dNTP-R, is used to replace some or all 5 of at least one of the corresponding deoxyribonucleotide triphosphates (dNTP) employed.
Each extension product formed is complernentary to a portion of the preselected nucleic acid sequence contained within the target nucleic acid and becomes a template for further primer binding. Thls process is then repeated as necessary in order to produce the desired amount of primer extension product for detection and/or measurement.
b) The resulting nucleic acid is rendered single-stranded by known methods, such as treatment with heat, chaotropic agents, or by raising or lowering the pH. The single-stranded nucleic acid so produced is then contacted with a capture probe which is attached to a solid support and allowed to 0 hybridize with it.
c) The amplified nucleic acid - capture probe complex is washed with appropriate buffers to remove unhybridized product from above and any unincorporated dNTP-R. The presence and quantity of the reporter 5 group is detected and/or measured and is proportional to the amount of amplified target nucleic acid. The amount of amplified target nucleic acid present, in turn, is proportional to the unamplified target nucleic acid originally present in the sample.
In an alternative detection method, a labeled antibody to the reporter group incorporated during the amplification process, is employed. It is brought into contact with the amplified product before or subsequent to capture of the ampli~ied pro~uct. The .

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label on the antibody can then be used to detect the presence of the amplifiPd product.
The term "PCR" as used hexein in referring ~o the process of amplifying tar~et nucleic acid sequences employing primer oligonucleotides to produc~ by enzymatic means a ~reatly increased number of copies of a small portion of the target nucleic acid is described in U.S. patent 4,683,202.
The term capture probe as used herein refers to an oligonucleotide which is complementary to a portion of a preselected sequence of the target nucleic acid and which is attached to a solid support. The capture probe cannot be complementary to either primer or to those portions of a primer extension product whose nucleic acid sequences axe defined by the primers.
Reporter group-containing deoxyribonucleotide triphosphates are known materials. Reporter groups of interest include fluorescent compounds such as fluorescein. In the alternative detection method, the reporter group has to be a moiety capable of forming a stable complex with an antibody.
Useful reporter groups in this invention include:
fluorescein, rhodamine, and other chromogenic or fluorogenic compounds.
The PCR targe~ amplification reaction requires approximately 20 to 30 repeat cycles in order to produce a sufficient quantity of the amplified target nucleic acid for further hybridization. Denaturation of the amplified nucleic acid can be accomplished by treatment with alkali, acid~ chaotropic agents, or heat, although the preferred me~hod is to place the amplified target nucleic acid in a boiling water bath for at least lO minutes followed by a chilled water bath ~4C) for at least two minutes.

The hybridization step can be accomplished by contacting the amplified sin~le-stranded target nucleic acid containing the reporter group(s) in solution with the capture probe, which is attached to a solid support, in an appropriate buffer, for a period of from 1 to 30 minutes. The length of the capture probe is determined by the ~ase of its synthesis, by the desired reaction kinetics, and by the identity of the primers, and preferably is an oligonucleotide of approximately 20 to 30 bases. The capture probe can be attached to the solid support by known means through sugar groups, preferably through either the 5'-terminal or the 3'-terminal sugar groups; or by attachment through a modified nucleotide base group.
A variety of solid supports can be utilized.
Among solid supports are included magnetic particles, such as the chromium dioxide particles disclosed by Lau et al., V.S. Patent 4,661,908, incorporated herein by reference, microtiter plates, and membranes.
To remove non-hybridized ampli~ied target nucleic acid and unincorporated reporter-group modified deoxyribonucleotide triphosphate, the immobilized target nucleic acid on the solid support can then be washed several times, for example, in the temperature ran~e of 25C - 37C, for approximately 5 to lO
minutes per wash cycle.
A variety of known detection methods can be utilized in the assay of this invention depending on the type of the reporter groups incorporated into the amplified product. When the reporter group is a chromophor or fluorophor, the ~ncorporated reporter group can be detected by known spectroscopic techniques. Alternativelyf a labeled antibody to the reporter group, incorporated durin~ the amplification , process, is brought into contact with the amplified product before or subsequent to capture of the amplified product by hybridiæa~ion to the capture probe. The label on the antibsdy can then be used to detect the presence of the amplified product subsequent to the washing step.
The Example below exemplifies the invention.

Detection of H~V I
A. Amplification of Target Nucleic Acid by PCR _ _ The procedure as described in U.S. Patent 9,683,202 and in a product bulletin for GeneAmp ~NA
Amplification Reagent Kit ~#N801-0043) can be followed utilizing the following specific conditions and reagents. A 103-nucleotide base sequence located within the GAG pl7 region of HIV I, incorporated into a plasmid tthe plasmid incorporating most of the HIV I
genome is designated pBH10-R3~, can be amplified using primers A and B as shown below:
5'-TGGGCAAGCAGGGAGCTAGG
Primer A
5'-TCTGAAGGGATGGTTGTAG~
Primer B
Aliquots of serial dilutions tlx10+7, lx10~6, lx10+5, lx10+4, lx10+3, lx10~2, lxlO+1, and zero copies~ of plasmid pBH10-R3 can be amplified using PCR. Each aliquot can be combined with a buffer 200 ~M in each of dATP, dTTP, dCTP, and dGTP and 10 ~M in succinyl-fluorescein dTTP, 1.0 ~M ln each of Primer A
and P~rimer ~, and containing 1 ~g oP human placental DNA/reaction and 2.5 units of a DNA polymerase enzyme in a total reaction volume of 100 ~1.

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~ ach reaction mixture can then be temperature cycled as described in the product bulletin thirty (30~ times.
This process is expected to result in the estimated increase in the number of target molecules by lx10+5 to lx10~6.

B. Hyb~ iQn l) Denaturation:
10 ~1 of amplified target DNA from each of the above aliquots can be combined with 30 ~l of H2O
in a 1.5-ml Eppendorf tube and placed in a boiling water bath for 10 minutes. The tubes can then be transferred to a chilled water bath ~4C) for two minutes and then centrifuged in a microcentrifuge for 10 seconds.
2) Hybridization:
The denatured samples can be split into two 20-~l portions ~in Eppendorf tubes) in order that duplicate samples could be run. 100 ~1 of hybridization mix, pre-equilibrated at 37C for 10 minutes, can then be added. ~ybridization buffer can be prepared by combining: 3 ml of 20X SSC, pH 7.0, 0.1 ml of Triton X-lO0, an alkylaryl polyether alcohol having 9-lO ethoxy units, 1.0 ml of deionized formamide, 6.375 ml of H20, and 25 ~l of 1.0 N HCl.
12 ~l o~ capture oligonucleotide bound to chromium dioxide particles ~12 ~g), prepared as described in U.S. 4,661,408, can then be added to the samples, incubated for 10 minutes ak 37C~ centrifuged in a ~: : microfuge ~or 5 seconds, and placed in a Corning ~ magnetic rack for two minutes at 25C. The pellets ; : can then b~ washed three times at 25C by adding 200 ;~ ~l of wash buffer containing lX SSC, pH 7.0, and 0.17 ~5 Triton X-100, mixing, placing the samples in a ~1 : :

' magnetic rack for 2 minutes, and removing the wash buffer.

3. PetectiQn Detection can be accomplished by adding 200 S ~1 of 10 mM Tris, pH 7.0, to each sample and placing them in boiling water ba~h for 10 minutes. The tubes can then be transferred to a chilled water bath ~4C) for two minutes and centrifuged in a microcentxifuge for 10 seconds. 200 ~1 portions can then be removed f~om each tube and the amplified nucleic acid product detected by direct fluorescence visualization.

Claims (4)

1. A nucleic acid assay for the detection and/or measurement of a preselected nucleic acid sequence in a sample suspected of including a nucleic acid containing said preselected sequence comprises the steps of:
(A) rendering the target nucleic acid single-stranded;
(B) amplifying at least one specific nucleic acid sequence contained within the preselected nucleic acid sequence in the presence of at least one deoxyribonucleotide triphosphate containing a reporter moiety in an amount up to the total replacement of the corresponding dNTP, by (1) treating the strands with two oligonucleotide primers, for each different specific sequence being amplified, under conditions such that for each different sequence being amplified an extension product of each primer is synthesized which is complementary to each nucleic acid strand, wherein said primers are selected so as to be sufficiently complementary to the different strands of each specific sequence to hybridize therewith such that the extension product synthesized from one primer, when it is separated from its complement, can serve as a template for synthesis of the extension product of the other primer;

(2) separating the primer extension products from the templates on which they were synthesized to produce single-stranded molecules; and (3) treating the single-stranded molecules generated from step (2) with the primers of step (1) under conditions that a primer extension product is synthesized using each of the single strands produced in step (2) as a template;
(4) repeating steps (2) and (3) to produce sufficient primer extension product for detection and/or measurement:
(C) rendering the product of step (B)(4) single-stranded;
(D) contacting the product of step (C) with an oligonucleotide which is attached to a solid support and which is complementary to a portion of a primer extension product not including the nucleic acid sequences defined by both primers;
(E) removing any unhybridized material; and (F) detecting and or measuring the reporter moiety immobilized on the solid support.

2. The assay of claim 1 wherein said preselected nucleic acid sequence is HIV I DNA.

3. The assay of claim 1 wherein said reporter moiety is a fluorescent moiety.

4. The assay of claim 1 wherein said reporter moiety is an antigenic moiety.