WO1993023573A1

WO1993023573A1 - Quantitation of viral rna by competitive polymerase chain reaction

Info

Publication number: WO1993023573A1
Application number: PCT/US1993/004548
Authority: WO
Inventors: David T. Scadden; Zhengyu Wang; Jerome E. Groopman
Original assignee: New England Deaconess Hospital
Priority date: 1992-05-15
Filing date: 1993-05-13
Publication date: 1993-11-25
Also published as: AU4248193A

Abstract

The claimed invention relates to a method for quantitating virus RNA in a biological sample using a competitive PCR reaction. The method employs an internal standard RNA which is included in the PCR reaction in order to control for variability in the amount of PCR product from reaction to reaction. The method is particularly useful for monitoring plasma levels of virus RNA. The invention also relates to a cloned nucleic acid sequence useful for producing the standard RNA used in the method claimed.

Description

QUANTITATION OF VIRAL RNA BY COMPETITIVE POLYMERASE CHAIN REACTION

BACKGROUND OF THE INVENTION

Currently available tools for measuring the human immunodeficiency virus type 1 (HIV-1) jLn vivo are limited. Although circulating CD4+ lymphocyte cell numbers, serum HIV-l p24 antigen, serum neopterin, serum beta-2 microglobulin, soluble CD8 and soluble interleu in-2 receptor levels have been proposed to be clinical indicators of HIV-1 disease activity, they are limited by sensitivity and specificity. (Osmond, D.H., et al. , AIDS j5:505-511 (1991); Anderson, R.E., et al., Arc . Intern. Med. 150.73-77 (1990); Fahey, J.L., et _i.₍ N. Enσl. J. Med. 3___:166-172 (1990); Lacey J.N., et ε_L. , AIDS 1:123- 127 (1987); Funches, D., et ___• » Immunol. Today 9;150-155 (1988); Goedert, J.J., et ___. , N. Enσl. J. Med. 321:1141- 1148 (1989)). Quantitative plasma cultures, which measure the concentration of circulating infectious viral particles, and quantitative cultures of limiting dilutions of HIV-l infected peripheral blood mononuclear cells (PBMC) are recently developed methods designed to better characterize the activity of the retrovirus (Ho, D.D., e_t al., N. Enσl. J. Med. 321:1621-1625 (1989); Coombs, R.W. , et l., N. Enσl. J. Med. 321:1626-1631 (1989)). However, these methods are lengthy, time consuming and expensive. Quantitation of HIV-l infected PBMC by PCR of PBMC DNA is a more rapid analysis and has been shown to correlate with the stage of the patient's illness (Schnittman, S.M., et al.. Ann. Int. Med. 113:438-443 (1990)). However, PCR of PBMC DNA provides the investigator with an estimate of the number of infected cells, but does not provide any direct information regarding changes in viral replicative activity, such as is induced by drug therapies or during the natural progression of the disease. An assay which could rapidly and accurately evaluate the concentration of circulating virus in patient serum would provide a useful means of directly measuring virus activity.

SUMMARY OF THE INVENTION

The invention described herein is a method for quantitating virus RNA of interest in a biological sample. In one embodiment, the invention is a method for measuring the concentration of HIV-l virus RNA in a biological sample, such as in the blood plasma of HIV-l-infected individuals. The method described takes advantage of the polymerase chain reaction (PCR) method of amplifying small amounts of DNA. The method also employs an internal standard RNA, which allows for control of the variability in the amount of end product produced by PCR. The internal standard RNA is reverse transcribed to produce DNA which is amplified as efficiently as the DNA transcription product of the viral RNA from the biological sample; the internal standard RNA transcription product (DNA) effectively competes with the transcription product of the RNA of interest during the polymerase chain reaction.

According to the method of the present invention, internal standard RNA and sample RNA are combined in a dilution series in which each member contains the same amount of sample RNA; the amount of standard RNA varies among the members of the series and is at least 10² copies. The range is generally 10² to 10⁸ copies and in one embodiment is 10² copies to 10⁷ copies. In the alternative, internal standard RNA and sample RNA are combined in a dilution series in which each member contains the same amount of standard RNA within the range described above and the amount of sample RNA varies among the members of the series. The RNA of interest and the standard RNA are then reverse transcribed in the presence of either random nucleotide hexamers or DNA primers which are specific to RNA sequences characteristic of both the RNA of interest and the standard RNA. Reverse transcription results in production of DNA transcripts of the RNA of interest, referred to as sample DNA, and DNA transcripts of the standard RNA, referred to as standard DNA. The DNA transcripts are then amplified by the polymerase chain reaction (PCR) method in the presence of detectable oligonucleotide primers which are specific for nucleic acid sequences characteristic of DNA sequences in both sample DNA and standard DNA.

This PCR reaction results in the production of a mixture of two species of double-stranded DNA in each member of the dilution series: one species of DNA derived from the internal standard RNA, and one species of DNA derived from the RNA of interest. During PCR, detectable oligonucleotide primers are incorporated into both DNA species produced. The number of copies of each species of double-stranded DNA in a given member of the dilution series after PCR depends on the relative number of copies of sample DNA and standard DNA in the mixture prior to PCR.

After PCR amplification, the PCR products resulting from amplification of sample DNA and standard DNA are separated on the basis of size, and the relative amount of primer incorporated into each species is determined for each member of the dilution series. The relative copy numbers of PCR end products representing the standard DNA and the sample DNA are dependent upon the relative copy numbers of each of the two DNA species prior to initiation of the PCR reaction. Therefore, a competition curve can be generated for each dilution series, and the point at which standard DNA and sample DNA compete equally in the PCR is extrapolated. The amount of internal standard RNA in the reaction mix which results in equivalent copy numbers of sample DNA and standard DNA after PCR can be calculated, and the amount of sample RNA extrapolated therefrom.

Any viral RNA in a biological sample can be quantit ted by the method of the present invention. For example, the method can be used to quantitate human immunodeficiency virus RNA, human T-cell lymphotropic virus RNA, simian immunodeficiency virus RNA, avian leukosis virus RNA, murine leukemia virus RNA, and feline leukemia virus RNA.

Internal standard RNA is RNA homologous to two distinct and separate sequences within a region of viral RNA characteristic of the virus of interest, and is a different size than the corresponding region of the virus of interest. Internal standard RNA may be a deletion mutant or an insertion mutant of a region characteristic of the virus of interest. For example, the internal standard RNA may be an RNA polymerase product of a DNA clone of an approximately 115 bp deletion/insertion mutant of a region of the HIV σaσ gene.

The invention described herein is also a method of monitoring levels of HIV or other RNA viruses in the blood plasma of infected individuals. The method involves obtaining a blood sample by phlebotomy, separating the plasma from the whole blood, and extracting RNA from the plasma. The RNA from blood plasma is then used as the RNA of interest in the PCR strategy already described, wherein a constant amount of RNA of interest is added to each member of a dilution series of internal standard RNA, reverse transcribed, amplified by PCR, separated from standard DNA, and the amount of RNA of interest in the initial mixture calculated as described above.

Use of the internal standard RNA of this method allows for control over the tube to tube variability in the amount of end product produced by PCR. As a result of this advantage, the method of this invention is useful for accurate quantitation of the concentration of virus in blood plasma or other biological samples. When using this method to quantitate the amount of HIV RNA in blood plasma, only a small quantity of blood is required for sample analysis, and the entire analysis can be completed within 48 hours of obtaining the sample. In addition to the advantages described above, the dynamic range of the assay is at least four orders of magnitude, which is substantially higher than that of other reported PCR-based strategies (Holodniy, M. , e_t al« , J. Infect. Diseases 162:862-866 (1991)). The wide dynamic range of the assay makes it particularly attractive for use in evaluating . anti-viral therapies where large individual differences may be anticipated in the concentrations of circulating virus, both before and during treatment.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1A is a graphic representation of the sample processing and analysis schema.

Figure IB is competitive PCR results of the wild type HIV-l (upper band) or K4 standard (lower band) at different ratios of wild-type:K4 RNA.

Figure 1C shows the ratio of wild-type:K4 RNA represented by lanes 4 and 5 of (B) , and an intermediate ratio, subjected to sequential three-fold dilutions prior to reverse transcription and ampli ication.

Figure 2A shows equivalent amplification of wild type (H9/IIIB) or K4 RNA at different PCR cycle numbers. Figure 2B depicts the relationship between the amount of DNA produced in a PCR, and the initial amount of K4 cRNA in the reaction mixture.

Figure 2C is an analysis of a single AIDS patient's plasma sample divided at the time of phlebotomy and independently analyzed.

DETAILED DESCRIPTION OF THE INVENTION

Evaluation of the therapeutic benefit of compounds used to treat HIV infection may be based on a number of clinical and laboratory parameters. Presently, the laboratory assays widely in use are indirect measures of the activity of the virus, such as T lymphocyte subsets, neopterin or beta-2 microglobulin or direct measurements of a virus product such as the HIV-l p24 antigen assay. The latter assay is limited by the high frequency of p24 antigen-negative patients. Improved direct measures of the infectious burden of HIV are desirable because of the nonspecificity of the indirect laboratory parameters and because of the potential time delay for indirect measures to reflect alterations in circulating virus. Methods which rapidly and accurately measure the circulating levels of free virus, as opposed to virus incorporated into circulating cells, would be useful to evaluate therapies directed at the viral replicative cycle. For example, monitoring the concentrations of circulating virus could be used to measure the effects on viral replication of inhibitors of reverse transcriptase, Tat or protease, or inhibitors of virus assembly or release. Described herein is a method of quantitating the amount of viral RNA in a biological sample. Because the claimed method makes use of an internal standard, it permits control of the variability in the amount of end product produced by PCR. The method of the present invention involves the steps of t obtaining viral RNA of interest from a biological sample; adding approximately the same amount of that RNA to each member of a dilution series of internal standard RNA; reverse transcribing the mixture to yield sample DNA and standard DNA; amplifying that mixture by PCR; separating the two major PCR amplification products from each other, measuring the relative amount of each produced, and extrapolating the amount of RNA of interest in the biological sample by calculating the amount of standard DNA necessary to compete equivalently with sample DNA in the PCR reaction.

In a preferred embodiment, the method of the present invention uses known laboratory techniques in conjunction with a mutant internal standard RNA template to evaluate the quantity of HIV-l RNA in the plasma of AIDS patients. This method requires only a small quantity of patient plasma (e.g., obtained through the phlebotomy of a single tube of blood) which can be processed and analyzed within a relatively short period of time (e.g., 48 hours). An internal RNA standard that is equivalently amplified during PCR is included in the analysis in order to avoid inaccurate quantitation of the sample material based on intrasample variability. Use of an external standard does not control for this variability, which may result in alterations in final product quantitation of up to 600% (Gilliland, G. , __ al« , J. Infect. Diseases 87:2725-2729 (1990)). With an internal standard, the method presented here has an average intrasample variability of 26. This relatively simple assay for quantitating plasma levels of HIV-l RNA provides a rapid tool for measuring changes in circulating virus in vivo. The method can be modified to apply to other RNA viruses without undue experimentation. For example, the modifications would include use of an appropriate standard RNA, and primers designed to be specific for nucleic acid sequences which occur both in the sample RNA of interest, and in the standard RNA. The method of the present invention is described in detail below.

Sample RNA The method described herein may be used to detect viral RNA from a variety of biological samples, and from a variety of RNA viruses. For example, because it measures unincorporated viral genomic RNA, the method can be used to quantitate genomic RNA of any replicating RNA virus in any tissue. The method is particularly useful for measuring the concentration of circulating virus particles or RNA in body fluid, such as in blood plasma, mucous, saliva or semen. In particular, the present method is useful to determine blood plasma levels of HIV in infected individuals, as shown herein.

In the present method, a biological sample is obtained and the RNA is separated from the sample by any of a variety of well-known methods. For example, the RNA may be isolated by precipitation with guanidine isothyocyanate _r followed by ultracentrifugation through 5.7M cesium chloride. The lower limit of sensitivity of the present method is on the order of 10 copies of RNA of interest per reaction, and the upper limit is at least 10⁶ copies per reaction. Accordingly, the amount of sample required for analysis by this method depends on the concentration of viral RNA in the sample to be tested and may vary over a wide range. For example, 0.5 ml of blood plasma containing between 10³ and 8.3 x 10* copies of HIV RNA per ml is sufficient for one reaction mixture, and, if four dilutions of internal standard RNA are used for an assay, then a total of 2 ml of blood plasma is sufficient for an assay by the present method. The amount of biological sample of other types, for example semen or mucous, may be easily determined. Internal Standard RNA

The internal standard RNA of the present method is an RNA with nucleic acid sequence homology to regions near the 5' and 3' ends of a nucleic acid sequence characteristic of the viral RNA of interest. DNA transcripts of the internal standard RNA are amplified by PCR with an efficiency substantially equal to that of DNA transcripts of the viral RNA of interest, and the PCR products of the two RNAs are separable on the basis of size. The same DNA primers are used for PCR amplification of DNA transcripts from both the internal standard RNA and the sample RNA of interest. Thus, the internal standard RNA must share nucleic acid sequence homology with sequences near the 5' and 3' ends of a region characteristic of the viral RNA of interest, and must be of a different size than the region of the RNA of interest with which it shares homology. For example, the internal standard RNA may be an RNA polymerase product of a cloned deletion, insertion or deletion/insertion mutant of a region of the virus of interest, wherein the insertion or deletion occurs internal to the cloned viral nucleic acid sequences, rather than at the 5' or 3' ends. In one embodiment, the internal standard RNA shares substantial sequence homology with sequences near the 5' and 3' ends of a highly conserved region of a retroviral genome, such as a portion of the σaσ region. In one embodiment of the present invention, the internal standard RNA is an approximately 175 base deletion/insertion mutant of a portion of the σaσ region between the sequences corresponding to the primers SK100 and SK104. In this embodiment, the internal standard RNA is approximately 115 bases smaller than the corresponding region in the wild- type virus. Establishinσ the Mixtures of the Dilution Series

A portion of the RNA from the biological sample is added to each member of a dilution series of mixtures, each containing a known amount of an internal standard RNA; each member of the dilution series contains the same amount of sample RNA, and the amount of standard RNA varies among the members of the series and is at least 10² copies. The range is generally between 10² and 10^s copies. Quantitation in this assay is dependent on determining the amount of internal standard RNA required for equivalent amplification of the DNA transcripts resulting from reverse transcription of a mixture containing internal standard RNA and sample RNA. Because the efficiency of PCR amplification of transcripts of both RNAs is equivalent in the present method, a mixture containing both internal standard RNA and sample RNA will result in essentially equivalent amounts of PCR product only when the ratio of internal standard RNA copy numbers to sample RNA of interest copy numbers approaches 1:1. Therefore, the range of the internal standard dilution series should cover the range of viral RNA copy numbers which may be found in the biological sample to be tested.

For example, the concentration of HIV RNA observed in the HIV-infected individuals described in the Examples below ranged from 10³ to 8.3 x 10* copies in 1 ml of plasma. In order to find an internal standard RNA dilution at which the copy numbers of standard RNA and of sample RNA of interest are nearly equal, a dilution series may be provided wherein the sample RNA from 0.5 ml of blood plasma is added to each of four members of a dilution series which contain, respectively, 10³, 10*, 10⁵ or 10⁶ copies of the internal standard RNA.

It will be observed that the method described herein would work as well if the dilution series was comprised of mixtures containing a series of dilutions of the viral RNA of interest, and a constant amount of internal standard RNA in each member of the series.

Reverse Transcription of Internal Standard RNA and Sample RN pf intres

Because the mixtures containing sample RNA and internal standard RNA are to be reverse transcribed into DNA before amplification by PCR, the mixtures are maintained under conditions appropriate for reverse transcription by methods well recognized in the art and described in the Methods Section below.

Polymerase Chain Reaction

The polymerase chain reaction is performed under conditions well recognized in the art. The conditions used in the Examples provided herein are listed in the Methods section below. The reaction is terminated while amplification is in log phase, for example, at approximately 30 cycles or less.

Separation of PCR Products

Separation of the two species of DNA produced by PCR amplification of sample DNA and standard DNA can be accomplished by a variety of known chromatographic methods such as agarose gel electrophoresis, acrylamide gel electrophoresis, affinity chromatography and chromatographic separation on the basis of size or charge. After separation, the PCR products may be quantitated by any of a variety of known methods. For example, the primers, which are incorporated into the PCR products, may be radiolabeled before incorporation and quantitated after separation of the PCR products by counting in a scintillation counter. Alternatively, the primers may be labeled with a fluorescent compound and the amount of primer incorporated into the PCR products determined by measuring fluorescence of the two types of PCR products.

Determining the Amount of Viral RNA in the Biological Sample as a Function of the Relative Amounts of PCR Products of Sample DNA and Standard DNA

During a polymerase chain reaction in which two species of DNA template are present, and in which the two species are amplified with equivalent efficiency using the same primers, the amount of PCR product resulting from each of the two species depends on the relative molar concentrations of the two templates present at the start of the amplification reaction. For example, if the molar concentration of sample DNA is greater than the molar concentration of standard DNA, then the amount of PCR product of the sample DNA will be relatively greater than the amount of PCR product of the standard DNA. Conversely, if the molar concentration of standard DNA is greater than the molar concentration of sample DNA, then the amount of PCR product of the standard DNA will be relatively greater than the amount of PCR product of the sample DNA. When the standard DNA and sample DNA are present in the reaction mix in close to equimolar amounts, the amount of PCR product produced from each DNA template will be close to equal. RNA from the blood plasma of an HIV-infected individual may be quantitated in this manner, and the effect of anti-viral therapies thereby monitored.

The method of the present invention can, for example, be used to monitor changes in blood plasma levels of HIV in infected individuals, as described below.

The relationship between the two PCR products varies linearly with changes in the relative molar amounts of the DNA templates before amplification. Accordingly, differences in the relative amounts of sample DNA and standard DNA among members of a dilution series, as previously described, will result in differences in the relative amounts of PCR end product among the members of the dilution series. The ratio of the two PCR products for each of the several members of the dilution series is determined and together will define a relationship from which the quantity of RNA of interest in the original sample can be determined (e.g., using linear regression analysis) .

The present invention will now be illustrated by the following examples, which are not intended to be limiting in any way.

METHODS Patients Samples 10-15 ml of blood was obtained by phlebotomy either from HIV-l infected individuals with an AIDS defining diagnosis or from normal controls. Blood was collected in heparinized tubes (Becton-Dickenson, Rutherford, NJ) and plasma harvested following centrifugation at 1200 x g for 10 minutes. 2.0 ml of plasma was mixed with 4.0 ml of 5M quanidine isothyocyanate (Bethesda Research Laboratories, Gaithersburg, MD) and 100 ug yeast tRNA and ultracentrifuged through 5.7 M cesium chloride (BRL) at 180,000 + g for 18 hours at 25^βC. The resultant RNA pellet was resuspended in 0.3M sodium acetate and ethanol precipitated. The RNA pellet was washed with 70% ethanol and resuspended in water or stored under ethanol at -80°C.

Preliminary observations suggest that there are no substantial differences in samples aliquoted upon arrival in the laboratory where one aliquot was processed immediately and one aliquot was processed after a three hour delay. No differences were observed between samples which were processed immediately, and plasma aliquots frozen at -80^βC. RNA Standard

The DNA clone used to produce the internal standard RNA was derived from a mututated σaσ region comprising the nucleic acid sequence between bases 1377 and 1667 of HIV-l _₂ (GenBank #K02007) and including the intact primer sites for the primers SKI00 and SK104 (Rayfield, M. , ________ l-, J. Infect. Diseases 158:1170-1176 (1988); Ou, C.Y. , e__ al«. Science 239:295-297 (1988)). An approximately 290 bp portion of the HIV-l σaσ region of HlV-l^_y._j was PCR amplified using the oligonucleotide primer pair

SK100/SK104 and the PCR protocol described below. The resultant fragment was filled in using Klenow (BRL) and blunt end ligated into the Smal site in the multiple cloning site of the plasmid vector pGEM-3Z (Promega, Madison, WI) . The gag-specific insert was mutated to produce a σaσ-related fragment with an insertion and an approximately 115 bp deletion internal to the region cloned, and to also include sequences specific for the primers SK100/SK104 at the 5' and 3' ends of the insert (SEQ ID NO:l). The resultant plasmid (K4) was determined, on the basis of size by agarose gel electrophoresis, to be a satisfactory template for PCR directed by the primer pair, SK100/SK104. Single-standard RNA was made using single-stranded K4 DNA as the template for T7 polymerase (Promega) using random nucleotide hexamers as primers.

This RNA is referred to as K4 cRNA or as internal standard RNA. The K4 cRNA was purified by ultracentrifugation through a 5.7M cesium chloride cushion, precipitated and resuspended, and the concentration determined by both spectrophotometry and incorporation of ³²P-uridine triphosphate (UTP) . Reverse Transcription and Polymerase Chain Reaction

RNA was reverse transcribed in the presence of random nucleotide hexamers in a 20 ul volume containing 0.5 mM deoxynucleotides, 75 mM magnesium chloride, 50 mM TRIS (pH 8.3), 10 mM dithiothreotol, 200 units recombinant Moloney murine leukemia virus reverse transcriptase (BRL) , 40 units RNasin (Promega) at 37^βc for 90 minutes. Ten ul of the reaction product was added to a 50 ul final volume solution containing 50 pMoles of SK100 and SK104, 0.1 pMole ³²P-end labeled SK104, 0.2 mM deoxynucleotides, 1.5 mM magnesium chloride, 50 mM potassium chloride, 10 mM TRIS, 0.1% gelatin and 1 unit Taq polymerase (Perkin-Elmer Cetus, Norwalk, CT) and subjected to cycles of 92^βC for 30 seconds, 55^βC for 30 seconds and 72^βC for 1 minute repeated 30 times with a 10 minute extension at 72°C following the final cycle using an Ericomp (San Diego, CA) thermocycler.

PCR Product Analysis Amplified DNA was electrophoresed through either 2% agarose or 6% acrylamide, visualized by exposure to ultraviolet light after ethidium bromide staining, and the resultant bands harvested and Cherenkov counted in a liquid scintillation counter (Beckman Instruments, Palo Alto, CA) . Figure 1A summarizes the methods used.

EXAMPLES

Example 1: Production of a DNA Clone of a Standard RNA A portion of the HIV-l gag region, delimited by sequences specific to the oligomers SK100 and SK104,

(Rayfield, M. , ___ al* , J. Infect. Diseases 158:1170-1176 (1988); Ou, C.Y., et a -. Science 239:295-297 (1988)) was cloned and a 113bp deletion/insertion mutant produced (K4). K4 was amplified by PCR using the SK100/SK104 primer pair as described in the methods section. PCR amplification of K4 yielded a PCR product which was readily distinguished from wild-type HIV-l by either acrylamide or agarose gel electrophoresis (Figure 1B,C). In Figure IB, a fixed amount of K4 cRNA was maintained in the samples corresponding to lanes 1-9. Three fold dilutions of RNA from HIV-l„_IB infected H9 cells were added sequentially from right to left in the samples corresponding to lanes 10 through 2. Lane 1 contained K4 cRNA alone (10* copies) and lane 10 contained H_V-l_mB infected H9 RNA alone (1 meg) .

Example 2: Evaluation of K4 cRNA as a Template For PCR

The K4 cRNA was evaluated as a template for PCR, as described in the Methods, using the SK100/SK104 primer pair and directly compared with the wild-type HIV-l genome represented by RNA derived from HIV-1„_|B infected H9 lymphoblastoid cells. Following reverse transcription, ³²P-end labeled SK104 was added to the PCR reaction mixture and the PCR product was analyzed by electrophoresis. The yield of DNA was quantitated at different cycle numbers of PCR by the amount of ³²P, measured as counts per minute, incorporated into the appropriate bands isolated from the electrophoretic gels. In the experiment represented in Figure 2A, H9/IIIB RNA or K4 cRNA (10* copies) were independently analyzed for incorporation of ³²P-labeled oligonucleotide primer (SK104) following reverse transcription and amplification by scintillation counting of isolated agarose gel slices of the electrophoresed reaction products. Figure 2A indicates that the rate of incorporation of ³²P-labeled primer is the same when either K4 or wild-type HIV-l_IIIB is used as the PCR template. These data were corroborated using K4 compared with independently or concurrently (in the same tube) amplified known quantities of plasmid DNA of the HIV-l clone HXB2 provided by Dr. Robert Gallo. K4 is amplified with an efficiency equal to that of wild-type virus and is, therefore, an appropriate template to use in a quantitative assay for wild-type HIV-l.

Example 3: Investigation of the Sensitivity and Range of the Present Method for Quantitatinσ HIV-l

Having demonstrated that K4 can be readily distinguished from wild-type HIV-l, and that it is amplified by PCR as efficiently as wild-type HIV-l, the sensitivity and range of the method remained to be demonstrated. Because the range of concentrations of infectious virus particles detected in the plasma of HIV-l infected individuals has been reported to be quite variable (Holodniy, M. , ejt al« , J. Infect. Diseases 163:862-866 (1991)), and because the range of concentrations of virus particles containing detectable viral RNA (not necessarily infectious virus) may vary over an even broader range, a wide dynamic range for the assay is desirable. Known amounts of K4 cRNA were diluted and used as starting material for the reverse transcription and PCR reactions. The resulting incorporation of ³²P- labeled tracer SKI04 primer for each starting concentration of K4 cRNA is indicated in Figure 2B. In the experiments shown in Figure 2B, known amounts of K4 cRNA were reverse transcribed and amplified for 30 cycles in the presence of ³²P labelled SK104 primer. The reaction products were subjected to agarose gel electrophoresis, the gel slices were isolated and scintillation counted. The number of K4 cRNA indicated is the log of copies per assay. The intrasample coefficient of variation (CV) ranged from 0.05 to 0.26 for the samples with 10³ to 10⁶ copies of K4 cRNA respectively. The sensitivity of the assay is limited to 10-100 copies of K4 in the starting material, but the dynamic range of the assay is at least four orders of magnitude.

Example 4: The Competitive Polymerase Chain Reaction In a competitive PCR assay, the relationship of end products of PCR to the starting quantities is dependent upon the ratio of the two starting templates at the time of initiation of the amplification protocol (Gilliland, G., __al., Proc. Natl. Acad. Sci. 87:2725-2729 (1990); Want, A.M., s£ al- , Proc. Natl. Acad. Sci.. USA 86:9717- 9721 (1989)). At ratios of sample template to standard template of less than or greater than 1:1 the amplification of the two templates is not equal. Figure IB represents PCR products of reactions containing a fixed amount of the K4 standard with increasing concentrations of sample HIV-l RNA. The variable intensity of the visible lower band (K4) indicates the change in amplification of the K4 template in the presence of changing concentrations of competing wild-type HIV-l RNA. To investigate whether the differential amplification of wild-type versus K4 template was due to a limiting concentration of substrate, dilutions of RNA of three fixed ratios of wild-type RNA to K4 RNA were added to the PCR reaction mixture and amplified. In Figure 1C, the series of lanes for each numbered set (1-3) represent the ethidium bromide stained, UV illuminated products of a thirty cycle amplification. Dilution of the starting concentration of RNA did not alter the relationship between the wild-type and K4 final products; differential amplification of the two RNAs occurred unless the ratio of wild-type RNA to K4 RNA approached 1:1. Exa ple 5: Quantitating the Amount of HIV-l RNA in Human Plasma

Having demonstrated that the starting ratio of wild- type RNA to K4 RNA must approach 1:1 in order for the RNAs to be amplified with equal efficiency, 2 ml human plasma specimens were processed and four 10-fold dilutions of K4 internal standard RNA were added to fixed amounts of plasma RNA (.5 ml:10³ copies to .5 ml:10⁶ copies). These dilutions were reverse transcribed, amplified and the resultant products analyzed as above. At equivalent levels of incorporation of radiolabeled primer, the ratio of wild-type RNA of interest to K4 internal standard RNA in the mixture before reverse transcription was assumed, based on the results described above, to be 1:1. The quantity of wild-type RNA of interest could thereby be extrapolated and the number of HIV-l RNA molecules derived.

To verify the reproducibility of the assay, duplicate samples from a single phlebotomy were independently processed and analyzed. The results of a single such analysis are represented in Figure 2C. In the experiment shown, the copy number of K4 cRNA molecules corresponding to the K4:sample ratio of 1:1 estimates the copy number of HIV-l RNA in 1 ml of the patient's plasma (8.6 ± 1.4 x 10*) . The results of eleven samples from eleven patients analyzed in this manner are reported in Table 1. The average intrasample CV for eleven samples from eleven patients analyzed was .26 ± .15. TABLE 1

15.6 ± 74 30 CMV, KS None

60.5 ± 9.5 30 Crypto, NHL None*

82.5 ± 17.5 110 KS None^*

6.7 ± 3.0 40 KS ddl

5.1 ± 0.7 40 KS ddl

65.0 ± 0.1 20 KS None

10 15.5 ± 3.5 200 KS ZDV, IFN

11 31.5 ± 10.5 80 KS, NHL ZDV

* (x 10³ copies/mm³ plasma) ** (cells/mm³)

(crypto: cryptococcal meningitis or fungemia, PCP: pneumocystis carinii pneumonia, CMV: cytomegalovirus viremia, KS: Kaposi's sarcoma, NHL: non-Hodgkin's lymphoma, ZDV: zidovudine, ddl: dideoxyinosine, IFN: interferon alpha, ^*: drug withdrawn within 4 weeks) To confirm that our results did not reflect cellular HIV-l, 40 cycles of PCR were performed on patient samples without first submitting the samples to reverse transcription. No amplified products were seen, which indicated that HIV-l proviral DNA was not being detected. Therefore, it is unlikely that cellular DNA could account for our results. The specificity for HIV-l of the bands assayed as described was supported by the absence of bands in control plasma and the same electrophoretic mobility of samples amplified with non-labeled primers and subsequently liquid hybridized with a radiolabeled SK19 probe. (Ou, C.Y., et al- , Science 239:295-297 (1988)). There were no false positives detected in eight control plasma samples.

EQUIVALENTS

Those skilled in the art will know, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein.

These and all other equivalents are intended to be encompassed by the following claims.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: New England Deaconess Hospital

(ii) TITLE OF INVENTION: Quantitation of Plasma HIV-l RNA By Competitive Polymerase Chain Reaction

(iii) NUMBER OF SEQUENCES: 1

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Hamilton, Brook, Smith _ Reynolds, P.C. CB) STREET: 2 Militia Drive

(C) CITY: Lexington

(D) STATE: MA (F) ZIP: 02173

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: PatentIn Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: 07/885,091

(B) FILING DATE: 15-MAY-1992

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Granahan, Patricia

CB) REGISTRATION NUMBER: 32,227

(C) REFERENCE/DOCKET NUMBER: NEDH91-04

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (617) 861-6240

(B) TELEFAX: (617) 861-9540 (2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 176 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

ATCAAGCAGC CATGCAAATC TTGATTCAAA AAAAAAAAAG CTGGAGGACT CATGTATGCT 60

GATTTCAAAA CTTACTACAA AGCTACAGGA ATCAAAACTG TGTTGTACTG GTATTAAGAC 120

AGTATCCAAA TTAAGACATT AAGTGTCCCT TAAGACATAA GACAAGGACC AAAGGG 176

Claims

C AIMS

(1) A method of quantitating viral RNA of interest in a biological sample, comprising the steps of:

(a) producing a series of mixtures, each member of the series comprising: i) a constant amount of RNA from the biological sample to be analyzed; and ii) an internal standard RNA which varies among the members of the series within a range of 10² to 10⁸ copies;

(b) reverse transcribing RNA present in the mixtures provided in (a) , thereby producing mixtures containing DNA transcripts from retroviral RNA of interest, referred to as sample DNA, and DNA transcripts from the internal standard RNA, referred to as standard DNA;

(c) combining each mixture produced in (b) with detectable oligonucleotide primers which are specific for nucleic acid sequences characteristic of DNA sequences in both sample DNA and standard DNA, and maintaining the resulting combination under conditions appropriate for amplifying sample DNA and standard DNA using polymerase chain reaction (PCR) , whereby detectable primers are incorporated into PCR products of sample DNA and into PCR products of standard DNA, in amounts relative to the copy numbers of each type of DNA in the mixtures produced in step (b) , thereby producing detectable PCR products of sample DNA and detectable PCR products of standard DNA; ^*

(d) separating, on the basis of size, detectable PCR products of sample DNA from detectable PCR products of standard DNA; (e) determining the amount of detectable PCR products of sample DNA and determining the amount of detectable PCR products of standard DNA;

(f) comparing the amount of detectable PCR products of sample DNA copies determined in step (e) with the amount of detectable PCR products of standard DNA copies determined in step (e) to determine the ratios of the two PCR products for each of the several members of the dilution series; (g) extrapolating the quantity of RNA of interest in the original sample from the curve described by the ratios determined in (f) .

(2) A method of Claim 1 wherein the viral RNA of interest is retroviral RNA.

(3) A method of Claim 2 wherein the retroviral RNA is selected from the group consisting of: human immunodeficiency virus RNA, human T-cell lymphotropic virus RNA, simian immunodeficiency virus RNA, avian leukosis virus RNA, rourine leukemia virus RNA, and feline leukemia virus RNA.

(4) A method Claim 2 wherein the standard RNA is a deletion mutant of a region characteristic of the retroviral RNA of interest.

(5) A method of Claim 4 wherein the retroviral RNA of interest is HIV RNA, and the internal standard RNA is a deletion mutant of a portion of the HIV gag region.

(6) A method of Claim 5 wherein the internal standard RNA is an RNA transcript of the plasmid K4 (SEQ ID NO: 1).

(7) A method of Claim 5 wherein the oligonucleotide primers used for PCR amplification are specific for sequences in the portions of the HIV oaσ region which are also present in the internal standard RNA.

(8) A method of Claim 5 wherein the biological sample is blood plasma.

(9) A method of quantitating viral RNA of interest in a biological sample, comprising the steps of:

(a) producing a series of mixtures, each member of the series comprising: i) a constant amount of an internal standard RNA within a range of 10² to 10⁸ copies; and ii) an amount of RNA RNA from the biological sample which varies among the members of the series within a range of 10² to 10⁸ copies;

(c) combining each mixture produced in (b) with detectable oligonucleotide primers which are specific for nucleic acid sequences characteristic of DNA sequences in both sample DNA and standard DNA, and maintaining the resulting combination under conditions appropriate for amplifying sample DNA and standard DNA using polymerase chain reaction (PCR) , whereby detectable primers are incorporated into PCR products of sample DNA and into PCR products of standard DNA, in amounts relative to the copy numbers of each type of DNA in the mixtures produced in step (b) , thereby producing detectable PCR products of sample DNA and detectable PCR products of standard DNA; (d) separating, on the basis of size, detectable PCR products of sample DNA from detectable PCR products of standard DNA;

(e) determining the amount of detectable PCR products of sample DNA and determining the amount of detectable PCR products of standard DNA;

(f) comparing the amount of detectable PCR products of sample DNA copies determined in step (e) with the amount of detectable PCR products of standard DNA copies determined in step (e) to determine the ratios of the two PCR products for each of the several members of the dilution series;

(g) extrapolating the quantity of RNA of interest in the original sample from the curve described by the ratios determined in (f) .

(10) A method of monitoring levels of HIV in the blood plasma of an HIV-infected individual, comprising the steps of:

(a) obtaining a blood sample from the individual; (b) separating the RNA from the blood plasma;

(c) producing a series of mixtures, each mixture of the series comprising: i) a constant amount of RNA from the blood plasma of the individual to be monitored; and ii) an internal standard RNA which varies among the members of the series within a range of 10² to 10⁸ copies; (d) reverse transcribing RNA present in the mixtures provided in (c) , thereby producing mixtures containing DNA transcripts from HIV RNA, referred to as sample DNA, and DNA transcripts from the internal standard RNA, referred to as standard DNA;

(e) combining each mixture produced in (d) with detectable oligonucleotide primers which are complementary to nucleic acid sequences characteristic of DNA sequences in both sample DNA and standard DNA, and maintaining the resulting combination under conditions appropriate for amplifying sample DNA and standard DNA using polymerase chain reaction (PCR) , whereby detectable primers are incorporated into PCR products of sample DNA and into PCR products of standard DNA, in amounts relative to the copy numbers of each type of DNA in the mixtures produced in step (d) , thereby producing detectable PCR products of sample DNA and detectable PCR products of standard DNA; (f) separating on the basis of size detectable

PCR products of sample DNA from detectable PCR products of standard DNA;

(g) determining the amount of detectable PCR products of sample DNA and determining the amount of detectable PCR products of standard DNA;

(h) comparing the amount of detectable PCR products of sample DNA copies determined in step (g) with the amount of detectable PCR products of standard DNA copies determined in step (g) to determine the ratios of the two PCR products for each of the several members of the dilution series; (i) extrapolating the quantity of RNA of interest in the original sample from the curve described by the ratios determined in (h) .

(11) A plasmid comprising a deletion mutant of a region of HIV-l gag which includes regions near it 5' and 3' ends which share sequence homology with viral RNA of interest.

(12) The plasmid K4 comprising the plasmid vector pGEM-3Z containing a gag-derived nucleic acid sequence (SEQ ID NO: 1) inserted into the Smal site of the vector, the nucleic acid insert comprising a deletion mutant of the nucleic acid sequence between approximately bases 1377 and 1667 of HIV-l which includes primer sites for the primer pair SKI00 and SK104.