TITLE OF THE INVENTION
METHOD AND KIT FOR DETECTING REPLICATION COMPETENT ADENOVIRUSES
FIELD OF THE INVENTION
[0001] The present invention relates to a method for detecting replication competent adenoviruses and kits therefore. More specifically, the present invention is concerned with a polymerase chain reaction ("PCR")-based method for detecting replication competent adenoviruses ("RCA").
BACKGROUND OF THE INVENTION
[0002] Adenoviruses are non-enveloped DNA viruses composed of an icosahedral capsid and an inner DNA protein core. The genome consists of a single linear, double-stranded DNA of approximately 36 kb covalently bound to the 55 kDa terminal protein (Rekosh et al., 1977; Chroboczek et al., 1992)).
[0003] During their productive infection cycle, the viral genes are expressed in two phases: an early phase and a late phase, which respectively occur before and after DNA replication. Early transcription is accompanied by a succession of complex splicing events. These gene products, which have regulatory functions, are encoded by four early transcriptional units named E1, E2, E3 and E4. During the late phase, adenovirus late genes encoding structural proteins are expressed at the onset of viral DNA replication, disturbing the host DNA and protein synthesis (Berk, 1986; Tooze, 1981 ; Shenk, 1996).
[0004] These viruses are among the most efficient gene delivery tools.
However, before they may be used for this purpose, adenoviruses have to be rendered replication-defective to prevent virus production and prevent their causing the various diseases with which they, are normally associated (i.e. Human adenoviruses have been associated with acute febrile respiratory infections, epidemic conjunctivitis and infantile gastroenteritis) (Horwitz, 1996). Hence, first-generation recombinant adenovirus vectors used for gene delivery/therapy have deletions in the E1 region (E1 deleted adenoviruses) to render the vector replication-defective, thus preventing virus production and lysis of the target cell. These first-generation vectors are unable to replicate since the essential E1 region has been replaced with foreign DNA. The E3 region has been
shown to be dispensable for the replication of the virus and was removed simply to provide space for the exogenous nucleic acid. Adenovirus ΔE1/E3 can be propagated by infecting the human embryonic kidney (HEK) cell line 293, which provides the E1 gene products in trans (Graham et al., 1977). However, it has been shown that adenoviruses ΔE1/E3 can acquire the E1 region from the HEK 293 genome by homologous recombination during multiple passages, leading to RCA (Lochmϋller et al., 1994; Hehir et al, 1996; Zhu et al., 1999). These RCAs could have deleterious effects in gene therapy by activating the immune system or by acting as a helper virus for the replication of the attenuated viral vector (Imler et al., 1995). Indeed, exacerbation of host inflammatory responses with significant tissue damage and pathogenicity has been assigned to RCA contaminations (Lochmϋller et al, 1994; Hehir et al, 1996; Zhu et al., 1999). Labor-intensive and expensive RCA screening tests are therefore required to detect the presence of RCAs among a population of replication-defective adenovirus recombinants.
[0005] Zhang et al, (1995) have developed a multiplex PCR-based method, using two pairs of primers in the same reaction to detect a 1.07 kb adenoviral E1A DNA fragment with co-amplification of a 0.86 kb E2B DNA fragment as an internal control. Zhang indicated that his method could be used to detect 1 wild-type virus in 109 recombinant adenoviruses (Zhang et al., 1995). In this method however, because the E1 DNA fragment is found not only in wild- type adenovirus and RCA, but also in 293 cells, an amplification of the fragment occurs not only in the presence of the wild-type or RCA molecules that the method seeks to detect but also when 293 cells are present, thus resulting in some non-specific amplification. Furthermore, later experiments demonstrating that multiple passages in a liquid phase as those used in the Zhang et al. method often produce RCAs cast doubts on the reliability of the results reported by Zhang et al. Indeed, the flaw in the procedure used to propagate the virus in that method may indicate that the number of RCAs detected in the dilution that Zhang labeled as a "1 wild-type virus in 109 recombinant adenoviruses" was probably higher than that, resulting in a lower detection level.
[0006] Lochmϋller et al. (1994) used primers located in E1 and E2 (E1-1 and E2-1 , respectively in Table 1) to amplify a 3.8 kb DNA fragment found exclusively in RCAs or wild-type viruses. The maximal sensitivity obtained by
hybridization of radiolabeled probe to the PCR product was 1 RCA in 106 recombinant adenoviruses (Lochmϋller etal., 1994).
[0007] Another method for detecting contaminant RCAs is based on the fact that only RCAs are able to grow in non-complementing cell lines such as HeLa or A549. Dion et al. (1996) have used a stock of recombinant adenoviruses to infect HeLa cells and, based on the amount of virus produced, they were able to determine the amount of RCAs present in the original stock. Their assay detected the presence of only a single RCA mixed within 109 recombinant adenoviruses. Although, the cell culture assay is the preferred method for the detection of RCAs, it is a cumbersome and lengthy procedure.
[0008] Screening for RCA has significantly increased the manufacturing costs of clinical recombinant adenovirus lots and has led to delays in the onset of clinical studies. Therefore there is a need for an improved sensitivity method of detection and quantification of RCAs against a large background of recombinant adenoviruses. More specifically, there remains a need for such a method that would be more sensitive than that which appears the most sensitive (allegedly, 1 RCA/109 recombinant adenoviruses).
[0009] The present invention seeks to meet these needs and other needs.
[0010] The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.
SUMMARY OF THE INVENTION
[0011] Broadly, the present invention relates to methods and kits for the detection of RCA which are more sensitive or less cumbersome than those of the prior art. The present invention provides a sensitive nested PCR-based method to detect RCAs and wild-type adenoviruses ("wtAd") in purified samples. This method is RCA and/or wild-type specific and the detection limit is as low as one copy of RCA among 3 x 1010 recombinant adenoviruses per nested-PCR reaction and 5 copies of RCA among 1011 recombinant adenoviruses per nested-PCR reaction.
[0012] The present invention also relates to kits for assessing the presence or absence of RCAs in recombinant adenoviruses productions.
[0013] More specifically, in accordance with the present invention, there is provided a method for detecting the presence or absence of replication competent adenovirus ("RCA") in a sample suspected of being contaminated with RCA, the method including performing a nested polymerase chain reaction ("PCR") to selectively amplify a targeted RCA nucleotide sequence within the sample, wherein the targeted RCA nucleotide sequence is a nucleotide sequence overlapping at least a portion of E1 and at least a portion of E2 of the adenovirus, the nested PCR comprising a) performing a first round of PCR amplification using an outer primer pair flanking a first targeted RCA nucleotide sequence, the outer primer pair consisting of a 5' outer primer and a 3' outer primer, to yield a first round PCR amplification mixture; b) performing a second round of PCR amplification from said first round PCR amplification mixture using an inner primer pair flanking a second targeted nucleotide sequence, internal to the first targeted nucleotide sequence, the inner primer pair consisting of a 5' inner primer and a 3' inner primer, to yield a second round PCR amplification mixture; and d) analyzing the second round PCR amplification mixture to detect the presence or absence of the second targeted nucleotide sequence wherein the presence of the second targeted nucleotide sequence indicates the presence of the RCA in the sample suspected of being infected with the RCA. In a specific embodiment, the adenovirus is selected from the group consisting of adenovirus serotype 5 and adenovirus serotype 2 and the first targeted RCA nucleotide sequence is comprised between about nucleotide 2901 and about nucleotide 5072. In a more specific embodiment, the 5' outer primer is the nucleotide sequence set forth in SEQ ID NO: 4, the 31 outer primer is the nucleotide sequence set forth in SEQ ID NO: 19, the 5' inner primer is the nucleotide sequence set forth in SEQ ID NO: 8 and the 3' inner primer is the nucleotide sequence set forth in SEQ ID NO: 17.
[0014] According to an other aspect of the present invention, any one of the 5' inner primer and a 3' inner primer is identical to the 5' outer primer and a 3' outer primer, respectively. In a more specific embodiment, the 5' outer primer is the nucleotide sequence set forth in SEQ ID NO: 4, the 3' outer primer is the nucleotide sequence set forth in SEQ ID NO: 19, the 5' inner primer is the nucleotide sequence set forth in SEQ ID NO: 4 and the 3' inner primer is the nucleotide sequence set forth in SEQ ID NO: 17. In an other more specific embodiment, the 5' outer primer is the nucleotide sequence set forth in SEQ ID NO: 4, the 3" outer primer is the nucleotide sequence set forth in SEQ ID NO: 19,
the 5' inner primer is the nucleotide sequence set forth in SEQ ID NO: 8 and the 3' inner primer is the nucleotide sequence set forth in SEQ ID NO: 19.
[0015] According to specific embodiments of the present invention, the polymerase used in the PCR may be selected from the group consisting of Taq™ and Arrow Taq™, the sample used is essentially constituted of purified DNA or of purified viruses.
[0016] According to a further aspect of the present invention, there is provided a kit for assessing the presence or absence of replication competent adenoviruses in a sample using a nested polymerase chain reaction, the kit comprising a) two nucleotide sequences for an outer pair of oligonucleotide primers consisting of a 5' outer primer and a 3' outer primer used in a first-round polymerase chain reaction, the outer primer pair flanking a first region overlapping at least a portion of E1 and at least a portion of E2 of the adenovirus; and b) at least one nucleotide sequence for an inner pair of oligonucleotide primers consisting of a 5' inner primer and a 3' inner primer used in a second- round polymerase chain reaction, the inner primer pair flanking a second region comprised within said first region. In a specific embodiment, the adenovirus is selected from the group consisting of adenovirus serotype 5 and adenovirus serotype 2 and the region is flanked by the outer primer pair is comprised between about nucleotide 2901 and about nucleotide 5072. In a more specific embodiment, the nucleotide sequence for an inner pair of oligonucleotide primers is any one of a 5' inner primer and a 3' inner primer and is identical to the 5' outer primer and a 3' outer primer, respectively. In an other more specific embodiment, the 5' outer primer is the nucleotide sequence set forth in SEQ ID NO: 4, the 3' outer primer is the nucleotide sequence set forth in SEQ ID NO: 19, the nucleotide sequence for an inner pair of oligonucleotide primers is a 3' inner primer having the nucleotide sequence set forth in SEQ ID NO: 17. In a further more specific embodiment, the 5' outer primer is the nucleotide sequence set forth in SEQ ID NO: 4, the 3' outer primer is the nucleotide sequence set forth in SEQ ID NO: 19 and the nucleotide sequence for an inner pair of oligonucleotide primers is a 5' inner primer having the nucleotide sequence set forth in SEQ ID NO: 8. In a further more specific embodiment, the 5' outer primer is the nucleotide sequence set forth in SEQ ID NO: 4, the 3' outer primer is the nucleotide sequence set forth in SEQ ID NO: 19, the 5' inner primer is the nucleotide
sequence set forth in SEQ ID NO: 8 and the 3' inner primer is the nucleotide sequence set forth in SEQ ID NO: 17. In an other embodiment, the kit further comprises a polymerase selected from the group consisting of Taq™ and Arrow Taq™.
[0017] According to a further aspect of the present invention, there is provided a composition of matter for assessing the presence or absence of RCA in a sample comprising: a) a first primer pair consisting of a 5' outer primer and a 3' outer primer and flanking a first region overlapping at least a portion of E1 and at least a portion of E2 of an adenovirus; and b) at least one inner primer selected from the group consisting of a 5' inner primer and a 3' inner primer for use in an inner primer pair, said inner primer pair flanking a second region comprised within said first region. In a specific embodiment, the 5' outer primer is the nucleotide sequence set forth in SEQ ID NO: 4, the 3' outer primer is the nucleotide sequence set forth in SEQ ID NO: 19, and the at least one inner primer is a 3' inner primer having the nucleotide sequence set forth in SEQ ID NO: 17. In a further specific embodiment, the 5' outer primer is the nucleotide sequence set forth in SEQ ID NO: 4, the 3' outer primer is the nucleotide sequence set forth in SEQ ID NO: 19, and the at least one inner primer is a 5" inner primer having the nucleotide sequence set forth in SEQ ID NO: 8. In an other specific embodiment, the 5' outer primer is the nucleotide sequence set forth in SEQ ID NO: 4, the 3' outer primer is the nucleotide sequence set forth in SEQ ID NO: 19, and the at least one inner primer is a 5' inner primer having the nucleotide sequence set forth in SEQ ID NO: 8 and a 3' inner primer having the nucleotide sequence set forth in SEQ ID NO: 17.
[0018] In a further aspect of the present invention, the method enables a detection sensitivity equal or higher than 1 RCA in 1x109, equal or higher than 1 RCA in 1x1010 or equal to 1 RCA in 3x1010.
[0019] In order to provide a clear and consistent understanding of terms used in the present description, a number of definitions are provided hereinbelow.
[0020] Unless defined otherwise, the scientific and technological terms and nomenclature used herein have the same meaning as commonly understood by a person of ordinary skill to which this invention pertains. Generally, the procedures for cell cultures, infection, molecular biology methods and the like are common methods used in the art. Such standard techniques can be found in
reference manuals such as for example Sambrook et al. (1989, Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratories) and Ausubel et al. (1994, Current Protocols in Molecular Biology, Wiley, New York).
[0021] The present description refers to a number of routinely used recombinant DNA (rDNA) technology terms. Nevertheless, definitions of selected examples of such rDNA terms are provided for clarity and consistency.
[0022] Nucleotide sequences are presented herein by single strand, in the
5' to 3' direction, from left to right, using the one-letter nucleotide symbols as commonly used in the art and in accordance with the recommendations of the IUPAC-IUB Biochemical Nomenclature Commission.
[0023] As used herein, "nucleic acid molecule", refers to a polymer of nucleotides. Non-limiting examples thereof include DNA (e.g. genomic DNA, cDNA), RNA molecules (e.g. mRNA) and chimeras thereof. The nucleic acid molecule can be obtained by cloning techniques or synthesized. DNA can be double-stranded or single-stranded (coding strand or non-coding strand [antisense]).
[0024] The term "recombinant DNA" as known in the art refers to a DNA molecule resulting from the joining of DNA segments. This is often referred to as genetic engineering. The same is true for "recombinant nucleic acid". Similarly, the term "recombinant adenovirus" or "rAd" refers to a nucleotide molecule resulting from the modification of the sequence of a natural "wild-type" adenovirus, as known in the art.
[0025] The term "DNA segment", is used herein, to refer to a DNA molecule comprising a linear stretch or sequence of nucleotides. This sequence when read in accordance with the genetic code, can encode a linear stretch or sequence of a ino acids which can be referred to as a polypeptide, protein, protein fragment and the like.
[0026] As used herein, the term "gene" is well known in the art and relates to a nucleic acid sequence defining a single protein or polypeptide. A "structural gene" defines a DNA sequence which is transcribed into RNA and can be translated into a protein having a specific amino acid sequence thereby giving rise to a specific polypeptide or protein. For instance, E1 , E2, E3 and E4 are
examples of genes of adenoviruses expressing proteins having regulatory functions in the early stage of expression of adenoviruses.
[0027] The term "vector" is commonly known in the art and defines a plasmid DNA, phage DNA, viral DNA and the like, which can serve as a DNA vehicle into which DNA of the present invention can be cloned. Numerous types of vectors exist and are well known in the art. The present invention is concerned more specifically with adenovirus vectors used in gene therapy, functional genomic and proteomic applications.
[0028] As used herein, a "primer" defines an oligonucleotide which is capable of annealing to a target sequence, thereby creating a double stranded region which can serve as an initiation point for DNA synthesis under suitable conditions. In the present invention, primers are designed to be specific for certain nucleotide sequences present only in replication competent vectors or in wild-type adenoviruses. In accordance with the present invention, the use of such a primer with the other necessary reagents would give rise to an amplification product only when a replication competent adenovirus or a wildtype adenovirus is present in the sample analysed.
[0029] Primers of the present invention may be of any suitable length. In general, the oligonucleotide probes or primers are at least 12 nucleotides in length, preferably between 15 and 24 molecules, and they may be adapted to be especially suited to a chosen nucleic acid amplification system. In specific embodiments of the present invention, they are 20 nucleotides in length. As commonly known in the art, the oligonucleotide primers can be designed by taking into consideration the melting point of hybridization thereof with its targeted sequence (see below and in Sambrook et al., 1989, Molecular Cloning - A Laboratory Manual, 2nd Edition, CSH Laboratories; Ausubel ef al., 1989, in Current Protocols in Molecular Biology, John Wiley & Sons Inc., N.Y.).
[0030] Primers of the invention can be utilized with naturally occurring sugar-phosphate backbones as well as modified backbones including phosphorothioates, dithionates, alkyl phosphonates and α-nucleotides and the like. Modified sugar-phosphate backbones are generally taught by Miller, 1988, Ann. Reports Med. Chem. 23:295; and Moran et al., 1987, Nucleic Acids Res., 14:5019. Primers of the invention can be constructed of either ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), and preferably of DNA. Primers of the
present invention are meant to include single-stranded primers comprised of nucleotide sequences including naturally occurring nucleotides and any variants thereof.
[0031] The terminology "primer pair" refers herein to a pair of oligonucleotide primers of the present invention, which are selected to be used together in amplifying a selected target nucleic acid sequence in an RCA or a wtAd by a PCR-based method. As commonly known in the art, the primers are designed to bind to a complementary sequence under selected conditions.
[0032] The terminology "naturally occurring nucleotides" refers to adenosine triphosphate, guanosine triphosphate, cytosine triphosphate, thymidine triphosphate, uridine triphosphate, and inosine triphosphate. The terms "any variants thereof" are meant to include any modified nucleotide and any analog thereof. Variants are well known in the art; examples thereof can be found in the WIPO Standard ST.25 (1998) Appendix 2, table 2, in a double-stranded or a single-stranded form. They include any nucleotides comprising modified bases of the form N6-(6-aminohexyl) (as in N6-(6-aminohexyl) dATP or N6-(6- aminohexyl) ATP), or comprising bases modified as 5'-thiol, 5'-phospho, 5'- methyl, 5'-biotinylated, 5'-amino, or 5'-fluoro (as in 5'-fluoro-deoxyadenosine).
[0033] Polymerase chain reaction (PCR) is carried out in accordance with known techniques. See, e.g., U.S. Pat. Nos. 4,683,195; 4,683,202; 4,800,159; and 4,965,188 (the disclosures of all four U.S. Patent are incorporated herein by reference). In general, PCR involves, a treatment of a nucleic acid sample (e.g., in the presence of a heat stable DNA polymerase) under hybridizing conditions, with one oligonucleotide primer for each strand of the specific sequence to be detected. An extension product of each primer which is synthesized is complementary to each of the two nucleic acid strands, with the primers sufficiently complementary to each strand of the specific sequence to hybridize therewith. The extension product synthesized from each primer can also serve as a template for further synthesis of extension products using the same primers or different primers as in the method of the present invention. Following a sufficient number of rounds of synthesis of extension products, the sample is analyzed to assess whether the sequence or sequences to be detected are present. For a review on PCR techniques (see PCR Protocols, A Guide to Methods and Amplifications, Michael et al. Eds, Acad. Press, 1990).
[0034] By the terms "nested PCR" are intended a two PCR amplification rounds process. In a first PCR amplification round, a pair of "outer" oligonucleotide primers ("outer primer pair"), consisting of a 5' outer primer and a 3' outer primer that flank a particular first "target" nucleotide sequence in the 5' and 3' position, respectively, are used to amplify that first sequence. In a second PCR amplification round, a pair of "inner" or "nested" oligonucleotide primers ("inner primer pair"), consisting of a 5' inner primer and a 3' inner primer, are used to amplify a smaller second "target" nucleotide sequence that is contained within the first target nucleotide sequence. The 5' and 3' inner primers flank the second target nucleotide sequence in the 5' and 3' positions, respectively. By "flanking primers" is intended primers that are complementary to segments on the 5' and 3' end portions of the double-stranded target nucleotide sequence that is amplified during the PCR process. The definition "nested PCR" includes herein an asymmetrical nested PCR. The terms "inner primer pair" therefore encompass the case wherein one inner primer of the pair is identical to one primer of the outer primer pair.
[0035] The term "aliquot" is meant to refer to a sufficient amount of amplification mixture yielded in a first round of PCR amplification to enable a detection of RCA, in accordance with the detection sensitivity of the present invention, should it be present in the initial sample.
[0036] The primers, nested PCR methods and kits of the present invention may be used to detect the presence or absence of RCAs or wtAds in any sample suspected of containing same. Such samples include samples containing nucleic acid isolated from any sample suspected of containing RCAs or wtAds. In particular, the primers, nested PCR methods and kits of the present invention may be used to detect RCAs or wtAds in mixtures containing a background of recombinant ΔE1/E3 adenovirus vectors that have been propagated in cell lines that provide the E1 gene product.
[0037] The general principle of the amplification rounds of the present invention is to assess the presence or absence of nucleic acid regions contained in RCAs or wtAds but absent in recombinant adenovirus ("rAd") vectors and in cell lines used to propagate these rAd vectors. The presence or absence of these nucleic acids therefore indicate the presence or absence of RCAs or wtAds. The identification of the amplification products may be carried out by methods known
in the art to detect amplified nucleotide sequences. These methods include, but are not limited to determination of size, restriction enzyme digestion pattern, subsequent cloning of amplification products, Southern blot hybridization with an oligonucleotide probe internal to the nucleotide sequence being amplified, or DNA sequencing.
[0038] The size of the amplification product or products may be determined by electrophoresis through a gel, preferably an agarose gel, simultaneously with molecular size standards of known base-pair length. The gel may be stained with ethidium bromide, which intercalates between base pairs and enables the visualization of DNA upon illumination with ultraviolet light. In this manner, the migration of amplification products from the first or second PCR amplification rounds for a distance equal with those of molecular size standards of approximately the base-pair length of the predicted first or second targeted nucleotide sequence, respectively, would confirm the presence of the RCAs or wtAd within the original sample.
[0039] The amplification products identity may also be determined through digestion with restriction endonucleases. Following digestion with restriction enzymes specific for known base-pair positions within the targeted nucleotide sequences, the base-pair length of the digestion products may be determined using gel electrophoresis and ethidium bromide staining as described above. Depending upon the base-pair location of the restriction enzyme cut within the PCR amplification products, digestion would yield two nucleotide sequence fragments of predicted size. In this manner, digestion products from the PCR amplifications product having migrated the same distance as molecular size standards of approximately the base-pair length of the predicted nucleotide sequence fragments would verify the presence of RCA(s) or wtAd within the original sample
[0040] Additional proof of sequence identity may be obtained by cloning of the amplification product. This product can be ligated into any conventional plasmid vector for subsequent cloning in Escherichia coli. Following an incubation period, plasmid DNA can then be isolated from transformed bacterial colonies, quantified with ultraviolet spectrophotometry, and incubated with a desired restriction enzyme that removes the cloned insert from the plasmid backbone. The DNA fragments in the restriction digest can then be analyzed by gel
electrophoresis as before to determine the presence of the predicted PCR amplification product.
[0041] Any method for the identification of PCR products available in the art can be used with the present invention. See particularly Sambrook el al., Molecular Cloning: A Laboratory Manual (2d ed.; Cold Spring Harbor Laboratory: Plainview, N.Y., 1989).
[0042] The types of detection methods in which probes can be used include Southern blots (DNA detection), dot or slot blots (DNA, RNA), and Northern blots (RNA detection). Labeled proteins could also be used to detect a particular nucleic acid sequence to which it binds. Other detection methods include kits containing probes on a dipstick setup and the like.
[0043] Although the present invention is not specifically dependent on the use of a label for the detection of a particular nucleic acid sequence, such a label might be beneficial, by increasing the sensitivity of the detection. Furthermore, it enables automation. Probes can be labeled according to numerous well known methods (Sambrook et al., 1989, supra). Non-limiting examples of labels include 3H, 1 C, 32P, and 35S. Non-limiting examples of detectable markers include ligands, fluorophores, chemiluminescent agents, enzymes, and antibodies. Other detectable markers for use with probes, which can enable an increase in sensitivity of the method of the invention, include biotin and radionucleotides. It will become evident to the person of ordinary skill that the choice of a particular label dictates the manner in which it is bound to the probe.
[0044] As commonly known, radioactive nucleotides can be incorporated into probes of the invention by several methods. Non-limiting examples thereof include kinasing the 5' ends of the probes using gamma 32P ATP and polynucleotide kinase, using the Klenow fragment of DNA Pol I of E. coli in the presence of radioactive dNTP (e.g. uniformly labeled DNA probe using random oligonucleotide primers in low-melt gels), using for example the SP6/T7 system to transcribe a DNA segment in the presence of one or more radioactive NTP, and the like.
[0045] In one embodiment, the present invention relates to a kit for detecting RCAs or wtAd. For example, a compartmentalized kit in accordance with the present invention includes any kit in which reagents are contained in
separate containers. Such containers include small glass containers, plastic containers or strips of plastic or paper. Such containers allow the efficient transfer of reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another. Such containers will include a container which will accept the test sample (DNA), containers which contain the first set of primers used in the assay, containers which contain the second set of primers, containers which contain control DNA, containers which contain the reagents used to amplify RCAs or wtAdδ present in samples (buffer, dNTPs, MgCI2, DNA polymerase) and containers which contain the reagents used to detect the extension products.
[0046] As used herein, the terminology "targeted RCA nucleotide sequence" is meant to refer to a region in RCAs that is sufficiently specific to permit RCAs to be identified in a background of other nucleic acid. While a region overlapping a portion of E1 and a portion of E2 is a preferred region to be targeted by the primer pairs in accordance with the present invention, the invention should not be so limited. Indeed, any region only found in wtAd and in RCAs (and not in rAd or in the cell lines used to propagate the rAds), at least a portion of which being indispensable for the virus replication, could serve as a target nucleotide region for the primer pairs of the present invention.
[0047] As used herein, the terminology "purified DNA" is meant to refer to
DNA which has been separated from a portion of cellular and/or viral components.
[0048] As used herein, the terminology "purified viruses" is meant to refer to propagated viruses extracted from the host cells where virus propagation occurred.
[0049] Other objects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of preferred embodiments thereof, given by way of example only with reference to the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0050] In the appended drawings:
[0051] Figure 1 : A) provides a schematic representation of the shared region of HEK 293 cells, of the recombinant adenovirus, of RCAs/wild-type adenovirus serotype 5; and of the amplification products resulting from the PCR and the nested-PCR; B) provides the position of primers according to specific embodiments of the present invention;
[0052] Figure 2: shows an ethidium-bromide stained agarose gel of a second round of PCR amplification of wtAdδ mixed with 1x1010 copies of pAd- Null DNA according to embodiments of the present invention. Lanes 1-8 correspond to the first round of amplification and lanes 10-17 to the second round. Lanes 1-6, and 10-15 contain 0.1 , 1 , 10, 100, 1000 and 10000 copies of wtAdδ, respectively; lanes 7 and 16 contain 0.1 ng of HEK 293A DNA, lanes 8 and 17 contain a negative control (absence of wtAdδ DNA); and lane 9 contains 1 kb DNA ladder (Invitrogen);
[0053] Figure 3: shows an ethidium-bromide stained agarose gel of a second round of PCR amplification of wtAdδ mixed with 1x1010 copies of Ad5CMVrev-GFP DNA generated and amplified in BMAdEI cells according to embodiments of the present invention. Lane 1 contains 1 kb DNA ladder (Invitrogen), lanes 2-9 contain 0.1, 1 , 2, 5, 10, 100, 1000 and 10000 copies of wtAdδ, respectively and lane 10 contains a negative control (absence of wtAdδ DNA); and
[0054] Figure 4: shows an ethidium-bromide stained agarose gel of a second round of PCR amplification of RCA in a preparation of AdδCMVrev- GFP generated in BMAdEI cells and amplified in 293 cells according to embodiments of the present invention. Lane 1 corresponds to 1 kb DNA ladder (Invitrogen); lanes 2-8 correspond to 1x1010, 1x109, 1x108, 5x107, 1x107, 5x106 and 1x106 copies of Ad5CMVrev-GFP, respectively; lane 9 corresponds to 0.1 ng of BMAdEI DNA; and lane 10 contains the negative control (absence of DNA).
DETAILED DESCRIPTION OF THE INVENTION
IO0551 According to one embodiment of the present invention, two sequential rounds of amplification using primers are performed to detect RCAs or wtAd in a sample. Two different primer pairs are used for the two rounds of amplification: an outer primer pair and an inner primer pair. A small aliquot of the
PCR product of the first round of amplification was used as a template for the second round of PCR amplification. The second amplification round used an inner primer pair located inside the first pair of primers. Both primer pairs were designed to amplify exclusively the E1-plX-E2 region of RCAs or wild-type adenovirus serotype 5 ("wtAdδ") (Fig. 1 ).
EXAMPLE 1
Primers
[0056] The primers used in the first and second rounds of PCR amplifications according to specific embodiments of the present invention are listed in Table 1 below along with their sequences and localizations on adenovirus serotype 5 ("Adδ") DNA. The positions of primers of the invention overlapping a portion of E1 and a portion of E2, are schematically illustrated by arrows in Figure 1 relative to the shared region of HEK 293 cells, recombinant adenovirus and RCAs/wtAdδ. The length of each PCR product is also represented. Different combinations of these primers were used to perform amplifications according to specific embodiments of the present invention as shown in Tables 2 and 3. For instance when E1-δ long (no. 4 in Table 1), E2-2 long (no. 19 in Table 1), E1-4 long (no. 8 in Table 1) and E2-1 nested long (no. 17 in Table 1) are used as δ' outer primer, 3' outer primer, δ' inner primer and 3' inner primer of the invention, respectively, the amplification product of the first PCR amplification round has a length of 1420 bp and that of the second PCR amplification round has a length of 1281 bp.
Table 1. Sequences of primers used for detecting RCAs ° Primer Sequence Position on wtAd5
E1-1 5'-GAC GCC CGA CAT CAC CTG TG-3' SEQ 1319-1338 ID NO:1
E1 -2 5'-CGC CCC AAA AGC AGG GCT TC-3' SEQ 2901-2920 ID NO:2
E1-3 5'-GGC ACA GCC CCG AAC CCT TA-3' SEQ 2870-2851 ID NO:3
E1-5 long 5'-TGT TTG AGC ATA ACA TAC TGA CCC 3169-3198 GCT GTT-3' SEQ ID NO:4
E1-5 5'-ACA TAC TGA CCC GCT GTT CCT TGC-3' 3181-3204
SEQ
ID NO:5
E1 -5 short 5'-CAT AC T GAC CCG CTG TT-3' SEQ ID 3182-3198
NO:6 E1 -4 5'-ACA CTA AGA TAT TGC TTG AGC CCG-3' 3257-3279
SEQ
ID NO:7 E1-4 long 5'-ACT AAG ATA TTG CTT GAG CCC GAG 3258-3287
AGC ATG-3' SEQ ID NO:8 E1-1 -nested 5'-ATG ACC ATG AAG ATC TGG AAG GTG 3318-3347 long CTG AGG-3' SEQ ID NO:9 0 E1 -1 -nested 5'-ATG AAG ATC TGG AAG GTG CTG AGG-3' 3324-3347
SEQ
ID O:10 1 E1 -nested long 5'-TAC AGA TTG AGG TAC TGA AAT GTG 3500-3529
TGG GCG-3' SEQ ID NO:11 2 E1 -nested 5'-TGA GGT ACT GAA ATG TGT GGG CG-3' 3507-3529
SEQ
ID NO.12 3 E1 -nested short 5'-CTG AAA TGT GTG GGC GT-3' SEQ ID 3514-3530
NO:13 4 E2-nested 5'-GTC CAA GAT GCA TCT CAT ATC CCC-3' 4404-4426
SEQ
ID NO:14 5 E2-nested long 5'-AAA AAT ACA GTC CAA GAT GCA TCT 4407-4436
CAT ATC-3' SEQ ID NO:15 6 E2-nested short 5'-AAG ATG CAT CTC ATA TC-3' SEQ ID 4407-4423
NO:16 7 E2-1 -nested- 5'-TAA GCT ACA TGA CAA ATT TCC CAA 4509-4538 long GTG CAC-3' SEQ ID NO:17 8 E2-1 -nested 5'-TCT AAG CTA CAT GAC AAA TTT CCC-3' 4517-4540
SEQ
ID O:18 E2-2 long 5'-AAT CTT GGA GGT CAC AAG GGC GTC 4559-4588
TCC AAG-3' SEQ ID NO:19 E2-2 5'-CTT GGA GGT CAC AAG GGC GTC TCC-3' 4562-4585
SEQ
ID NO:20 1 E2-3 5'-GTG ATC CCA GAA ATA TCTTCG CCC-3' 4639-4662
SEQ
ID NO:21 E2-3 long 5'-ATG ACG TTA GTG ATC CCA GAA ATA 4642-4671
TCT TCG-3' SEQ ID NO:22 E2-3 short 5'-TCC CAG AAA TAT CTT CG-3' SEQ ID 4642-4658
NO:23 E2-1 5'-CGG CGA GCG CCT TCT GGC GG-3' SEQ 5053-5072
ID NO:24
Extraction of adenoviral DNA
[0057] The adenoviral preparation (1 x1012 vp) was digested with proteinase K (1 mg/ml proteinase K, 0.5% SDS and 5 mM EDTA) for 2 hours at
37°C. NaCI was added to a final concentration of 100 mM. DNA from the digested virus was extracted with phenol-chloroform-isoamyl alcohol (2δ:24:1 , v/v/v), followed by a chloroform-isoamyl alcohol (24:1, v/v) extraction. It was then precipitated by the addition of 1/10 volume of sodium acetate 3M, pH δ.2 and 2.δ volume of 100% ethanol. Viral DNA was resuspended in δO μl of sterile distilled water. DNA concentration and purity was determined by spectrophotometry.
Calculation of the molecular weight of single copies of pAd-Null and of wtAdδ
[0058] The molecular weights of pAd-Null and of wtAdδ were calculated to determine precisely the amount of each molecule in the PCR reactions. The plasmid pAd-Null has 33,09δ bp (A=7,6δ2, C=9,460, G=9,122, T=6,861). Since this molecule is a supercoiled double stranded DNA, the complementary strand was included in the calculation of the molecular weight. Therefore, the amount of each base will be: A=14,δ13, C=18,δ82, G=18,δ82 and T=14,δ13. The molecular weight of pAd-Null was then determined by multiplying the amount of each base by its corresponding molecular weight, and adding the Avogadro's number.
(A x 313.149 g/mole) + (C x 289.129 g/mole) + (G x 329.189 g/mole) + (T x 304.189 g/mole) + 34"
6.022045 x 1023 molecules/mole
* This value is added for phosphorylated linear double stranded DNA
[0059] According to this formula, the molecular weight of a single molecule of pAd-Null was found to correspond to 3.396 x10'17 g. The same formula was used to calculate the molecular weight of wtAdδ. It is a linear double stranded-DNA of 3δ,93δ bp (A=8,367, c=10,073, G=9,761 , T=7,734). The molecular weight of a single molecule of wtAdδ has been calculated to be 3.687x10"17 g. The amount of pAd-Null and wtAdδ per reaction was then determined by using both the concentration and the molecular weight.
General PCR Amplification Conditions using Taq™ DNA polymerase
[0060] The two rounds of PCR amplification were accomplished in a δO μl reaction mixture containing 2 mM MgCl2, 0.2 μM of each primer, 0.1 mM dNTP and 2 unit of Taq DNA polymerase (Qbiogene Inc. Montreal, Quebec, Canada) with a thermal cycler 480™ (Applied Biosystems Inc., Foster City, California, USA). The amplification cycles parameters for the first amplification round consisted of 300 sec at 94°C followed by 2δ cycles of 30 sec at 94°C, 60 sec at 70°C, 60 sec at 72°C, followed by an extension of 10 min at 72°C. The amplification cycles parameters for the second amplification round consisted of 300 sec at 94°C followed by 30 cycles of 30 sec at 94°C, 60 sec at 70°C, 60 sec at 72°C, followed by an extension of 10 min at 72°C. These PCR conditions were optimized for the utilization of primers of a length of 30 nucleotides. Otherwise, the same PCR conditions were used for 17-24 nucleotides primers except for the annealing temperature which was δ0°C instead of 70°C.
General PCR Amplification Conditions using Arrow Taq™ polymerase
[0061] The two rounds of PCR amplification were accomplished in a δO μ\ reaction mixture containing 2 mM MgCl2, 0.2 μM of each primer, 0.1 mM dNTP and 2 unit of Arrow Taq™ DNA polymerase (Qbiogene Inc. Montreal, Quebec, Canada) with a thermal cycler 480™ (Applied Biosystems Inc., Foster City, California, USA). The amplification cycles parameters for the first amplification round consisted of 300 sec at 94°C followed by 30 cycles of 4δ sec at 94°C, 90 sec at 70°C, 90 sec at 72°C, followed by an extension of 10 min at 72°C. The amplification cycles parameters for the second amplification round consisted of 300 sec at 94°C followed by 3δ cycles of 4δ sec at 94°C, 90 sec at 70°C, 90 sec at 72°C, followed by an extension of 10 min at 72°C. These PCR conditions were optimized for the utilization of primers of a length of 30 nucleotides. The Arrow Taq™ was demonstrated to possess more specificity and sensitivity than the Taq™ polymerase
Amplification and Detection of wtAdδ DNA
[0062] The wtAdδ DNA, was used as a substitute for RCA contamination. The wtAdδ was diluted to obtain a range representing 0 to 108
copies. A first round of PCR amplification was performed as described above, for the various primer pairs described in Table 2. A portion of the PCR products of these first rounds (10 μl) was separated on a 1% agarose gel, stained with ethidium bromide and then visualized under ultraviolet light.
Table 2. Detection Sensitivity of a First PCR Amplification Round with Primer Pairs of the Present Invention
Approximate number of copies
Amplification N° Primer pairs detected
1 E1-1-NESTED/E2-1-NESTED 107
2 E1-2/E2-1 107
3 E1-1/E2-1 >105
4 E1-2/E2-2 >105
5 E1-2/E2-3 105
6 E1-5/E2-2 105
7 E1-1/E1-3 10s
8 E1-1-NESTED/E2-3 105
9 E1 -NESTED/E2-NESTED 105
10 E1-1-NESTED/E2-1 -NESTED 105
11 E1-4/E2-2 104
12 E1-4/E2-3 104
13 E1-4/E2-2 104
14 E1-4/E2-1-NESTED 104
15 E1-1-NESTED/E2-2 104
16 E1-2/E2-1 -NESTED 104
17 E1-5/E2-3 103
18 E1-5/E2-1 -NESTED 103
The second round of PCR amplification (or nested-PCR) was performed using an aliquot of 2 μl of the amplification products of the first round of PCR amplification. The amplification products of the first PCR amplification round used as templates for the second PCR amplification round are listed in the column entitled "Outer Primer Pairs" of Table 3. They are, those used in Amplifications no. 3, 2, 11 and 17, respectively of Table 2. Ten microlitres of the amplification product of the second round of amplification were separated on a 1% agarose gel, stained with ethidium bromide and then visualized under UV light.
Table 3. Detection Sensitivity of a Second PCR amplification Round with Primer Pairs of the Present Invention
Approximate number of copies Amplification N° Inner Primer Pairs Outer Primer Pairs detected
19 E1-2/E2-3 E1-1/E2-1 104
20 E1-5/E2-3 E1-2/E2-1 100
21 E1 -NESTED/E2-NESTED E1-4/E2-2 10
22 E1-NESTED/E2-NESTED E1-5/E2-3 1
23 E1-4/E2-NESTED E1-5/E2-3 1
24 E1-4/E2-1 -NESTED E1-5/E2-3 1
25 E1-4/E2-2 E1-5/E2-3 1
Comparison of Primer Pairs Performance
[0063] The primer pairs E1-δ/E2-3 and E1-57E2-1 -NESTED detected around 1 ,000 copies of wtAdδ DNA in the first PCR amplification round of experiments described above (see for example 17 and 18 in Table 2). In a second round of PCR amplification, using the amplification product of the outer primer pair E1-δ/E2-3 as a template, the inner primer pairs E1-NESTED/E2- NESTED, E1-4/E2-NESTED, E1-4/E2-1 -NESTED AND E1-4/E2-2 detected a single copy of wtAdδ (see for example "Amplifications No." 22- 2δ in Table 3).
[0064] The fact that the different inner primer pairs used in amplifications no. 22, 23, 24 and 2δ (Table 3) with the outer primer pair E1-6/E2- 3 had similar detection sensitivities (i.e. were able to detect a single copy of wtAdδ) is interpreted as indicating that the choice of the outer primer pair used has more impact on the PCR amplification sensitivity of the present invention than that of the inner primer pair used. However, a number of outer primer pairs can be used in the nested PCR of the present invention without substantially decreasing its detection sensitivity. For instance, the outer primer pairs used in amplifications no. 17 and 18 in Table 2 achieved the same detection sensitivity. The choice of an inner primer pair seems to be even less limiting for the detection sensitivity of the nested-PCR. Any inner primer pair within the region covered by the outer primer pairs of the invention could thus be used without substantially modifying the detection sensitivity of the nested PCR of the present invention.
EXAMPLE 2 Asymmetrical nested PCR
[0065] Two asymmetrical PCR reactions were performed with primers of the present invention with parameters described in Example 1. The first one was performed with the outer primer pair E1-5 long/E2-2 and the inner primer E2- 1 -nested long. This reaction was able to detect 100 copies of wtAdδ in a
background of 1 x 1010 of pAd-Null. The second one was performed with outer primers E1-δ long/E2-2 long and inner primer E1-4 long. This reaction was able to detect 1 copy of wtAdδ in a background of 1 x 1010 of pAd-Null. Asymetrical PCR reactions can thus be used effectively within the scope of the present invention.
EXAMPLE 3
Determination of the performance of an outer primer pair of the prior art with inner primer pairs of the present invention
[0066] The Lochmuller primer pair, namely E1-1 (SEQ ID NO: 1) and
E2-1 (SEQ ID NO: 24), respectively in Table 1 were used with inner primer pair of the present invention E1-4 long(SEQ ID NO: 8)/E2-1 -nested-long (SEQ ID NO:
17). They achieved a detection sensitivity of 1 ,000 copies wtAdδ in 1x1010 copies of pAd-Null. This reduced detection sensitivity as compared to the best combination of primers of the present invention is presumably due to the distance between of the outer primers and the resulting length of the amplification product.
EXAMPLE 4
Determination of optimal primer pairs to avoid non-specific amplification in the presence of recombinant adenovirus
[0067] To improve the specificity of the PCR-based assay, first PCR amplification round reactions were performed in the presence of 1x1010 copies of pAd-Null, acting as recombinant adenovirus (rAd) DNA without wtAdδ contamination. pAd-Null is a plasmid containing the total genome of a rAd that does not express any exogenous protein. It is schematically illustrated in Figure
1A. The amplification products of this experiment (10 μl) were separated on a 1% agarose gel, stained with ethidium bromide and then visualized under UV light.
Table 4 indicates for each pair of primers tested whether the amplification round generates non-specific amplification. Primer pairs E1-δ long(SEQ ID NO: 4)/E2-2 long(SEQ ID NO: 19) and E1-4 long(SEQ ID NO: 8)/E2-1 -nested-long (SEQ ID
NO: 17) generated a relatively small amount of non-specific amplification products and were therefore selected as outer and inner primer pairs, respectively, for further exemplary experiments.
Table 4. Determination of optimal inner and outer primer pairs to avoid nonspecific amplification in the presence of recombinant adenovirus DNA.
Relative amplification of non-
Amplification N° Primer pairs specific product (+)
26 E1-5 long/E2-3 long
27 E1-5 long/E2-2 long
28 E1-5 long/E2-1 nested long
29 E1-5 long/E2-nested long ++
30 E1-4 long/E2-3 long ++
31 E1-4 long/E2-2 long
32 E1-4 long/E2-1 nested long +
33 E1-4 long/E2-nested long ++
34 E1-1 nested long/E2-3 long +++++
35 E1-1 nested long/E2-2 long +++++
36 E1-1 nested long/E2-1 nested long +++++
37 E1-1 nested long/E2-nested long +++++
38 E1 nested long/E2-3 long +++++
39 E1 nested long/E2-2 long +++++
40 E1 nested long/E2-1 nested long +++++
41 E1 nested long/E2 nested long +++++
42 E 1 -5-short/E2-nested-short ++++
43 E1-5-short/E2-1 -nested ++++
44 E1-5-short/E2-2 +++
45 E1-5-short/E2-3 short +++
46 E1-4/E2-nested ++++
47 E1 -4/E2-nested-short ++++
48 E1-4/E2-1-nested ++++
49 E1-4/E2-2 ++++
50 E1-4/E2-3 short +
51 E1-1 nested/E2-nested +++
52 E1-1 nested /E2-nested-short ++++
53 E1-1 nested /E2-1 -nested +++
54 E1-1 nested /E2-2 +++
55 E1-1 nested /E2-3 short +++
56 E1 nested/E2-nested +++
57 E1 nested /E2-nested-short +++
58 E1 nested /E2-1 -nested +++
59 E1 nested /E2-2 +++
60 E1 nested /E2-3 short
61 E1 nested short E2-nested +
62 E1 nested short/E2-nested-short +
63 E1 nested short/E2-1 -nested +
64 E1 nested short/E2-2 ++
65 E1 nested short/E2-3 short +
Detection of wtAdδ in the presence of pAd-Null (recombinant substitute) or AdδCMVrev-GFP (recombinant adenovirus)
[0068] To improve the sensitivity and the specificity of the PCR-based assay, PCR (first PCR amplification round) and nested-PCR (second PCR amplification round) reactions were performed in the presence of pAd-Null, acting as recombinant adenovirus or AdδCMVrev-GFP (this plasmid is schematically illustrated in Figure 1A) amplified on an E1 -complementing cell line which significantly reduces the presence of RCA (BMAdEI cells, U.S. Pat. No.
δ,891 ,690). Indeed, the BMAdEI cells are devoid of homologous sequence to the adenoviral backbone. All DNA samples used in the first round of PCR amplification were mixed with 1x1010 copies of pAd-Null or AdδCMVrev-GFP. The outer and inner primer pairs E1-δ long/E2-2 long and E1-4 long/E2-1 -nested long, respectively, were used for the first and the second rounds of PCR amplification. The amplification was performed as described above except for the addition of pAd-Null or AdδCMVrev-GFP DNA in the first round of amplification.
[0069] The amplification products of both rounds of PCR amplification with the pAd-Null appear in the ethidium-bromide stained agarose gel of Figure 2. In Figure 2, first-round amplifications were performed in the presence of pAd-Null acting as recombinant adenovirus. Both rounds of amplification were carried-out using Taq DNA polymerase.
[0070] A 1281 bp amplification product was identified after the second round of amplification in the amplified mixture of a single copy of wtAdδ among 1x1010 copies of pAd-Null (lane 11 , Figure 2), no amplification product was observed in the amplified mixture of 0.1 copy of wtAdδ (lane 10, Figure 2), in the amplified mixture of HEK 293 DNA with 1x1010 copies of pAd-Null (lane 16, Figure 2); or in the negative control (1x1010 copies of pAd-Null) (lane 17, Figure 2). Additional experiments demonstrated that a single copy of wtAdδ could be detected in a background of 3 x 1010 copies of pAd-Null (data not shown).
[0071] The amplification products of the second round of PCR amplification with AdδCMVrev-GFP, generated and amplified in BMAdEI cells, appear in the ethidium-bromide stained agarose gel of Figure 3.. Both rounds of amplification were carried-out using Arrow Taq™ polymerase.
[0072] Detection sensitivities comparable to those obtained with wtAdδ in a background of pAd-Null were achieved with a wtAdδ in a production of AdδCMVrev-GFP. Indeed, one copy of wtAdδ was detected among 1x1010 copies of AdδCMVrev-GFP (lane 3, Figure 3). Again, no amplification product was observed in the amplified mixture of 0.1 copy of wtAdδ among 1x1010 copies of AdδCMVrev-GFP (lane 2, Figure 3), or with the negative control (1x1010 copies of AdδCMVrev-GFP) (lane 10, Figure 3). These experiments suggest that 1 copy of wtAdδ can be routinely detected among 1x1010 copies of either pAd-Null or recombinant adenoviruses (AdδCMVrev-GFP).. It should be noted that non-
specific amplification could be observed in the second amplification round depending on the homology of the DNA cloned into the recombinant adenoviruses. This non-specific amplification does not hamper however an appropriate determination of RCA contamination.
[0073] These results therefore demonstrate that the sensitivity of the method of the present invention is the same whether it is applied to a background of substitute recombinant (i.e. pAd-Null) or to a real adenovirus recombinant production (AdδCMVrev-GFP).
Detection of RCAs in a production of AdδCMVrevGFP [0074] AdδCMVrev-GFP was generated on a BMAdEI cell line.
Once constructed, the recombinant adenovirus was expanded through 5 passages on HEK 293 cells and was then purified. The concentration of viral DNA (number of copies/μl) was determined using the above-described formula. To determine the amount of RCA present in this preparation, various amounts of viral DNA were used (1x106, δx106, 1x107, δx107, 1x108, 1x109 and 1x1010 copies). The outer and inner primer pairs E1-δ long/E2-2 long and E1-4 long/E2- 1 -nested long, respectively, were used for the first and the second rounds of PCR amplification, and performed as described above. The amplification products of the second round of PCR amplification appear in the ethidium-bromide stained agarose gel of Figure 4. Both rounds of amplification were carried-out using Arrow Taq™ polymerase.
[0075] As shown in Figure 4, the amount of RCAs in this preparation of recombinant adenovirus was determined by the method of the present invention to be close to 1 RCA in 1x107 AdδCMVrevGFP. No amplification occurred beyond this dilution (HEK 293 DNA and absence of DNA).
Application of the method of the present invention to other adenovirus serotypes
[0076] Although the detection method of the present invention was demonstrated with adenovirus serotype δ, it readily applies to other adenovirus serotypes having a significant homology with Adδ. The full
nucleotide sequence of Adδ may be found in Genebank under the access number M73260.
[0077] Indeed, primer pairs of the present invention could usefully hybridize to RCAs originating from the production of recombinant adenoviruses of other serotypes. For instance, the sequence of outer primers E1-5 long (SEQ ID NO: 4) and E2-2 long (SEQ ID NO: 19) of the present invention possess identities of 96.6% with the corresponding sequences in adenovirus serotype 2 (Ad2); similarly, E1-4 long ((SEQ ID NO: 8) and E2-1- nested-long (SEQ ID NO: 17) are identical to corresponding sequences in Ad2. The full nucleotide sequence of Ad2 may be found in Genebank under the access number j01917.
[0078] Although the present invention has been described hereinabove by way of preferred embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims.
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