Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-10-05
2001-05-15
Whisenant, Ethan (Department: 1655)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S024300, C435S006120, C435S091200
Reexamination Certificate
active
06232455
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to HIV. In particular the invention relates to oligonucleotides and methods for detecting HIV 1 and HIV 2.
BACKGROUND OF THE INVENTION
Viruses classified as HIV contain RNA as their genetic information and the infectivity of HIV depends upon the virus's ability to insert its genetic information into the DNA of a host. In order to insert its genetic information and therefore successfully infect a host, an HIV virus must convert its genetic material (RNA) into DNA so that the HIV genetic information is compatible with that of the host. Apparently, HIV is successful at converting its RNA into DNA, given the prevalence of AIDS. However, while the virus may successfully convert RNA into DNA, the conversion is seldom accurate. In other words, the DNA copy of the viral RNA is not always exact and the DNA copy can diverge from the viral RNA by several base pairs. Hence, while a host initially may be infected with a single virus particle, after several rounds of replication, the host may be infected with a genetically diverse population of viruses.
Although HIV is not uniformly classified, it is generally accepted that HIV 1 and HIV 2 are different viruses. Within each of these viral classifications are several groups or subtypes. For example, within the classification HIV 1, there is “group M”, which is further classified into subtypes A-F, and “group O”. HIV 2, on the other hand, contains subtypes A-E. Subtypes of HIV 1 and HIV 2 are broken down even further into categories to numerous to mention in this context. However, it is worth mentioning that all of these divisions are based upon the genetic variance between the viruses and, according to taxonomic theory, many of these viruses are the progeny of a single virus. Hence, the numerous HIV types and subtypes demonstrate the highly mutable nature of HIV and the genetic variability of the HIV genome.
The genetic variability of the virus can be attributed to the inefficiency with which the virus converts its RNA into DNA, as mentioned above. Another theory concerning the genetic variability of the virus is that hosts can be infected with multiple different populations of HIV (which as mentioned above, can arise out of an infection by a single virus) and through the course of replication and packaging of the viral genetic information, pieces of one viral genome can be recombined with pieces of another viral genome. Hence, upon packaging of the recombined genome, a genetically distinct virus is formed. Regardless of the manner by which the virus mutates, it is clear that viruses known as HIV have genomes that are highly mutable and are therefore constantly changing. This presents those searching for methods of detecting the virus based upon its genetic information with a constantly moving target.
Although it is known that certain regions of the HIV genome are conserved, this is not to say that these regions are immune from mutation particularly if mutations in these regions do not effect the structure of a protein encoded by these regions. Hence, developing reagents and methods for detecting HIV based upon its genetic information is a continuing challenge.
SUMMARY OF THE INVENTION
The present invention provides reagents useful for detecting HIV (that is the various subtypes of HIV 1 and HIV 2) based upon the genetic information of these viruses. In particular, the reagents are in the form of primer and probe sets which can be employed according to nucleic acid amplification procedures to specifically and sensitively detect various subtypes of HIV 1 and HIV 2. Preferably, the primer/probe sets herein provided comprise two primer sequences and one probe sequence and are employed according to a reverse transcriptase (RT) PCR format.
Primer/probe sets of the invention which can be employed to detect HIV 1 are designated herein as primer/probe set 1 (SEQ. ID. NOs. 2, 3 and 4); primer/probe set 2 (SEQ. ID. NOs. 5, 6 and 7); primer/probe set 3 (SEQ. ID. NOs. 8, 9, 10 and 11); and primer/probe set 4 (SEQ. ID. NOs. 12, 13 and 14). Primer/probe sets which can be employed to detect HIV 2 are designated herein as primer/probe set 5 (SEQ. ID. NOs. 16, 17 and 18); primer/probe set 6 (SEQ. ID. NOs. 19, 20 and 21 or 22); primer/probe set 7 (SEQ. ID. NOs. 23, 20 and 21); and primer/probe set 8 (SEQ. ID. NOs. 16, 24 and 18).
The method for detecting HIV will generally comprise the steps of (a) forming a reaction mixture comprising nucleic acid amplification reagents, at least one primer/probe set mentioned above, and a test sample containing an HIV target sequence; (b) subjecting the mixture to amplification conditions to generate at least one copy of a nucleic acid sequence complementary to the target sequence; (c) hybridizing the probe to the nucleic acid sequence complementary to the target sequence, so as to form a hybrid comprising the probe and the nucleic acid sequence complementary to the target sequence; and (d) detecting the hybrid as an indication of the presence of HIV in the test sample.
The preferred RT PCR format will comprise the same steps as mentioned above but the amplification reagents will comprise an enzyme having reverse transcriptase activity. In addition, according to any of the methods provided herein, step (b) can be repeated multiple times to increase the number of target sequence copies. It will be understood by those skilled in the art that step (b) can be repeated through thermal cycling the reaction mixture.
DETAILED DESCRIPTION OF THE INVENTION
The primer/probe sets provided herein comprise two primers and at least one probe. These primer/probe sets can be employed according to nucleic acid amplification techniques. Hence, the primers in any particular primer/probe set can be employed to amplify a target sequence. In most cases, the probe hybridizes to the copies of the target sequence generated by one of the primers and generally facilitates detecting any copies of the target sequence generated during the course of the amplification reaction. All of the primer/probe sets can be employed according to nucleic acid amplification procedures to specifically and sensitively detect HIV 1 group M and group 0, as well as the various subtypes of HIV 2. Primer/probe sets for detecting HIV 1 subtypes are presented below in Table 1 and primer/probe sets for detecting HIV 2 subtypes are presented in Table 2 below.
TABLE 1
Primer/
SEQ. ID.
Probe Set
Sequence (5′-3′)
NO.
1
ATTCCCTACAATCCCCAAAGTCAAGGAGT
2
CCTGCACTGTACCCCCCAATCC
3
ACAGCAGTACAAATGGCA
4
2
GGAGCAGAAACTTTCTATGTAGATGG
5
CATATTGTGAGTCTGTTACTATGTTTACT
6
TAGGAAAAGCAGGATATG
7
3
GGTACGTATTAGTAGGACCTACACCTGT
8
GGCCATTGTTTAACTTTTGGGCCATCCA
9
ATTAGTCCTATTGAAACTGT
10
ATAAGCCCCATCGCC
11
4
CCTAGTATAAACAATGAGACACCAGG
12
GATCCTACATACAAGTCATCCATGTA
13
GGATGGAAAGGATCACCA
14
TABLE 2
Primer/
SEQ. ID.
Probe Set
Sequence (5′-3′)
NO.
5
ACTGATGGCAGTTCATTGCATGAATTTTAAAAG
16
TTCCACAGCTGATCTCTGCCTTCTCTG
17
CAGAACAAGAAATACAATTC
18
6
CCTCAATTCTCTCTTTGGAAAAGACC
19
AAATGTTGATTGGGGTATCTCCTGTC
20
CCAAAAATAGTAGTAGGGGG
21
ATAGTAGCAGGAATAGA
22
7
CAATAGTAGCAGGAATAGAGTTAGG
23
AAATGTTGATTGGGGTATCTCCTGTC
20
CCAAAAATAGTAGGGGG
21
8
ACTGATGGCAGTTCATTGCATGAATTTTAAAAG
16
CACAGCTGATCTCTGAATTCTGTAATAGAC
24
CAGAACAAGAAATACAATTC
18
As alluded to above, primers included in the primer/probe sets can be used to prime synthesis of copies of an HIV 1 target sequence in the case of SEQ ID NOs. 2, 3, 5, 6, 8, 9, 12, and 13; and copies of an HIV 2 target sequence in the case of SEQ ID NOs. 16, 17, 19, 20, 23 and 24. The remaining SEQ ID NOs. (SEQ ID NOs. 4, 7, 10, 11, 14, 18, 21, and 22), hybridize with the amplification products of either or both of the primer sequences found in the same primer/probe set. For example, primer/probe set 6 is specific for HIV 2 insofar as SEQ. ID. NOs. 19 and 20 prime synthesis of the HIV 2 target sequence and SEQ. ID. NOs. 21 and 22 hybridize with the amplification products produced by SEQ. ID. NOs. 19 and 20. Hence, the probe sequences are also specific for the various subtypes of HIV 1 or HIV 2.
Primer sequences g
Abravaya Klara
Cygan Esping Claudia A.
Gorzowski Jacek J.
Hoenle Robert J.
Kroeger Paul E.
Abbott Laboratories
Whisenant Ethan
Yasger Paul D.
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