Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-12-17
2001-10-23
Jones, W. Gary (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S022100, C536S024300
Reexamination Certificate
active
06306602
ABSTRACT:
FIELD OF THE INVENTION
The present invention is concerned with oligonucleotides that can be used in the amplification detection of human Cytomegalovirus (HCMV) mRNA. Furthermore a method for the diagnosis of HCMV disease is provided.
BACKGROUND OF THE INVENTION
Human Cytomegalovirus is an ubiquitous Herpes-type virus, having a double stranded DNA genome of about 240,000 nucleotides in length that infects 40-80% of humans before puberty. A prominent feature common to all herpesviruses is their establishment of lifelong persistence after infection and their ability to cause recurrent infection after reactivation (Stevens, J. G. Microbiol. Rev. 53, 318-332., 1989). HCMV also becomes latent after primary infection which often occurs without clinical symptoms. Even recurrent infection in most cases goes asymptomatic or leads to only mild disease in the immunocompetent host. However, in congenitally infected infants and immunocompromised patients, such as allograft recipients (Meyers, J. D., et al., J. Infect. Dis. 153, 478-488., 1986) or AIDS patients (Drew, W. L. J Infect. Dis 158, 449-456., 1988; Drew, W. L. Clin. Infect. Dis 14, 608-615., 1992), where the fine balance between the immune system and the latently existing virus is disturbed, HCMV may cause severe and sometimes life-threatening disease, including retinitis, gastrointestinal disorders, and encephalitis (Drew, 1992). Early administration of antiviral drugs like ganciclovir and foscarnet can have significant beneficial effects on the prognosis of a patient (Jahn, G. et al., Intervirology 35, 60-72., 1993; Schmidt, G. M. et al., N. Engl. J Med. 324, 1005-1011., 1991). Therefore, with the availability of clinically effective antiviral therapy, early and sensitive diagnosis is of significant importance.
CMV specific antibodies, in particular IgM antibodies, can be used as a marker for CMV infection, but are of limited value when it comes to discrimination between latent and active infections. Most viral detection methods currently employed do not unambiguously allow for prediction of whether a given infection will be symptomatic. Furthermore serological methods are indirect and often lack sensitivity. Viral culture is a more direct diagnostic parameter for CMV viremia. Although CMV culture from blood cells appeared to be indicative for an active CMV infection, the method does not enable rapid diagnosis and is technically difficult. Moreover, viral culture does not necessarily correspond to HCMV disease. A reliable relation between virus isolation from peripheral leukocytes and the appearance of clinical symptoms may not exist in some immunosuppressed patients (Delgado, R. et al., J Clin. Microbiol. 30, 1876-1878., 1992). Also urinary or pharyngeal shedding of the virus frequently occurs without clinical symptoms and organ involvement. Amplification of HCMV DNA in peripheral leukocytes by polymerase chain reaction (PCR), although a very sensitive technique for CMV viremia, is not usable as a marker of clinically symptomatic HCMV infection either. Due to the high sensitivity of enzymatic amplification, occasionally HCMV DNA was detectable in peripheral leukocytes without HCMV-related disease. Latent viral genomes may be detected by this technique or a patient may remain HCMV-DNA positive over a prolonged period of time after the disease has resolved (Jahn, G. et al., 1993; Zipeto, D. et al., J Clin. Microbiol. 30, 527-530., 1992; Delgado et al., 1992).
At the moment, the method of choice for the early diagnosis of acute symptomatic HCMV infection is the antigenemia assay based on immunological detection of the structural protein pp65 by using specific antibodies (Storch, G. A., et al., J. Clin. Microbiol. 32, 997-1003., 1994; Gerna, G. et al., J. Infect. Dis. 164, 488-498., 1991; Gerna, G., et al., J Clin. Microbiol. 30, 1232-1237.98., 1992). However, a matter of concern employing this method is its sensitivity. The number of pp65-positive cells in the early course of infection may be very low. Furthermore, in expressing cells stability of the pp65 antigen appeared to be limited (Chou, S., Curr. Opin. Infect. Dis. 5, 427-432., 1991) and sensitivity can be reduced due to the application of monoclonal antibodies rather than a pool of anti-pp65 antibodies that would recognize different epitopes of the protein.
Since viral replication requires transcription of mRNA species, the use of HCMV mRNA detection as a marker for active CMV infection was investigated (Bitsch, A. et al., J Infect. Dis 167, 740-743, 1993.
Recently, HCMV infections were examined on the transcript level using RNA amplification (Bitsch, A. et al., 1993; Meyer, T. et al., Mol. Cell Probes. 8, 261-271., 1994; Gerna, G., et al., J Clin. Microbiol. 30, 1232-1237.98, 1993; Gerna, G., et al., J Clin. Microbiol. 30, 1232-1237.98, 1992). In principle, like detection of viral antigens, analysis of viral transcripts expressed in association with viral replication should allow reliable diagnosis of symptomatic infections.
More recently, the detection of certain mRNA's of HCMV i.e. based on IEA (immediate early antigen) and the matrix tegument protein pp67 mRNA have been described in U.S. Ser. No. 08/628,654. However, the novel oligonucleotides of the present invention have several advantages over the oligonucleotides as disclosed in the previous application. Comparison studies are presented in the experimental part of the description.
The sensitivity and reliability (robustness) of CMV mRNA detection is greatly dependent on the selection of the oligonucleotides used in the amplification, since there is sequence variation among strains of CMV potentially in every region of the genome. Ideally, primer selection should be based on knowledge of interstrain variability in candidate primer sequences and the consequences of mismatching at primer sites. (Chou S., J. of Clin. Microbiol., 2307-2310, 1992).
Therefore, the need exists for suitable oligonucleotides including nucleic acid sequences that can be used as in the amplification and subsequent detection of all strain variants of CMV.
SUMMARY OF THE INVENTION
The present invention is related to the detection of a certain late HCMV mRNA and provides oligonucleotides suitable for use in the amplification and subsequent detection of this mRNA. The binding sites of the oligonucleotides according to the present invention are located in the matrix tegument protein pp67 encoding gene sequence (UL65), which is expressed during the late phase of CMV infection.
Preferred oligonucleotides according to the present invention, correspond to part of a nucleic acid sequence encoding HCMV pp67, said oligonucleotide being 10-35 nucleotides in length and comprising at least a fragment of 10 nucleotides of a sequence selected from the group consisting of:
5′-GGGTCGATTCAGACTGA-3′ [SEQ.ID.No.: 10];
5′-CTGGAGATATATGTTGACCA-3′ [SEQ.ID.No.: 11],
5′-GGATTCGGACTTTCCGTTCGA-3′ [SEQ.ID.No.: 8], and
5′-CCAAAAAGCTAGCCGTCACG-3′ [SEQ.ID.No.: 12], or its complementary sequence.
A preferred embodiment of the present invention is directed to an oligonucleotide linked to a promoter sequence.
DETAILED DESCRIPTION OF THE INVENTION
An example of oligonucleotide pairs according to the present invention includes the following oligonucleotide sequences:
5′-aattctaatacgactcactatagggagaGGGTCGATTCAGACTGA-3′ [SEQ ID NO: 9] or
5′-aattctaatacgactcactatagggagaGGGTCGATTCGAGACCGA-3′ [SEQ ID NO: 5] in combination with 5′-GACCTGATATCCCTCCATATA-3′ [SEQ ID NO: 7]. (The T7 promoter sequence is shown, but may be replaced by any other suitable promoter sequence.) A probe that may be used for the detection of the amplificate generated using this pair may comprise an oligonucleotide consisting essentially of the following sequence: 5′-GGATTCGGACTTTCCGTTCGA-3′ [SEQ ID NO: 8]. Probes comprising said sequence are also part of the present invention.
For the pp67 gene sequence interstrain variations between CMV
Clasina Timmermans Eveline Catharina Anna
Sillekens Peter Theodorus Gerardus
Akzo Nobel N.V.
Jones W. Gary
Myers Bigel & Sibley & Sajovec
Tung Joyce
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