Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1998-01-08
2001-12-18
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S091300, C435S006120, C536S023100, C536S023720, C536S024300, C536S024330
Reexamination Certificate
active
06331417
ABSTRACT:
This invention relates to a method for detecting and quantifying the presence of human herpesvirus 7 (HHV-7) in a sample, and to nucleic acid sequences useful in such a method.
HHV-7 is a member of the family Herpesviridae and is related to but genetically distinct from human herpesviruses 6A and 6B (HHV-6A and HHV-6B). HHV-7 was first isolated in 1989 and subsequently found to be present in the saliva of from 75% to over 90% of healthy adult humans (Frenkel et al (1990): PNAS 87, 748-752; Wyatt et al (1991): J. Virol. 65(11), 6250-6265).
HHV-7 has been found to cause an exanthem subitum-type illness in some children, one symptom of this illness being the development of rashes and acute febrile illness. At present, other disease symptoms attributable to HHV-7 have not been identified but, by analogy with other human herpesviruses, HHV-7 can be expected to cause disease, especially in immunocompromised hosts, such as organ transplant recipients. Therefore, it is desirable to be able to detect and quantify the presence of HHV-7 in samples of human tissue and bodily fluids. Until now, methods of detecting HHV-7 have been serological in nature, relying on the use of antibodies to HHV-7 proteins. These methods are, however, unsatisfactory. This is because they fail to quantify HHV-7, or have low specificity for HHV-7 with respect to other viruses such as HHV-6A/B.
The inventors have identified a sequence of HHV-7 DNA that is unique to this virus and not found in other related viral genomes although it codes for a protein that has some sequence homology with the product of KA3L gene of HHV6. The unique sequence is a 193 base pair fragment of HHV-7 DNA, and was identified by digestion of purified HHV-7 DNA with a restriction endonuclease, followed by cloning the restriction fragments into a plasmid vector. Using the sequence of this 193 base pair “target” fragment, it has been possible to synthesise a variety of primers that hybridise to it over part of its length. These can be used in Polymerase Chain Reaction (PCR), including nested PCR reactions, to amplify the 193 base pair fragment.
The inventors have also prepared a mutant DNA control sequence based on the 193 base pair fragment. This construct has internal substitutions that provide an additional restriction site (for the restriction endonuclease SmaI) that is absent from the target sequence. Primers suitable for construction of the mutant sequence by PCR amplification have also been synthesised.
The mutant control sequence can therefore act as a target sequence mimetic for the 193 base pair fragment in any PCR reaction performed on a sample containing the 193 base pair sequence and the control sequence. This forms the basis of a method of detecting and quantifying the presence of the 193 base pair fragment in samples, for example samples of human tissue or bodily fluids, and thus quantifying HHV-7 in these samples. A known amount of the control sequence is added to the sample; a pair of primers is also added and PCR is performed. As the target and control sequences are very similar, and preferably identical at the primer binding sites, one pair of primers allows the amplification of both the 193 base pair target sequence and the control sequence by PCR. Cleavage of the mutant sequence with the restriction endonuclease SmaI, which does not cleave the 193 base pair wild-type sequence, followed by quantification of the cleavage products then allows the amount of the target sequence present to be determined as the amount of the control sequence added is known. This, in turn, allows the presence of the HHV-7 virus in the sample be quantified as each HHV-7 genome comprises one target sequence.
As the target sequence is unique to HHV-7, this method can be used to quantify HHV-7 accurately in samples, thus potentially providing information as to the likelihood of the sample donor suffering from the symptoms caused by the virus.
Accordingly, the invention provides:
An isolated nucleic acid molecule comprising (i) a primer portion consisting of a contiguous sequence of from 10 to 50 nucleotides capable of hybridising to (a) the target nucleic acid molecule represented by SEQ ID NO:1 or (b) to the complementary stand thereof; and optionally (ii) a further portion comprising from 1 to 25 nucleotides joined to and immediately 5′ to the 5′ end of the primer portion;
a pair of nucleic acid molecules as defined above wherein one nucleic acid molecule has a primer region of type (a) as defined above and the other nucleic acid molecule has a primer region of type (b) as defined above; and wherein the two primers in combination are capable of amplifying the target nucleic acid molecule represented by SEQ ID NO:1 or a section thereof in a polymerase chain reaction;
a pair of nucleic acid molecules as defined above that hybridise respectively to the target nucleic acid sequence of SEQ ID NO:1 and its complementary stand in such positions that the 5′ end of the each primer region hybridises at a location that is from 0 to 50 nucleotides 3′ to the 5′ end of the sequence of SEQ ID NO:1 or the complementary strand thereto;
A method of determining the amount of a target nucleic acid having the sequence shown in SEQ ID NO:1 in a sample which method comprises:
(i) mixing the sample with a predetermined amount of control nucleic acid;
(ii) bringing the mixture formed in (i) into contact with at least one nucleic acid primer as defined above that is capable of hybridising to the target nucleic acid and at least one primer as defined above that is capable of hybridising to the control nucleic acid;
(iii) performing a nucleic acid amplification reaction, which reaction requires the presence of the primers defined in (ii) to amplify the target nucleic acid sequence or a section thereof and the control nucleic acid or a section thereof;
(iv) determining the relative quantities of the amplified control and target nucleic acids; and
(v) calculating from the determination of (iv) the amount of target nucleic acid in the sample;
An isolated nucleic acid sequence selected from the sequences shown in SEQ ID NO:1 and SEQ ID NO:2, or the complementary strands thereof; and
A kit for quantification of human herpesvirus 7 (HV-7) in a sample by means of a method as defined above which kit comprises:
(i) a control nucleic acid as defined above;
(ii) one or more pairs of primers as defined above that are suitable for amplifying both the target and control nucleic acid sequences.
REFERENCES:
patent: WO-A-9201816 (1992-02-01), None
patent: WO-A-9400140 (1994-01-01), None
patent: WO-A-9410344 (1994-05-01), None
Journal Of Medical Virology, vol. 45, Mar. 1995, pp. 282-283, Portolani M. et al: “Isolation of human Herpesvirus 7 from an infant with Febrile Syndrome”.
Archives Of Virology, vol. 136, 1994, pp. 183-190, Yalcin S. et al: “Human herpesvirus 6 and human herpesvirus 7 infections in renal transplant recipients and healthy adults in Turkey”.
Pediatrics, vol. 95, No. 2, Feb. 1995, pp. 187-190, Asano Y. et al: “Clinical features and viral excretion in an infant with primary human herpesvirus 7 infection”.
Hepatology, vol. 19, No. 5, May 1994, pp. 871-875, Naito M. et al: “Serum hepatitus c virus RNA quantity and histological features of hepatitis c virus carriers with persistently normal ALT levels”.
Genetic Analysis Techniques And Applications, vol. 11, No. 1, 1994, pp. 1-6, Clementi M. et al: “Competitive polymerase chain reaction and analysis of viral activity at the molecular level”.
Medical Sciences, vol. 89, Nov. 1992, pp. 10552-10556, Proc. Natl. Acad. Sci. USA, Berneman Z. et al: “Human herpesvirus 7 is an T-lymphtropic virus and is related to, but significantly different from, human herpesvirus 6 and human cytomegalovirus”.
Medical Sciences, vol. 87, Jan. 1990, pp. 748-752, Proc. Natl. Acad. Sci. USA, Frenkel N. et al: “Isolation of a new herpesvirus from human DC4+T cells”.
Journal Of Virology, Nov. 1991, pp. 6260-6265, Wyatt L. et al: “Human Herpesvirus 7: Antigenic Properties and Prevalence in Children and Adults”.
Emery Vincent C
Griffiths Paul
Guzo David
Leffers, Jr. Gerald G.
Nixon & Vanderhye P.C.
Royal Free Hospital School of Medicine
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