Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
1999-08-13
2001-12-25
Whisenant, Ethan (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S006120, C536S023100, C536S024300
Reexamination Certificate
active
06333150
ABSTRACT:
FIELD OF THE INVENTION
The present invention is directed to an isothermal transcription based assay for the detection and genotyping of dengue virus. The present invention is also directed to oligonucleotides for amplifying dengue RNA and for type-specific probes used in the detection of the amplification product.
BACKGROUND OF THE INVENTION
Dengue virus infection is an arthropod-borne viral disease which has very high morbidity and mortality in humans. Dengue is a member of Flaviviridae, and utilizes an 11 kb single stranded, positive RNA genome. The genome encodes 3 proteins and contains additional non-coding regions at the 5′ and 3′ ends.
There are currently four different dengue virus subtypes known, which are distributed among geographically distinct tropical and subtropical regions. All four subtypes can cause an array of maladies, ranging from an acute, self limited illness (dengue fever, DF) to the more severe and potentially fatal dengue hemorrhagic fever or dengue shock syndrome (dengue hemorrhagic fever, DHF). As many as 50 million human cases occur annually, with an estimated 10,000 infant deaths due to the hemorrhagic form of dengue. Immunity to one serotype does not protect against infection by the others. In fact, sequential infection by another serotype substantially increases the probability of developing DHF (Rico-Hesse et al, Virology 230, 244-251 (1997)). For this reason it may be critical to be able to determine the genotype of the dengue virus for management of the disease.
Diagnosis and management of the disease, as well as vector surveillance and epidemiological studies, would be facilitated by a rapid and sensitive assay for the specific type of dengue involved. Extensive cross reactions among the flaviviruses and the existence of four distinct dengue virus serotypes makes serotype identification difficult. Currently, the most reliable method for identification involves isolation of the virus in a sensitive host followed by serotype identification using reference antisera or monoclonal antibodies. This method usually takes two or more weeks, however. Alternatively, a four-fold or greater increase in antibody titer by standard serological tests can be used, but generally requires paired samples. An ELISA for the detection of dengue virus-specific IgM in patient serum has also been used in diagnosis; however, this assay has been shown to be of very limited sensitivity.
As a consequence of the limitations of the above methods, several attempts have been made to devise RT-PCR based assays for the detection and genotyping of dengue virus infection (Henchal et al,
Am. J. Trop. Med. Hyg
., 45(4), 1991, pp. 418-428; Lanciotti et al,
Journal of Clinical Microbiology
, 30(3), March 1992, p. 545-551; Chungue et al,
Journal of Medical Virology
, 40:142-145,1993; Vorndam et al,
Journal of Virological Methods
, 48(1994) 237-244; and Sudiro et al,
Am. J. Med. Hyg
., 56(4), 1997, pp. 424-429). These assays have relied on alternative strategies, including universal or specific primer amplification and probe detection, differential nested amplification, and RFLP analysis of the PCR products. To date, isothermal amplification methods for dengue RNA have not been reported.
SUMMARY OF THE INVENTION
The present invention provides isothermal transcription based amplification assays for the detection and genotyping of dengue virus. The detection assay may use primer pairs and probes for the envelope gene of dengue virus type 2. In another embodiment of the invention, primer pairs and probes from the 3′ non-coding region of the virus are used for the detection and genotyping of dengue virus.
Amplification in an isothermal transcription based amplification system is achieved through the coordinated activities of three enzyme activities (reverse transcriptase, RNase H, and RNA polymerase) and two DNA oligonucleotides (referred to herein as primers) specific for the target sequence. The method starts with an RNA template and alternately synthesizes DNA and RNA. Using an RNA template, a primer, and reverse transcriptase, an RNA/DNA hybrid is generated. The RNA is degraded from the hybrid by the RNase H activity. A double stranded DNA is then generated by the reverse transcriptase using another primer, and then the double stranded DNA is used as template for large amounts of RNA synthesis by the RNA polymerase. One of the primers has, in addition to the sequences complementary to the template, additional sequences necessary for generating an RNA polymerase promoter and transcription initiation site which can be used by the RNA polymerase. The single stranded RNA product can be readily detected through the hybridization of an appropriately labeled oligonucleotide DNA probe, with or without an additional probe which can be used to immobilize the amplification product. Detection of an amplification product indicates that the target molecule (RNA) is present in the sample.
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E. Chungue et al.,Journal of Medical Virology, 40:142-145, 1993.
R. Lanciotti et al.,Journal of Clinical Microbiology, 30:3:545-551, 1992.
V. Vorndam et al.,Journal of Virological Methods, 48:237-244, 1994.
E. Henchal et al.,Am. J. Trop. Med. Hyg., 45(4):418-428, 1991.
T.M. Sudhiro,Am. J. Trop. Med. Hyg., 56(4):424-429, 1997.
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Wattre, Jan.-Feb. 1997, Annales De Biologi Clinique, vol. 55, (1), p. 25-31. (Abstract).
Hurteau Gregory J.
Lee Eun Mi
Romano Joseph W.
Akzo Nobel N.V.
Rothwell Figg Ernst & Manbeck p.c.
Whisenant Ethan
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