Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-04-05
2001-08-07
Whisenant, Ethan (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S023100, C536S024300
Reexamination Certificate
active
06270977
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the use of a nested polymerase chain reaction (PCR) method for detection of viral organisms, more particularly to highly specific PCR primers and optimized reaction conditions for detecting the pseudorabies virus.
BACKGROUND OF THE INVENTION
Pseudorabies virus is a herpes virus belonging to the genus alphaherpesvirinae. Although most warm-blooded species can serve as a host, either through natural or experimental means, this virus primarily resides within the swine population. In its active state, pseudorabies virus causes a disease that is generally fatal to young pigs. Those animals surviving infection become lifelong carriers, harboring the virus in an inactive, noninfectious state. The latent virus can be reactivated to its infectious state within these carriers and spread to other susceptible animals. This inactivation-reactivation cycle leads to perpetuation of the virus within a swine herd.
The polymerase chain reaction (PCR) method (Mullis and Faloona (1987)
Meth. Enzymol
. 155:335-350) provides for the enzymatic amplification of rare DNA sequences and/or minute quantities of DNA, enabling detection of rare DNA sequences not possible by other methods. The technique has been successfully utilized to detect a number of viral agents, such as human immunodeficiency virus (HIV) (Kwok et al. (1987)
J. Virol
. 61:1690-1694; Laure (1988)
Lancet
. 2:538-541; Murakawa et al. (1988) DNA 7:287-295; Ou et al. (1988)
Science
239:295-297); human papillomavirus (Shihata (1988)
J. Exp. Med
. 167:224-230); HSV (Rowley et al. (1990)
Lancet
. 335:440-441); human rhinovirus (Gama et al. (1988)
Nucleic Acids Res
. 16(19):9346); hepatitis B (Larzul et al. (1988)
J. Virol. Methods
20:227-237); human T-cell leukemia (Kwok et al. (1988)
J. Infect. Dis
. 158:1193-1197; Bhagavati et al. (1988)
N. Engl. J. Med
. 318:1141-1147); and pseudorabies virus (Maes et al. (1997)
Vet. Microbiol
. 55 (1-4): 13-27).
The PCR technique is currently the preferred method for detecting the presence of latent pseudorabies virus in animals in the absence of detectable infectious virus (Cheung (1995)
Am. J. Vet. Res
. 56(1):45-50). Initially this method was restricted to trigeminal ganglia tissues, which were believed to be the primary site of the latent virus. More recently, standard PCR and nested PCR methods have been used to demonstrate that tonsilar mucosal cells also harbor the latent virus (Cheung (1995)
Am. J. Vet. Res
. 56(l):45-50; Brown et al. (1995)
Am J. Vet. Res
. 56(5):587-594).
Detecting the latent virus in tonsilar tissues even with present PCR methods available in the art remains difficult. The frequency of viral DNA and RNA in tonsilar tissues is lower than that seen for trigeminal ganglion tissues. Yet use of tonsilar tissues is preferable, as suspected carriers of the virus do not have to be euthanized to obtain the sample.
Greater specificity for the latent pseudorabies virus is needed to enable more efficient detection, particularly in tonsilar tissues. To this end, the present invention provides for highly specific PCR primers that are used in an optimized nested polymerase chain reaction to detect a highly sensitive region of the pseudorabies virus gII gene.
SUMMARY OF THE INVENTION
The present invention provides for a highly sensitive nested polymerase chain reaction (PCR) method for detecting the presence or absence of the pseudorabies virus. The method targets a region of the pseudorabies virus genome referred to as the gII gene, which encodes a glycoprotein essential for the virus's replication in the host. Nucleotide sequences for highly specific primers derived from the gII region are also disclosed. These primers are used in the nested polymerase chain reaction to amplify targeted nucleotide sequences within the gII gene.
The method of the present invention comprises: a) isolating a purified sample nucleic acid mixture from tissue suspected of being infected with the pseudorabies virus; b) mixing said sample with highly specific oligonucleotide primers derived from a 674 base-pair region of the pseudorabies virus gII gene to amplify targeted nucleotide sequences within the isolated nucleic acid mixture; and c) analyzing the amplified nucleotide sequences to detect the presence or absence of targeted nucleotide sequences comprising a specific region of the gII gene, where the presence of said targeted nucleotide sequences indicates the presence of the virus.
Kits useful in the practice of the invention are also provided.
REFERENCES:
patent: 4683202 (1987-07-01), Mullis
patent: 5449765 (1995-09-01), Schreurs et al.
patent: 5487969 (1996-01-01), Eberle et al.
patent: 5545523 (1996-08-01), Batt et al.
patent: 5798265 (1998-08-01), Springer et al.
patent: 6068974 (2000-05-01), Klann
Robbins, et al., “The Pseudorabies Virus gII Gene Is Closely Related to the gB Glycoprotein Gene of Herpes Simplex Virus”Journal of Virology, vol. 61, No. 9, Sep. 1987, pp. 2691-2701.
Maes, et al., “Polymerase Chain Reaction Amplification of Pseudorabies Virus DNA from Acutely and Latently Infected Cells”Veterinary Microbiology, vol. 24, 1990, pp. 281-295.
Andrew K. Cheung, “Detection of the Large Latency Transcript of Pseudorabies Virus by RNA-PCR and Its Potential in Diagnosis”Journal of Veterinary Diagnostic Investigation, vol. 6, 1994, pp. 483-486.
Brown, et al., “Detection of Pseudorabies Viral DNA in Tonsillar Epithelial Cells of Latently Infected Pigs”American Journal of Veterinary Research, vol. 56, No. 5, May 1995, pp. 587-594.
Andrew K. Cheung, “Investigation of Pseudorabies Virus DNA and RNA in Trigeminal Ganglia and Tonsil Tissues of Latently Infected Swine”American Journal of Veterinary Research, vol. 56, No. 1, Jan. 1995, pp. 45-50.
Maes, et al., “Recent Developments in Latency and Recombination of Aujeszky's Disease(Pseudorabies)Virus”Veterinary Microbiology, vol. 55, 1997, pp. 13-27.
L.W. Enquist, PubMed Nucleotide Query, Accession No. M17321, Nov. 1987, pp. 1-3 (Computer-readable sequence), as published in Robbins, et al., “The Pseudorabies Virus gII Gene Is Closely Related to the gB Glycoprotien Gene of Herpes Simplex Virus”Journal of Virology, vol. 61, No. 9, Sep. 1987, pp. 2691-2701.
Alston & Bird LLP
Encelle, Inc.
Whisenant Ethan
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