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Diagnostic assays for parvovirus B19

a parvovirus and diagnostic assay technology, applied in the field of parvovirus diagnostic assays, can solve the problems of unreliable serological diagnosis, impracticality of serological based tests, and difficult laboratory detection and isolation of the virus

Inactive Publication Date: 2005-10-06
CHIRON CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0012] The present invention is based on the discovery of unique primers and probes for use in nucleic acid-based assays, as well as on the development of a sensitive, reliable nucleic acid-based diagnostic test for the detection of parvovirus B19 DNA in biological samples from potentially infected individuals. The techniques described herein utilize extracted sample DNA as a template for amplification of conserved genomic regions of the B19 sequence using transcription-mediated amplification (TMA), as well as in a 5′ nuclease assay, such as the TaqMan™ technique. The methods allow for the detection of B19 DNA in viremic samples having viral titers as low as 103 virus particles / ml. Accordingly, infected samples can be identified and excluded from transfusion, as well as from the preparation of blood derivatives. The probes and primers described herein are also useful in, for example, standard hybridization methods, as well as in PCR-based techniques, nucleic acid sequence-based amplification (NASBA) and in assays that utilize branched DNA molecules.

Problems solved by technology

Human parvovirus B19 cannot be grown in conventional cell cultures making laboratory detection and isolation of the virus extremely difficult.
In addition, IgM-based diagnostic tests cannot detect the virus during the viremic stage of infection and once IgM antibodies are synthesized, they can remain in circulation for several months after the end of viremia.
The high prevalence of B19 antibodies in the normal population together with the fact that high viremia usually persists for only one week, make the use of serological based tests impractical.
In addition, in immunocompromised patients, serological diagnosis may be unreliable.
However, DNA hybridization techniques are time consuming and limited in use and PCR is impractical for screening large numbers of samples.

Method used

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  • Diagnostic assays for parvovirus B19
  • Diagnostic assays for parvovirus B19
  • Diagnostic assays for parvovirus B19

Examples

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Effect test

example 1

Parvovirus B19 Nucleic Acid Extraction for PCR

[0157] Human serum samples that had previously tested positive for human parvovirus B19 by either IgM or PCR tests were obtained from commercial sources and used to isolate DNA for subsequent PCR experiments. Samples were stored at −80° C. until used.

[0158] DNA was extracted from 0.2 mL of serum using the QIAamp DNA Blood Mini Kit (QIAGEN, Valencia, Calif.) following the manufacturer's specifications with the following considerations. Carrier DNA was added to the lysis buffer to enhance nucleic acid binding and yield. In particular, an amount of 5.6 μg per sample of poly-adenylic acid 5′ (Sigma, St. Louis, Mo.) or poly-da (Roche, Indianapolis, Ind.) was added. Additionally, parvovirus B19 DNA was eluted with 200 μL of buffer AE (Qiagen) instead of water.

example 2

Detection of Parvovirus B19 Nucleic Acid-Positive Samples by PCR

[0159] Two different PCR procedures were used to amplify parvovirus B19 fragments. One method, described in detail below, was used to amplify fragments of approximately 700 bp, 370 bp and 214 bp (see, FIG. 1). High Fidelity Expand PCR (Roche) was used to amplify fragments of approximately 4.7 kb. The approximately 700 bp fragment corresponds to nucleotide positions 2936-3635 of the parvovirus B19 genome described in Shade et al., J. Virol. (1986) 58:921-936. The approximately 370 bp occurs within the 700 bp fragment at nucleotide positions 3073-3442. The approximately 4.7 kb fragment is a 4677 nucleotide fragment corresponding to nucleotide positions 217-4893 of Shade et al., J. Virol. (1986) 58:921-936.

[0160] In order to amplify the B19 fragments of approximately 700 bp, 370 bp and 214 bp, the primers shown in Table 1 were used.

TABLE 1PCRGenomicPrimerSequenceproductregionVP-5AGGAAGTTTGCCGGAAGTTC370 bpVP1(SEQ ID NO:...

example 3

Cloning of Parvovirus B19 DNA Fragments

[0162] The PCR fragments were cloned into TOPO-TA vectors (Invitrogen, Carlsbad, Calif.). Cloning into these vectors is highly facilitated when the amplified DNA contains a single deoxyadenosine (A) at its 3′ end. Accordingly, a catalytic reaction to add the 3′ (A) overhead was used. The reaction mix contained 1.25 mM of dATP, 0.5 units of Taq polymerase (Perkin Elmer, Boston, Mass.) and proceeded at 72° C. for 15 min.

[0163] PCR fragments were cloned into the pCR2.1-TOPO vector using Invitrogen's TA cloning kit (TOPO™ TA CloningR Kit with One Shot TOP10 Electrocompetent Cells) following the manufacturer's specifications. Bacterial cells were incubated at 37° C. on Luria Broth plates containing ampicillin at 100 μg / ml, 0.66 mM IPTG and 0.033% X-Gal. A number of white colonies were inoculated in 4 mL of Luria-Broth ampicillin (100 μg / ml) and incubated overnight at 37° C. with shaking. Three mL of the overnight cultures were used to prepare plas...

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Abstract

Human parvovirus B19 primers and probes derived from conserved regions of the parvovirus B19 genome are disclosed. Also disclosed are nucleic acid-based assays using the primers and probes.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation of U.S. patent application Ser. No. 10 / 187,253, filed Jun. 28, 2002, from which application priority is claimed under 35 USC §120, and is related to provisional patent application Nos. 60 / 302,077, filed Jun. 28, 2001; 60 / 365,956, filed Mar. 19, 2002; and 60 / 369,224, filed Mar. 29, 2002, from which applications priority is claimed under 35 USC § 119(e)(1), all of which applications are incorporated herein by reference in their entireties.TECHNICAL FIELD [0002] The present invention pertains generally to viral diagnostics. In particular, the invention relates to nucleic acid-based assays for accurately diagnosing parvovirus B19 infection and to primers and probes for use in these assays. BACKGROUND OF THE INVENTION [0003] Human parvovirus B19 is a member of the family Parvoviridae, genus Erythrovirus and is a small 22-nm icosahedral nonenveloped virus with a linear single-stranded DNA molecule of approxi...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68C12N15/09C12N15/35C12Q1/70G01N33/569
CPCC12Q1/701C12N15/1131C12Q2600/112C12Q2600/166A61P31/12C12Q1/6888C12Q1/68C12Q1/6813C12Q1/70
Inventor PICHUANTES, SERGIOSHYAMALA, VENKATAKRISHNA
Owner CHIRON CORP
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