Compositions and methods for the rapid differential detection of zika virus

A technology for Zika virus and virus genome, which is applied in the field of compositions and methods for rapid differential detection of Zika virus, and can solve problems such as methods or assays without differential detection of arboviruses, difficulties in differential diagnosis and the like

Active Publication Date: 2019-01-04
THE TRUSTEES OF COLUMBIA UNIV IN THE CITY OF NEW YORK
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Zika virus and dengue virus infections, as well as infections from other viruses, namely West Nile virus (a flavivirus) and chikungunya virus (an alphavirus), present with similar clinical symptoms, making differential diagnosis difficult
[0007] To date, there are no methods or assays for the differential detection of these four related arboviruses

Method used

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  • Compositions and methods for the rapid differential detection of zika virus
  • Compositions and methods for the rapid differential detection of zika virus
  • Compositions and methods for the rapid differential detection of zika virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0137] Example 1 - Primers and Probes for Assays

[0138] The following probes and primers were used in the examples.

[0139] Table 2 - Primers and Probes

[0140]

[0141]

[0142] All primers and probes are generic. Zika primers and probes target the 3'UTR of ZIKV genomic RNA (SEQ ID NO: 1-3). West Nile virus primers and probes target the NS5 gene of WNV genomic RNA (SEQ ID NO: 4-6). Dengue virus primers and probes target the 3'UTR of DENV genomic RNA (SEQ ID NO: 7-10). The gene target of the Chikungunya virus primers and probes was the NSP2 gene (SEQ ID NO: 11-13) of the CHIKV genomic RNA.

[0143] SEQ ID NO: 14-16 are control primers and probes. The gene target is human ribonuclease P subunit p30 mRNA.

Embodiment 2

[0144] Example 2 - Reagents and materials for multiplex assays

[0145] Probes and primers listed in Table 2 are supplied in lyophilized form in one vial of each virus to be prepared in 100 μl H 2 Recovery in O. Vials labeled ZIKV-MIX contained primers and probes SEQ ID NO: 1-3. Vials labeled WNV-MIX contained primers and probes SEQ ID NO: 4-6. Vials labeled DENV-MIX contained primers and probes SEQ ID NO: 7-10. Vials labeled CHIK-MIX contained primers and probes of SEQ ID NO: 11-13. Vials labeled RP-MIX contained primers and probes of SEQ ID NO: 14-16. See Tables 1 and 2.

[0146] Additional reagents included assay controls in labeled vials, as shown in Tables 1 and 3.

[0147]All assay controls should be included on each plate and tested concurrently with the clinical samples. These assay controls include extraction control reagents, positive controls for each virus, etc.

[0148] The extraction control was a human sample control (HSC, extraction control) and was a...

Embodiment 3

[0162] Example 3 - Nucleic acid extraction and storage

[0163] manufacturer to The instructions generally follow the extraction process and machine interface operation.

[0164] Briefly, a sample input volume of 250 μL and an elution volume of 50 μL total nucleic acid were used.

[0165] Thaw previously collected and frozen biological samples (including urine and serum) on ice. Pipette 250 μL sample and mix with 750 μL Lysis buffer was mixed and incubated at room temperature for 10-15 min (standard ratio 1:3 sample:lysis buffer).

[0166] 50μL Magnetic silica beads were added to the sample + lysis buffer mixture and mixed well by pipetting sample and magnetic silica beads up and down. Transfer samples to in the sample box. The magnetic silica cartridge is loaded into the machine along with the pipette cartridge and the sample is processed.

[0167] When the run is complete, transfer 50 µL of the eluted total nucleic acid to a clean reaction tube for the PCR assa...

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Abstract

This invention relates to compositions and methods for the differential detection of multiple viruses using a one-step assay. The viruses to be detected include Zika, West Nile, dengue (genotype 1-4)and chikungunya viruses. In particular, the invention relates to a method of and assay for differential detection of the viruses using specific primers and probes designed to detect and differentiatebetween the viruses.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to US Patent Applications Serial No. 62 / 309,770, filed March 17, 2016, and Serial No. 62 / 431,550, filed December 8, 2016, which are hereby incorporated by reference in their entirety. field of invention [0003] The present invention relates to compositions and methods for the differential detection of multiple viruses using a one-step assay. Viruses to be tested include Zika virus, West Nile virus, dengue virus (genotypes 1-4), and chikungunya virus. In particular, the invention relates to methods for the differential detection of viruses using specific primers and probes designed to detect and differentiate between viruses. Background of the invention [0004] Zika virus (ZIKV) is an emerging mosquito-borne virus affecting several major continents, including Africa, Asia and the Americas, and is considered a global emergency by the World Health Organization (WHO). ZIKV infection is ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/686C12Q1/70
CPCC12Q1/701C12Q2600/112C12Q2600/16Y02A50/30C12Q1/6806C12Q1/686C12Q1/6888C12Q2600/166
Inventor W·I·利普金N·米什拉T·布里斯R·托卡兹
Owner THE TRUSTEES OF COLUMBIA UNIV IN THE CITY OF NEW YORK
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