Methods for detecting norovirus

A detection method and virus gene technology, applied in the direction of viruses, viral peptides, antiviral agents, etc., can solve problems such as the difficulty in developing broad-spectrum protective human norovirus vaccines

Pending Publication Date: 2019-07-30
QUEST DIAGNOSTICS INVESTMENTS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Significant viral diversity makes it difficult to develop a broadly protective human norovirus vaccine

Method used

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  • Methods for detecting norovirus
  • Methods for detecting norovirus
  • Methods for detecting norovirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0158] Example 1: Using SIMPLEXA TM Direct real-time RT-PCR detection of norovirus genogroups

[0159] Real-time RT-PCR amplification and detection: Raw formed and unformed stool samples were suspended in sample buffer. For each reaction on a direct amplification plate (Focus Diagnostics, Inc., Cypress, California, USA), 50 μL or mg of each untreated stool sample was suspended in 2 mL of sample buffer (or similar ratio, That is, 25 μL or mg in 1 mL of sample buffer). Subsequently, 50 μL of each stool-sample buffer mixture was loaded into the sample port without a separate front-end sample preparation step. Load 50 µL of norovirus direct reaction mix consisting of the following: PCR buffer, DNA polymerase, reverse transcriptase, RNase inhibitor, BSA, dNTPs, magnesium chloride, potassium chloride into the reaction port , primers consisting of SEQ ID NO: 5, 6, 9 and 10 (each primer has a concentration of 750 nM), probes consisting of SEQ ID NO: 7, 8 and 11 (each probe has a ...

Embodiment 2

[0172] Example 2: Norovirus SIMPLEXA TM Comparison of direct real-time RT-PCR results with conventional real-time RT-PCR

[0173] Use Simplexa TM Norovirus direct assay evaluated norovirus in 144 de-identified clinical stool samples (47 GI positive, 43 GII positive and 54 norovirus negative from FocusDiagnostics: Reference Laboratory) previously tested using routine real-time RT-PCR. Such as virus. Simplexa TM The results of the norovirus direct assay were compared with the previous real-time RT-PCR results to determine the positive and negative coincidence rates. Conventional real-time RT-PCR employs a separate nucleic acid extraction step prior to the amplification step of real-time RT-PCR.

[0174] Table 3: Norovirus GI Concordance of Stool Samples

[0175]

[0176] a 5 samples were previously tested using conventional real-time RT-PCR and subsequently using Simplexa TM Norovirus direct assay to test. Using the CDC Norovirus Taqman assay in combination with Ro...

Embodiment 3

[0180] Example 3: Effects of Endogenous and Exogenous Interferants on the Sensitivity of Norovirus Multiplex Assays

[0181] Evaluation of the impact of potential endogenous and exogenous interfering substances on the sensitivity of a norovirus multiplex assay.

[0182] The interference group was artificially designed as GI.1 or GII.4 with 3 times the LoD concentration. Spike each substance into norovirus GI.1 or GII.4 artificial samples and use Simplexa TM Norovirus direct assay test. The concentrations of each interferent are listed in Tables 5-6. The set was created using pooled formed negative fecal matrices or pooled unformed negative fecal matrices.

[0183] Table 5: Exogenous Interferants Tested in Human Fecal Matrix

[0184]

[0185]

[0186] Table 6: Endogenous Interferants Tested in Human Fecal Matrix

[0187]

[0188] Results. No interference was detected in both formed and unformed fecal matrices with any of the exogenous interferents tested (listed...

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Abstract

The present disclosure provides methods and compositions for determining whether a patient exhibiting acute gastroenteritis will benefit from treatment with therapeutic agents that inhibit Norovirus genogroup I (GI) or Norovirus genogroup II (GII). The methods disclosed herein are based on detecting Norovirus genogroup I (GI) and Norovirus genogroup II (GII) in a stool sample without extracting viral nucleic acids from a clinical specimen prior to performing real-time reverse transcription PCR. Kits for use in practicing the methods are also provided.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of and priority to US Application No. 62 / 345,331 filed June 3, 2016, the contents of which are hereby incorporated by reference in their entirety. technical field [0003] The present disclosure provides methods and compositions for determining whether a patient with acute gastroenteritis would benefit from treatment with a therapeutic agent that inhibits norovirus genogroup I (GI) or norovirus genogroup II (GII). The method of the present technology is based on the detection of norovirus genogroup I (GI) and norovirus genogroup II in stool samples without extraction of viral nucleic acid from clinical samples prior to real-time reverse transcription PCR (RT-PCR) (GII). The disclosure also provides kits for practicing the method. [0004] Background of the invention [0005] The following description of the background of the disclosure is provided only to assist the reader in unders...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686A61K39/125C07K14/08A61P31/14
CPCC12Q1/68C12Q1/70A61P31/14C12N7/00C12Q1/686C12N2770/16021A61K39/00G01N2333/08G01N33/56983C12Q1/701C12Q2521/107C12Q2600/106C12Q2600/112
Inventor P·李R·黄K·拉莫斯J·陈M·泰布
Owner QUEST DIAGNOSTICS INVESTMENTS INC
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