Oligonucleotide primer for effective detection of hepatitis C virus (HCV) and its use

A hepatitis C virus and oligonucleotide technology, applied in the field of sequence improvement, can solve the problems of indistinguishable infection, undetected HCV infection, and unsuccessful development

Inactive Publication Date: 2006-01-18
ORTHO-CLINICAL DIAGNOSTICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Positive EIA results can be confirmed by a recombinant immunoblot assay (RIBA), but neither EIA nor RIBA assays can distinguish between past and current infections
Due to the typically low titers of infectious viruses, a method for the direct analysis of viral proteins has not been successfully developed
Furthermore, antibody-based assays did not detect HCV infection typically after 2-3 months of exposure

Method used

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  • Oligonucleotide primer for effective detection of hepatitis C virus (HCV) and its use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0144] Example 1: Detection of HCV in Biological Samples: Comparison of 5' HCV Amplification Primers and Roche Amplicor HCV Identification.

[0145] The following experiments were performed to compare the HCV identification of the present invention and the Roche Amplicor system.

[0146] A method:

[0147] 1. Sample preparation:

[0148] RNA was prepared from plasma samples using Pure Script RNA Isolation Reagent (Gentra Systems, Minneapolis MN). Modifications to the manufacturer's protocol for body fluid preparation included using 40 g of glycogen instead of 20 g of glycogen as a carrier to aid in the precipitation of viral RNA. Also, in most cases, after precipitating RNA with isopropanol and washing the RNA pellet with ethanol, the RNA pellet was resuspended in RT buffer mix rather than in the RNA hydration solution provided by the manufacturer.

[0149] 2. Reverse transcription

[0150] Add 100u of recombinant Moloney murine leukemia virus (M-MLV) reverse ...

Embodiment 2

[0176] Embodiment 2: Analysis of the sensitivity of 5' NCR HCV detection method.

[0177] Use the 'NCR primer pair of the present invention to carry out the following experiments to check the sensitivity of the HCV detection method.

[0178] 1. Three patient samples were diluted according to the theoretical copy number of HCV RNA and analyzed for HCV RNA using the Roche Amplicor system and the primer pairs of the present invention. The results are shown in Table 9 below:

[0179] Table 9

theoretical copy

number / ml

Roche

Amplicor

5'NC

131F / 294R

JJCD Password

A1

500

+

+

A2

100

+

+

A3

10

-

+

A4

5

-

+

A5

1

-

-

B1

500

+

+

B2

100

+

+

B3

10

+

+

B4

5

+

+

B5

1

+

+

C1 ...

Embodiment 3

[0185] Example 3: Detection of HCV in Biological Samples: Comparison of 3' NCR HCV Amplification Primers and Roche Amplicor HCV Assay.

[0186] 150 plasma samples from Brazil that had previously tested positive for HCV antibodies were identified by Roche Amplicor and examined with the 3'NCR primers of the present invention. Reverse transcription and amplification were performed as described in Example 1. Reverse transcription was performed with primers whose sequence was 5'GTATCAGCACTC-3', and amplification was performed with X1F27 / 57R27 primer pair. Each reaction contained an internal positive control (IPC) of plasmid DNA. The amplified product was detected with a capture probe 3X30PRB25 using the Sure Cell system. The product of each reaction was run on a gel to confirm the results. Negative controls (interspersed with clinical samples) containing negative plasma samples and water (no RNA) were both involved and were negative in RT-PCR. A comparison of the results ob...

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Abstract

Described herein are methods and kits for the detection of hepatitis C virus RNA is biological samples obtained from human subjects. The invention includes novel amplification primers and probes useful in the amplification of DNA derived from hepatitis C virus RNA, and kits and methods which incorporate the novel primers.

Description

technical field [0001] The present invention relates to an improved method for the detection of nucleic acid sequences in biological samples, in particular sequences derived from infectious microorganisms. Background technique [0002] Hepatitis C virus (HCV) is a parenterally transmitted virus responsible for most cases of post-transfusion hepatitis and most cases of sporadic (or population-acquired) hepatitis worldwide. It is estimated that more than 1% of the world's population is infected with HCV. Acute hepatitis, chronic hepatitis, cirrhosis, and hepatocellular carcinoma are all associated with HCV infection. [0003] HCV is currently classified as a separate genus, Hepacivirus in the family Flaviviridae. Its genome consists of a positive-strand RNA molecule of approximately 9,500 nucleotides, containing a large single open reading frame (ORF) encoding a polyprotein precursor of approximately 3,000 amino acids. This large open reading frame is preceded by a 5'-nonco...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/09C12Q1/686C12Q1/70C12R1/92
CPCC12N2770/24211C12Q1/686C12Q1/707C12Q1/6888C12Q2565/125
Inventor J·M·林南K·M·戈曼
Owner ORTHO-CLINICAL DIAGNOSTICS
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