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Assay for the detection of nucleotide substitutions in the genome of the Sabin oral poliovirus vaccine virus

A polio and genome technology, applied in the field of kits containing the above primers, can solve the problems of expensive, non-existent fast and convenient tests, etc.

Inactive Publication Date: 2016-03-30
INDIAN COUNCIL OF MEDICAL RES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] Complete genome sequencing and whole-genome microarrays enable the determination of the precise nucleotide (A, T, G, or C) of all known polio attenuating loci, but there is still no rapid and convenient assay to perform as described above in a single Alternative to costly and complex technology that simultaneously detects mutations in all known attenuating sites of poliovirus 1 (six sites), 2 (three sites) and 3 (2 sites) in assays

Method used

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  • Assay for the detection of nucleotide substitutions in the genome of the Sabin oral poliovirus vaccine virus
  • Assay for the detection of nucleotide substitutions in the genome of the Sabin oral poliovirus vaccine virus
  • Assay for the detection of nucleotide substitutions in the genome of the Sabin oral poliovirus vaccine virus

Examples

Experimental program
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Embodiment 1

[0116] Preparation of DNA amplicons from three serotypes of poliovirus

[0117] Poliovirus of each serotype (type 1, 2 or 3) was obtained separately. Poliovirus samples, cDNA, were synthesized from frozen infected cultures using antisense PCR primers and DNA amplicon generation in a single multiplex reverse transcription and PCR reaction (Roche). PCR products (DNA amplicons) were purified by PCR purification kit (Quagcn).

[0118] RT-PCR - For RT-PCR analysis, direct frozen infection cultures (1fll) and 67mM Tris / HCl (pH 8.8), 17mM Ammonium Sulfate, 6mM EDTA, 2mM MgCh, 200Mm each dNTP, 1mM dioxane in a total volume of 50fll were taken directly. Thiothreitol, 1 mM of each primer, 10 U placental RNase inhibitor (Roche), 3 U avian myeloblastosis virus (AMV) reverse transcriptase (Roche), and 5 U Taq DNA polymerase (Roche AppliedScience). Reactions were incubated at 50°C for 30 minutes, followed by 3 minutes at 94°C. 35 thermal cycles of 94 °C for 30 s, 42 °C for 30 s, 72 °C fo...

Embodiment 2

[0120] Single Nucleotide Polymorphism Assay (SNP Assay) for Mutation Identification

[0121]Ten SNP reactions were performed in a final volume of 10III containing SNaPshot multiplex ready-to-use reaction mix (5lll), primer mix (3lll) (final concentration, 0.15-0.6Ilmolll) and template (2llll) prepared by QIAquickPCR as described above. Purification kit (Qiagcn) purified multiplex PCR product composition. Multiplex PCR products amplified from viral RNA were pooled and purified on the same column prior to the SNaPshot reaction. The cycling program consisted of 25 cycles of 96 °C for 10 s, 50 °C for 5 s and 60 °C for 30 s. Extension products were purified by inactivating the enzyme by incubating with 1 U shrimp alkaline phosphatase for IS-min at 37°C, followed by incubation for IS-min at 75°C. The purified product (0.5111) was mixed with 9III formamide and 0.5III GeneScan-120LIZ size standard (Applied Biosystems) and separated by capillary electrophoresis (ABIPRISM3130 GeneticA...

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Abstract

The present invention provides a multiplex SNP assay method and primers for the specific and simultaneous detection of mutations in the genomes of live attenuated oral poliovirus vaccine (Sabin) viruses, vaccine-like and vaccine derived polio viruses of all the three serotypes. Importantly, the inventive primers and the assay eliminate the need for costly, time-consuming and complex procedures like complete genome sequencing. This novel assay is able to test several samples in a very short time. Therefore, the assay is very simple, fast and cost-effective. A kit comprising the novel primers is also provided.

Description

Field of invention: [0001] The present invention generally belongs to the field of biotechnology, and in particular relates to a method for detecting mutations at specific sites in the genome of live attenuated oral poliovirus vaccine (Sabin) virus, vaccine-like and vaccine-derived polioviruses New primers, and kits containing the above primers. Background of the invention: [0002] Dr Albert B Sabin developed the live attenuated oral poliovirus vaccine for immunization against paralytic polio. Through meticulous laboratory work, he was very careful to isolate mutant virus strains from the disease that produced wild-type poliovirus of all three serotypes. These attenuated (non-neurotoxic) poliovirus strains were used to produce the live attenuated oral polio vaccine, which has been used worldwide for more than 50 years. Sabin also developed a test to evaluate poliovirus neurotoxicity (NVT) in monkeys. NVT has become the standard quality control test used to demonstrate at...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70
CPCC12Q1/701C12Q2600/156C12Q2600/16C12Q2600/166
Inventor J·M·达什潘德S·S·南迪D·K·沙玛
Owner INDIAN COUNCIL OF MEDICAL RES