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Kit and special primer for detecting GI.1 type norovirus in clinical sample

A GI.1, kit technology, applied in the field of biology, can solve the problems of high cost and long time consumption

Active Publication Date: 2021-07-30
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, obtaining norovirus subtype information is mainly based on the acquisition of the whole norovirus genome, which is time-consuming and expensive, and there is no kit on the market that can directly obtain norovirus subtype information

Method used

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  • Kit and special primer for detecting GI.1 type norovirus in clinical sample
  • Kit and special primer for detecting GI.1 type norovirus in clinical sample
  • Kit and special primer for detecting GI.1 type norovirus in clinical sample

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1, acquisition of special primers for detection of GI.1 type norovirus

[0037] A variety of primers were designed after a large number of sequence analysis, and the performance of the primers was verified through preliminary experiments, and finally the primer pair with the best performance for identifying GI.1 Norovirus was obtained.

[0038] Primer pair I is as follows (target sequence is 116bp):

[0039] Upstream primer F1 (sequence 1): 5'-TTGTAGTTGCAGGGCGAGTT-3';

[0040] Downstream primer R1 (SEQ ID NO: 2): 5'-CAATGGCAGATTTGGGAGTG-3'.

[0041] Primer pair II is as follows (target sequence is 209bp):

[0042] Upstream primer F2 (sequence 3): 5'-GACCTGCCCTAGTCCTGATT-3';

[0043] Downstream primer R2 (SEQ ID NO: 4): 5'-CCAAACGACCGTCCAAAGTA-3'.

[0044] Each primer was artificially synthesized separately.

Embodiment 2

[0045] Embodiment 2, application primer pair detection positive plasmid

[0046] 1. Preparation of positive plasmid

[0047] Positive plasmid I is a circular plasmid, as shown in sequence 5 of the sequence listing.

[0048] Positive plasmid II is a circular plasmid, as shown in sequence 6 of the sequence listing.

[0049] 2. Application of primer pairs to detect positive plasmids

[0050] 1. Dilute positive plasmid I with double distilled water to obtain template solution I; in template solution I, the concentration of positive plasmid I is 4.105×10 8 / μL. Dilute positive plasmid II with double distilled water to obtain template solution II; in template solution II, the concentration of positive plasmid II is 3.94×10 8 copies / μL.

[0051] 2. Take the template solution, carry out PCR amplification, and then carry out 1.5% agarose gel electrophoresis.

[0052]PCR amplification reaction system (25 μL): DreamTaq Green PCR Master Mix (2X) 12.5 μL, upstream primer 1 μL, downst...

Embodiment 3

[0066] Embodiment 3, the detection of stool sample GI.1 type norovirus

[0067] Human stool samples: Samples 1-5 are norovirus-positive human stool samples from the intestinal tract clinic of Haidian Hospital; samples 6-10 are norovirus-negative human stool samples from the intestinal tract clinic of Haidian Hospital.

[0068] 1. Preparation of template solution

[0069] Human feces samples were taken, total RNA was extracted using a kit (QIAGEN RNeasyPower Microbiome Kit), and cDNA was obtained by reverse transcription. The cDNA is in the form of a solution, which is the template solution.

[0070] 2. Conduct detection

[0071] Take the template solution, use primer pair I or primer pair II to carry out PCR amplification respectively, and then carry out 1.5% agarose gel electrophoresis.

[0072] PCR amplification reaction system (25 μL): DreamTaq Green PCR Master Mix (2X) 12.5 μL, upstream primer 1 μL, downstream primer 1 μL, template solution 1 μL, sterile water 9.5 μL. ...

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Abstract

The invention discloses a kit and a special primer for detecting GI.1 type norovirus in a clinical sample. The invention provides a primer pair II. The invention further provides a primer combination which is composed of a primer pair I and a primer pair II. The invention also provides a primer pair I. The primer pair II is composed of a single-stranded DNA molecule as shown in a sequence 3 in the sequence table and a single-stranded DNA molecule as shown in a sequence 4 in the sequence table. The primer pair I is composed of a single-stranded DNA molecule as shown in a sequence 1 in a sequence table and a single-stranded DNA molecule as shown in a sequence 2 in the sequence table. The method can be used for directly detecting the GI.1 type norovirus in excrement, the detection time can be greatly shortened, and the detection cost is reduced. The application prospect is wide, and a new thought is provided for detecting sample norovirus typing information.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a kit and special primers for detecting GI.1 norovirus in clinical samples. Background technique [0002] Norovirus (norovirus) belongs to the family Caliciviridae and is a type of single positive-sense RNA virus with a genome size of 7.5-7.7k, a diameter of about 27-37nm, and no envelope. The genome of norovirus contains three open reading frames (openreading frame, ORF), ORF1 encodes non-structural polyprotein including RNA-dependent RNA polymerase (RdRp), ORF2 encodes the main capsid protein (VP1), ORF3 Encodes a minor capsid protein (VP2). According to the amino acid homology of the main capsid protein VP1 of norovirus, it is divided into 10 genotypes (GI-GX), and the main types that cause human infection are GI and GII. Different genotypes can be further divided into different subtypes, among which GI type is divided into 9 subtypes (GI.1-GI.9), and GII type is divided into 27 s...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12N15/11C12R1/93
CPCC12Q1/701C12Q1/686C12Q2565/125
Inventor 朱宝利毛梦雨律娜张瑞芬
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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