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New method for rapid detection of G genotype of group A rotavirus by VP7 gene

A group A rotavirus, rotavirus technology, applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the rotavirus VP7 sequence variation, can not accurately determine the rotavirus G genotype, etc. problem, to achieve the effect of good specific sequence and obvious difference

Inactive Publication Date: 2011-10-26
PEOPLES HOSPITAL OF XINJIANG UYGUR AUTONOMOUS REGION
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Problems solved by technology

However, with the passage of time, the VP7 sequence of rotavirus has undergone great variation, and the original classic primers can no longer accurately determine the G genotype of rotavirus

Method used

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  • New method for rapid detection of G genotype of group A rotavirus by VP7 gene
  • New method for rapid detection of G genotype of group A rotavirus by VP7 gene

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Embodiment Construction

[0015] The present invention is further described with reference to examples.

[0016] 1. Extraction of viral RNA and amplification of virus type-specific fragments

[0017] (1) Add the same volume of PBS saline solution to 100ul of the sample, centrifuge at a speed of 3000g for 30s, and take the supernatant. Extract the total viral nucleic acid according to the viral nucleic acid extraction kit;

[0018] (2) Add 10ul H to 2ul template RNA 2 O (DEPC treatment), denaturation at 95°C for 3 minutes, ice bath;

[0019] (3) RT-PCR amplification reaction system: 5uL of the above denatured template RNA, 0.25uL PrimeScript RTase (200U / uL, TaKaRa), 0.25uL RNase Inhibitor (40U / uL), 0.5ul Ex Taq HS (5U / uL ), 1uL dNTP Mixture (10mmol / L each), 2ul MgCL 2 (25mmol / L), 10X Buffer 2.5ulL, 1uL primer ① (10umol / L), 1ul G typing mixed downstream primers (including primer ②, primer ③, primer ④, primer ⑤ and primer ⑥ 10u mol / L each), RNase Free H 2 O make up 25uL.

[0020] (4) RT-PCR reactio...

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Abstract

The invention provides a new method for rapid detection of G genotype of group A rotavirus by VP7 gene, comprising the steps of: 1) designing six nucleotide sequences capable of specifically amplifying different G genotypes of group A rotavirus as the primers for detecting the different G genotypes; 2) extracting virus nucleic acid from a sample and conducting one-time RT-PCR (reverse transcription-polymerase chain reaction) amplification with the six primers, and performing an electrophoresis detection to a product of PCR with 2% agarose gel, dyeing the product with ethidium bromide, and taking a picture by means of a gel imaging system; 3) judging the G genotype of group A rotavirus: when the size of the PCR product is respectively 332bp, 600bp, 98bp, 471bp and 248bp, the sample respectively contains G1, G2, G3, G4 and G9 rotaviruses, thus establishing a criteria for judging the G genotype of group A rotavirus.

Description

technical field [0001] The invention relates to designing specific primers on the VP7 gene of group A rotaviruses through sequence comparison, and by amplifying gene sequences of different fragments and lengths, it can economically and quickly determine the new genotype of group A rotavirus G. Detection method. Background technique [0002] Group A rotavirus (hereinafter referred to as rotavirus) is one of the main pathogens causing diarrhea in infants and young children. It mainly infects the villous epithelial cells of the small intestine, thereby affecting the normal absorption of salt, xylose and water in the intestine, causing diarrhea. Rotavirus is mainly prevalent in autumn and winter every year. The route of infection is the fecal-mouth route. The clinical manifestations are low-grade fever, vomiting and acute watery diarrhea. The course of the disease generally lasts for 7 days, which can lead to dehydration, electrolyte imbalance and malnutrition. In severe cases, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68
Inventor 杨学磊白杰李玉静杨学彤
Owner PEOPLES HOSPITAL OF XINJIANG UYGUR AUTONOMOUS REGION
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