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Enterovirus PCR (Polymerase Chain Reaction) quality control serum armored RNA and preparation method

A technology for enteroviruses and quality control products, applied in botany equipment and methods, biochemical equipment and methods, positive-sense single-stranded RNA viruses, etc., to achieve stable performance and simple operation

Inactive Publication Date: 2018-11-13
中华人民共和国大榭出入境检验检疫局
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Various hemorrhagic fever virus detection kits currently on the market in China generally use synthetic RNA or DNA fragments, and RNA templates are easily degraded by RNase enzymes in the environment

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0012] Embodiment 1: Obtaining and identification of Enterovirus 2B gene armored RNA

[0013] Enterovirus ENV-2B gene amplification: QiaGen RNeasy Mini Kit (purchased from Qiagen) was used to extract RNA from clinical CODEHOP positive samples, and the method was referred to the kit instructions. Enterovirus CODEHOP universal primers E4559 (commercially available) / FE4937 (commercially available) were used to amplify positive samples, and this pair of primers can be used for the detection of Enterovirus. The primer sequences are shown in SEQ ID NO:3 and 4 in the sequence listing, and the reaction system is: 2 X 1 step buffer 10µL, PrimerScript 1 setp Enzyme Mix 0.8µL, primer E4559 / FE4937 (20pmol) 1µL, RNA to be detected 1µL, supplement RNase free water to 20 µL. Reaction conditions: 42°C for 30min, 94°C for 2sec, 94°C for 30s, 56°C for 30sec, 72°C for 1min (35cycle), 72°C for 10min. 1.5% agarose gel electrophoresis was used to observe the effect of PCR amplification. Use TaKa...

Embodiment 2

[0014] Example 2: Identification of enterovirus armored RNA

[0015]Take 20 µL of CODEHOP virus, use 100U RNase A and 2U DNaseI, and act at 37°C for 1 hour, use the QiaGen RNeasyMini Kit extraction kit (purchased from Qiagen) to extract RNA, and use enterovirus primers to perform PCR and RT-PCR amplification respectively. The PCR reaction system: 10 X PCR Buffer 2µL, dNTP (2.5mM each) 1.6µL, rTaq (5U / µL) 0.4µL, Mgcl2 1.2µL, primer E4559 / FE4937 (20pmol) 1µL, DDW 11.8µL, extracted nucleic acid 1µL. Reaction conditions: 94°C for 5min, 94°C for 30sec, 52°C for 30sec, 72°C for 1min (35cycle) and 72°C for 7min. RT-PCR reaction system: RNase Free Water 20.2μl, 5×RT-PCR Buffer 10μl, 10mM dNTP Mixture 2μl, EnzymeMix 2μl, RNase Inhibitor 1μl, primer E4559 / FE4937 (20pmol) 1μL, template 1μL. Reaction conditions: 42°C for 30min, 94°C for 2sec, 94°C for 30s, 56°C for 30sec, 72°C for 1min (35cycle), and 72°C for 10min. The effect of PCR amplification was observed by electrophoresis.

Embodiment example 3

[0016] Implementation Case 3: Stability Verification of Armored RNA Quality Control

[0017] 1. Storage stability test under different environmental conditions: use RNase A and DNaseI to digest the crude virus-like particle product completely, and place it at 37°C, 4°C, -20°C for 15d, 30d, 45d, 60d, 75d , 90 days later, and the sample was stored at -70°C as a control, the QiaGen RNeasy Mini Kit was used to extract RNA, and the experimental results were detected by agarose gel electrophoresis after amplification with enterovirus primers;

[0018] 2. Digestion experiments with different quality control enzymes: 4 copies prepared from human intestinal EV71 virus RNA and CoxA16 virus RNA preserved in the laboratory were divided into two groups for experiments. One group was added with 100U RNase A and 2U DNaseI, and reacted at 37°C for 1 hour. The other group was left untreated. Use RNA extraction kit to extract RNA, use UV spectrophotometer to measure concentration, dilute to th...

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PUM

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Abstract

The invention discloses an enterovirus PCR (Polymerase Chain Reaction) quality control serum armored RNA and a preparation method. An expression vector of the armored RNA is a pET32a-MS2-CP-ENV-2B plasmid; the expression vector, namely the pET32a-MS2-CP-ENV-2B plasmid, is transferred into escherichia coli prokaryotes expression strain BL21 to induce expression; after the expression vector is ultrasonically disrupted, precipitates are centrifugally collected to obtain virus-like particles of the enterovirus PCR quality control serum armored RNA; and in the expression vector, namely the pET32a-MS2-CP-ENV-2B plasmid, the nucleotide sequence of the MS2-CP is as shown in SEQ ID NO:1 in the sequence list, and the nucleotide sequence of the ENV-2B is as shown in SEQ ID NO:2 in the sequence list.During storage, the armored RNA cannot be degraded by RNase enzyme and is an enterovirus PCR quality control serum with stable performance. The preparation method of the armored RNA is easy to operate, safe and effective.

Description

technical field [0001] The invention relates to a detection technology of enterovirus, in particular to an enterovirus PCR quality control product armored RNA and a preparation method thereof. Background technique [0002] Enterovirus belongs to Picornaviridae family and Enterovirus genus (Enterovir-us), 64 serotypes are known to infect human body, including poliovirus, Coxsackie virus, Echo virus And some new enteroviruses and hepatitis A viruses, etc. For example, hand, foot and mouth disease and herpetic pharyngitis caused by enterovirus 71 (EV71) broke out in Taiwan in 1998. The number of infected people exceeded 100,000. Edema and hemorrhage, acute flaccid paralysis and myocarditis and death; in recent years, some studies have shown that diabetes is also related to enteroviruses. The existing enterovirus detection technology, molecular biology technology represented by PCR technology, has the advantages of rapidity, sensitivity, and low cost, but nucleic acid detectio...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/66C12N15/41C07K14/085
CPCC07K14/005C12N15/66C12N15/70C12N2770/32322C12N2770/32323
Inventor 胡群马思杰
Owner 中华人民共和国大榭出入境检验检疫局
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