Plasmid standard molecule for hepatitis a virus quantitative detection

A technology for quantitative detection of hepatitis A virus, which is applied in the determination/inspection of microorganisms, the use of vectors to introduce foreign genetic material, and resistance to vector-borne diseases, etc. It can solve the problems that the standard plasmid of hepatitis A virus has not been reported, and the detection method of hepatitis A virus has not yet been formulated. , to improve the accuracy of the

Inactive Publication Date: 2014-07-02
NAT INST OF METROLOGY CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, a unified and feasible detection method for hepatitis A virus has not been formulated so far at home

Method used

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  • Plasmid standard molecule for hepatitis a virus quantitative detection
  • Plasmid standard molecule for hepatitis a virus quantitative detection
  • Plasmid standard molecule for hepatitis a virus quantitative detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1: Standard Molecular Construction

[0021] 1. Target gene sequence search and primer design:

[0022] By consulting the relevant information of hepatitis A virus, the 247bp single-copy conserved region (SEQ ID NO: 1) encoding VP1-VP3 capsid protein in the genome of hepatitis A virus was selected as the specific target fragment of the standard molecule of hepatitis A virus plasmid.

[0023] According to the relevant literature (Brooks, Gersberg et al.2005) of consulting hepatitis A virus, design the primer pair of 247bp conserved region gene fragment of coding VP1-VP3 capsid protein in the genome of amplifying hepatitis A virus (provided by Shanghai Yingwei Jieji Biotechnology Co., Ltd. company), the sequences are shown in SEQ ID NO:2 and SEQ ID NO:3 (Table 1).

[0024] 2. Amplification of target DNA fragments Standard molecule construction:

[0025] Extract viral RNA: Add Trizol and chloroform to the EP tube containing hepatitis A virus (the volume ratio of e...

Embodiment 2

[0031] Example 2: Specificity Validation and Substitutability Studies

[0032] 1. Specific verification of hepatitis A virus:

[0033] In order to verify the specificity of standard molecules, plasmid molecules were amplified with different virus detection primers.

[0034] Select three commercial or experimental viruses, including: specific detection primers for HIV, influenza virus, and hepatitis C virus to amplify the plasmid molecules, and the results are as follows: image 3 .

[0035] It can be seen from the figure that only the hepatitis A virus detection primer can perform specific amplification and is positive;

[0036] The detection results of other virus primers have no amplification curve, which is the same as that of the blank control.

[0037] This proves that the fragment gene of the conserved region of hepatitis A virus VP1-VP3 has good specificity for other primers, so the plasmid molecule has detection specificity.

[0038] 2. Real-time fluorescent quantita...

Embodiment 3

[0043] Embodiment 3: the quantification result of plasmid standard molecule

[0044] The way of determining the value of the plasmid standard molecule: the value of the result of the present invention is analyzed by the method of digital PCR to draw the identified value of the plasmid standard molecule as 1.24 × 10 11 copy number. After uncertainty evaluation, the expanded uncertainty (U) of the plasmid molecular reference material is 1.4×10 10 .

[0045] The identification value of the plasmid standard molecule is analyzed by digital PCR method to obtain its identification value, that is, the copy number.

[0046] Deemed values ​​are those obtained with the highest degree of accuracy and for which all known or suspected sources of deviation have been fully investigated or considered. The identified value of the plasmid molecule was obtained by digital PCR analysis, and the results showed that the identified value of the plasmid standard molecule was 1.24×10 11 Copy, relat...

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Abstract

The invention relates to a plasmid standard molecule for hepatitis a virus quantitative detection. The plasmid standard molecule contains a sequence as shown in SEQ ID NO: 1, wherein the sequence contains a 247bp conserved domain gene segment for coding VP1-VP3 capsid proteins in a hepatitis a virus genome; and the 247bp conserved domain gene segment is applied to specific and quantitative detection of the hepatitis a virus and a sample containing the hepatitis a virus. Real-time fluorescent quantitative PCR (polymerase chain reaction) amplification experiment proves that the plasmid standard molecule provided by the invention can substitute hepatitis a virus genome as a positive standard substance for hepatitis a virus nucleic acid quantitative detection; and the standard substance does not need any treatment, only needs to be used with a to-be-detected sample in a detection process, so that content of the hepatitis a virus in the to-be-detected sample can be provided.

Description

technical field [0001] The invention belongs to the technical fields of biological analysis testing, clinical medical testing and environmental monitoring, and in particular relates to a plasmid standard molecule for quantitative detection of hepatitis A virus. Background technique [0002] Hepatitis A virus (HAV) infection can cause acute viral hepatitis. It is an important food-borne enterovirus, belonging to the family Picornaviridae Hepavirus. Decahedral symmetrical structure with a diameter of 27nm-32nm, which contains a positive-strand RNA genome with a length of about 7.5kb. Hepatitis A virus is mainly transmitted through fecal-oral transmission, and ingestion of food and water contaminated by Hepatitis A virus is the most important cause of Hepatitis A transmission. [0003] Hepatitis A virus exists widely in food and environment. Although it is not easy to cause chronic infection, it can often cause outbreaks and cause heavy losses to the country and people's lives...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/63C12R1/93
CPCC12Q1/706C12Q2600/166Y02A50/30
Inventor 隋志伟乔煜婷傅博强张玲王晶刘瑛颖董莲华
Owner NAT INST OF METROLOGY CHINA
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