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Primer combination and application for adenovirus detection and sequencing

A technology for detecting primers and primer combinations, which is applied in the fields of molecular biology and medical testing, can solve the problems of false detection and misjudgment, incomplete coverage, and inaccurate detection, and achieve the effect of obvious effect, accurate judgment and convenient detection.

Active Publication Date: 2019-08-20
中国人民解放军西部战区总医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there is no effective rapid detection and sequencing method for human adenovirus (human adenovirus, HAdV)
Or the coverage is not comprehensive, the detection is not accurate, and it is easy to lead to false detection and misjudgment

Method used

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  • Primer combination and application for adenovirus detection and sequencing
  • Primer combination and application for adenovirus detection and sequencing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] This embodiment provides a detection primer for adenovirus, the detection primer includes a first primer combination, the first primer combination is the 1st-12th primer pair, and the base sequences of the 1st-12th primer pair are respectively as SEQ ID No.1- 24.

[0056] The use of the above-mentioned adenovirus detection primers is as follows:

[0057] sample acquisition

[0058] Sample: The sample of adenovirus type 55 was collected from a case of respiratory tract infection in Lhasa, Tibet in 2016.

[0059] Virus isolation was performed on throat swab samples using the adenovirus-sensitive cell line Hep-2 cells in a biosafety level 2 (BSL2) laboratory.

[0060] experimental method:

[0061] 1.1 Hep-2 cells were seeded in 24-well plates at a seeding density of 1.5×10 5 one / well, cultivated for 24h;

[0062] 1.2 Wash the cells 3 times with PBS, add 500 μl of 2% DMEM medium to each well, and add 30 μl of throat swab specimens from confirmed patients to each well; ...

Embodiment 2

[0081] This embodiment provides an adenovirus detection kit, which includes the first primer pair provided in Example 1, and the base sequences of the first primer pair are respectively shown in SEQ ID No. 1-2.

[0082] Of course, this kit also includes reagents that can directly perform PCR reactions, including PCR reaction buffer, TaqDNA polymerase, dNTPs and Mg 2+ at least one of the

[0083] For the use of the detection kit, refer to the method in Example 1.

Embodiment 3

[0085] This embodiment provides a sequencing primer for adenovirus, the sequencing primer includes the second primer combination and the first primer combination provided in Example 1, the second primer combination includes the 13th-24th primer pair, the base of the 13th-24th primer pair The base sequences are respectively shown in SEQ ID No.25-48.

[0086] The use of the sequencing primers provided in this example is as follows:

[0087] The first part, template preparation for sequencing samples

[0088] Sample template preparation refers to the sample extraction and adenovirus DNA extraction methods provided in Example 1.

[0089] The second part is the sequencing of adenovirus DNA, the method is as follows:

[0090] 1.1 For the 1st to 24th pair of primers, set 4 reaction tubes for each pair of primers, named A reaction tube, T reaction tube, C reaction tube and G reaction tube respectively;

[0091] 1.2 Add 1 μL of template adenovirus DNA, 1 μL of high-fidelity Taq DNA ...

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Abstract

The invention provides an adenovirus detection and sequencing primer combination and application and belongs to the field of molecular biology and medical examination. The adenovirus detection primer provided by the invention can specifically detect adenoviruses, application of the adenovirus detection primer in preparation of kits is convenient for detection of the adenoviruses, and the using method is simple, convenient and rapid. The invention further provides a sequencing primer employing adenoviruses. The sequencing primer is capable of sequencing adenoviral genomes in a full coverage manner. Application of the sequencing primer in preparation of adenovirus sequencing is convenient for rapid sequencing of the adenoviruses and capable of helping to accurately judge types of the adenoviruses, and the effect is obvious.

Description

technical field [0001] The invention relates to the fields of molecular biology and medical testing, in particular to a combination of primers for detection and sequencing of adenoviruses and their application. Background technique [0002] Human adenovirus (human adenovirus, HAdV) is a kind of non-enveloped double-stranded DNA virus, which is divided into 7 serological groups A-G, among which group B adenovirus infection has become an important factor of acute respiratory diseases. Group B can be divided into subgroups B1 and B2. B1 mainly includes types 3, 7, 16, 21, and 51; B2 includes types 14, 34, 35, and 55. [0003] The genome sequencing of the adenovirus strains that broke out in my country will help to understand the evolutionary characteristics and epidemic trends of the virus, and help to prevent and control the adenovirus type 55. [0004] At present, there is no relatively effective rapid detection and sequencing method for human adenovirus (human adenovirus, H...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11C12R1/93
Inventor 刘媛王文博邹自英范泉水熊杰
Owner 中国人民解放军西部战区总医院
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