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Rapid detection kit for human adenoviruses

A detection kit and technology for human adenovirus, which are applied to the determination/inspection of microorganisms, biochemical equipment and methods, microorganisms, etc. High accuracy, high sensitivity and high specificity

Inactive Publication Date: 2014-11-05
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The purpose of the present invention is to solve the problems of long detection time of human adenovirus in the prior art, low sensitivity and incapable of quantitative detection

Method used

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  • Rapid detection kit for human adenoviruses
  • Rapid detection kit for human adenoviruses
  • Rapid detection kit for human adenoviruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Adenovirus Detection in Micro Samples

[0044] 1. DNA extraction from sputum samples: 7 copies of a small amount of sputum samples obtained (Jiangsu Provincial Center for Disease Control and Prevention), each with 5 μl and corresponding 5 times the volume of DNA extraction solution: including 50mM Tris-HCl at pH 8.0, pH 8.0 in 100 mM EDTA, 100 mM NaCl, 1% SDS, 0.5 mg / ml proteinase K. Mix evenly, place in a 55-degree metal bath at 55°C for 30 minutes, heat and shake for 10 minutes, then add an equal volume of analytical pure isopropanol and mix evenly, place at room temperature for 2 minutes, centrifuge at 12,000 rpm for 2 minutes, discard the supernatant, and add 20ul sterile Dissolve in water to make template DNA, set aside.

[0045] 2. Primers were designed using primer primer5 and synthesized by GenScript Biotechnology

[0046] Adenovirus amplification primer: upstream primer sequence Adv-F: as shown in SEQ ID NO.1;

[0047] Downstream primer seque...

Embodiment 2

[0062] The kit is used for quantitative determination of human adenovirus in samples.

[0063] 1. Sample preparation: Take 2 small amount of sputum samples (Jiangsu Provincial Center for Disease Control and Prevention), 6 μl each and dilute to 1 ng / ul with TE buffer.

[0064] 2. Use the human adenovirus rapid detection kit provided by the present invention to prepare according to the ratio of sample and reagent provided in Example 1.

[0065] At the same time, according to the same ratio, the negative blank control, positive control, and standard detection were performed with the human adenovirus rapid detection kit provided by the present invention.

[0066] Negative blank control: the reaction system does not include template DNA, which is sample 4.

[0067] Positive control test: 2 ul of the PUC19 recombinant vector inserted with a 100 bp fragment was added to the PCR reaction system to replace the template DNA, which was sample 3.

[0068] 3.PCR amplification

[0069] S...

Embodiment 3

[0081] Sensitivity detection of human adenovirus in purified DNA samples

[0082] Dilute the DNA sample to 5, 5*10, 5*10 with TE buffer 2 、5*10 3 、5*10 4 Copies / ml 5 concentration difference groups, 6ul for each group, using 5 concentration difference groups as templates.

[0083] According to the reaction conditions and reaction system of Example 1, carry out the quantitative PCR reaction, measure the CT value, as shown in Table 1, "+" indicates that the absorbance value has obvious changes with the increase of the number of cycles, "-" indicates little change, In this way, the sensitivity of the system provided by the invention to detect human adenovirus is determined. Through comparison, it is obtained when the concentration of the reaction solution is ≥5*10 2 copies / ml, the absorbance value will change significantly with the increase of the number of cycles, and the detection sensitivity can reach 500copies / ml.

[0084] 4. Result analysis: After the reaction is finish...

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Abstract

The invention belongs to the technical field of virus detection and provides an extracting solution for extracting human adenovirus DNA (deoxyribonucleic acid), a PCR (polymerase chain reaction) reaction solution for detecting human adenoviruses, a PCR primer sequence for detecting human adenovirus DNA, a rapid detection kit for human adenoviruses and a method for quantitatively detecting the quantity of human adenoviruses. The rapid detection kit for human adenoviruses comprises the extracting solution for extracting human adenovirus DNA, the PCR reaction solution for detecting human adenoviruses and the PCR primer sequence for detecting human adenovirus DNA, and further comprises an adenovirus positive control and an amplification primer sequence for detecting the positive control. The adenovirus detection sensitivity of the kit disclosed by the invention can reach 500copies / ml. The quantitative linear range is 500-5.0E+08copies / ml, so that the kit is suitable for detecting the adenoviruses in a trace sample and provides a reliable basis for early diagram of adenovirus infection.

Description

technical field [0001] The invention belongs to the technical field of virus detection, and relates to a rapid detection kit for human adenovirus, in particular to an extraction solution for extracting human adenovirus DNA, a PCR reaction solution for detecting human adenovirus, and a human adenovirus DNA detection solution. PCR primer sequence, human adenovirus rapid detection kit and method for quantitative detection of human adenovirus. Background technique [0002] Adenovirus is a non-enveloped particle with a diameter of 70-90nm, composed of 252 capsomers arranged in an icosahedron. The diameter of each shell particle is 7-9nm. Inside the capsid is a linear double-stranded DNA molecule, containing about 35,000 bp, with an inverted repeat sequence of about 100 bp at each end. [0003] There are 52 known human adenoviruses, which are named adl~ad52, and the most detailed study is ad2. The adenovirus genome is transcribed to produce mRNA, and there are at least 5 known ...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/70C12Q1/68C12N15/11C12R1/93
Inventor 许雯黄佳莹曹林
Owner NANJING AGRICULTURAL UNIVERSITY
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