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B-actin-contained dual isothermal nucleic acid amplification method for rapidly detecting type-3 human adenovirus

An isothermal nucleic acid amplification and adenovirus technology, which is applied in the field of molecular biology, can solve the problems of poor DNA polymerase activity, incorrect mixture, false negatives, etc., and achieve easy wide-scale application, good specificity, and broad market prospects Effect

Active Publication Date: 2019-08-09
中国疾病预防控制中心病毒病预防控制所 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it could also mean that the reaction was inhibited due to incorrect temperature, incorrect RAA reaction mix, poor DNA polymerase activity, or the presence of inhibitory substances in the sample matrix, resulting in a false negative result

Method used

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  • B-actin-contained dual isothermal nucleic acid amplification method for rapidly detecting type-3 human adenovirus
  • B-actin-contained dual isothermal nucleic acid amplification method for rapidly detecting type-3 human adenovirus
  • B-actin-contained dual isothermal nucleic acid amplification method for rapidly detecting type-3 human adenovirus

Examples

Experimental program
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Effect test

Embodiment 1

[0040] Example 1 Design and determination of specific primer pairs, target probes, and internal reference probes suitable for isothermal nucleic acid amplification detection of type 3 adenovirus

[0041] Download the whole genome sequence of all HAdV-3 viruses, perform sequence comparison, find a conserved region with high homology, and determine a target sequence suitable for detecting HAdV-3, and its nucleotide sequence is shown in SEQ ID NO.6. Design multiple specific primers and target probes in conserved regions.

[0042] RAA primer design principles: First, the primer length, RAA primers are longer than typical PCR primers, generally required to be 30-35bp; second, the primer sequence, avoid repeated G at the 5' end (3-5bp), preferably C or T; preferably G and C at the 3' end (last 3 bases); GC content should not be greater than 70% or less than 30%; avoid formation of secondary structures, primer dimers, etc. between primers. RAA probe design principles: RAA fluorescen...

Embodiment 2

[0067] Example 2 Detection of type 3 adenovirus containing double isothermal nucleic acid amplification method with internal reference (1)

[0068] 1. Sample source and RNA extraction of HAdV-3

[0069] The virus samples were samples containing HAdV-3 live virus collected from the lavage fluid of different patients collected by the Hunan Provincial Center for Disease Control and Prevention. The DNA was extracted using Tianlong Extraction Kit, and the DNA extraction equipment was Tianlong Automatic Nucleic Acid Extractor.

[0070] 2, primer and probe adopt the primer and the probe (SEQ ID NO.1-4) that are applicable to the detection HAdV-3 virus of isothermal nucleic acid amplification method determined in embodiment 1, wherein target probe marks HEX fluorescent group, The internal reference probe is labeled with the FAM fluorophore.

[0071] 3. Prepare the amplification system: prepare the isothermal nucleic acid amplification system in a 200 μL centrifuge tube according to t...

Embodiment 3

[0077] Example 3 Detection of type 3 adenovirus containing double isothermal nucleic acid amplification method with internal reference (2)

[0078] For the method, see Example 2, the difference is that in the 50 μL isothermal nucleic acid amplification system, the concentrations of the forward and reverse primers are 300 nM, respectively, and other parameters and steps are the same as in Example 2. The results showed that the amplified fluorescent signal began to appear after 2 min, and the difference was that the peak value of the amplification curve of the target sequence decreased slightly. Such as Figure 3A-Figure 3B shown. By repeating the above embodiments, the same amplified fluorescent signal can be obtained with good repeatability.

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Abstract

The invention provides a b-actin-contained dual isothermal nucleic acid amplification method for rapidly detecting type-3 human adenovirus, and belongs to the technical field of isothermal nucleic acid amplification. The method comprises the steps that firstly, due to type-3 human adenovirus (HAdV-3) gene sequencing and comparing, a specific primer pair and probe for detecting HAdV-3 are designed,a b-actin probe is designed, the nucleotide sequences are shown in SEQ ID NO.1-4, on the basis, a b-actin-contained dual isothermal nucleic acid amplification system is built, and a kit for detectingthe type-3 adenovirus is constructed. The method is implemented under the isothermal condition, type-3 adenovirus and b-actin DNA amplification can be achieved at the same time within 5-20 min, sensitivity is high, specificity is good, the false negative and valid effects are removed by adding b-actin, the method is more suitable for being applied to detection of a large number of samples, clinicapplication is convenient, and the method is applicable to rapid detection of the type-3 adenovirus.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to target sequences, specific primers and target probes for detecting type 3 human adenoviruses, and the invention also relates to double isothermal Method and kit for nucleic acid amplification detection of type 3 adenovirus. Background technique [0002] Human adenovirus (HAdV) often causes respiratory system infection, diarrheal disease, eye infection, reproductive tract and urinary system infection, etc. Viruses of different subgenus can cause different diseases, among which A, F and G mainly cause digestive tract infection Mainly respiratory tract infection, B, C and E mainly cause respiratory tract infection, subgenus D often causes infection of ocular conjunctival tissue. Subgenus B and C are common pathogens that cause acute respiratory infections worldwide. Among them, the main type of adenovirus infection outbreaks in my country is HADV-3 of subgenus B, which is also the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6844C12Q2600/166C12Q2600/16C12Q2522/101C12Q2545/101C12Q2521/507C12Q2563/107C12Q2537/143Y02A50/30
Inventor 马学军申辛欣王智宏应清界张益王佶李鑫娜王瑞欢
Owner 中国疾病预防控制中心病毒病预防控制所
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