Amplification method of internal reference containing double isothermal nucleic acid for rapidly detecting type-7 adenovirus

An isothermal nucleic acid amplification and adenovirus technology, applied in the field of molecular biology, can solve problems such as design difficulties, achieve good specificity, easy to popularize and apply on a large scale, and good specificity.

Active Publication Date: 2019-07-26
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

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Problems solved by technology

Because the primers and probes required by the RAA method are long in length, they are easy to form dimers and ha

Method used

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  • Amplification method of internal reference containing double isothermal nucleic acid for rapidly detecting type-7 adenovirus
  • Amplification method of internal reference containing double isothermal nucleic acid for rapidly detecting type-7 adenovirus
  • Amplification method of internal reference containing double isothermal nucleic acid for rapidly detecting type-7 adenovirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Design and determination of specific primer pairs, target probes, and internal reference probes suitable for isothermal nucleic acid amplification detection of type 7 adenovirus

[0042] Download the whole genome sequence of all HAdV-7 viruses, perform sequence comparison, find a conserved region with high homology, and determine a target sequence suitable for detecting HAdV-7, and its nucleotide sequence is shown in SEQ ID NO.6. Design multiple specific primers and target probes in conserved regions.

[0043] 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

[0068] Example 2 Detection of type 7 adenovirus containing double isothermal nucleic acid amplification method with internal reference (1)

[0069] 1. Sample source and RNA extraction of HAdV-7

[0070] The virus samples were samples containing HAdV-7 live virus collected from the lavage fluid of different patients 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.

[0071] 2, primer and probe adopt the primer and the probe (SEQ ID NO.1-4) that are applicable to the detection HAdV-7 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.

[0072] 3. Prepare the amplification system: prepare the isothermal nucleic acid amplification system in a 200 μL centrifuge tube according to the followi...

Embodiment 3

[0078] Example 3 Detection of type 7 adenovirus containing double isothermal nucleic acid amplification method with internal reference (2)

[0079] 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 peak value was high. As shown in Figure 3. By repeating the above embodiments, the same amplified fluorescent signal can be obtained with good repeatability.

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Abstract

The invention provides an amplification method of internal reference containing double isothermal nucleic acid for rapidly detecting type-7 adenovirus and belongs to the technical field of isothermalnucleic acid amplification. According to the amplification method, a specific primer pair and a probe for HAdV-7 detection are designed first through human adenovirus type-7 (HAdV-7) gene sequencing and alignment, an internal reference probe is designed, and the nucleotide sequences of the specific primer pair and the probe are respectively as shown in SEQ ID NO.1-4, an internal reference containing double isothermal nucleic acid amplification system is established, and a kit for detecting adenovirus type 7 is further constructed. The method is carried out under isothermal conditions, the amplification of the type-7 adenovirus and the internal reference DNA can be realized within 5-20min, the sensitivity is high, the specificity is good, false negative and invalid results are eliminated due to the addition of an internal reference, the method is more suitable for the detection of a large number of samples, the clinical application is facilitated, and the method is suitable for the rapid detection of the type-7 adenovirus.

Description

technical field [0001] The present invention relates to the field of molecular biology, in particular to target sequences, specific primers and target probes for detecting type 7 adenovirus, and the present invention also relates to the use of specific primers, target probes and internal reference probes for double isothermal nucleic acid Method and kit for amplifying and detecting type 7 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. Airborne droplet transmission is the main transmission route of adenovirus, and it can also be transmitted through fecal-or...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/701C12Q1/6844C12Q2521/507C12Q2522/101Y02A50/30
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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