PCR primers, PCR method and kit for detecting mouse adenovirus k87 strain

A mouse adenovirus and kit technology, applied in biochemical equipment and methods, resistance to vector-borne diseases, measurement/inspection of microorganisms, etc., can solve problems that have not yet been retrieved, and achieve simple, easy-to-operate and specific result analysis Good sex, not easy to misjudge the effect

Active Publication Date: 2022-08-09
SUZHOU XISHAN BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In 2014, Wang Ji et al. established a RT-PCR detection method for mouse adenovirus (MAdV) for the E1B gene, which was applied to the detection of MAdV in experimental animals such as gerbils and mice. Among them, the smallest mouse adenovirus DNA can be detected. The template concentration is 1.67pg / μl, but this method needs to judge the result according to the position of the target band after electrophoresis
No PCR methods have been retrieved for the detection of MAdV-2

Method used

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  • PCR primers, PCR method and kit for detecting mouse adenovirus k87 strain
  • PCR primers, PCR method and kit for detecting mouse adenovirus k87 strain
  • PCR primers, PCR method and kit for detecting mouse adenovirus k87 strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1. Materials and Methods

1.1

[0066] Virus and nucleic acid extraction:

[0067] Mouse Adenovirus K87 Strain, Mouse Parvovirus, Mouse Sendai Virus, Mouse Hepatitis Virus, Mouse Norwalk Virus, Mouse Cytomegalovirus, Mouse Adenovirus FL Strain, Mouse Pneumonia Virus, Reo Arc 10 kinds of viral nucleic acids, such as virus type III and mouse pox virus, were provided by our laboratory for the specificity test of PCR method.

1.2

[0068] DNA extraction from clinical samples:

[0069] 20 mouse feces were collected, and the nucleic acid was extracted with a viral genome DNA / RNA extraction kit (Tiangen Biochemical Technology Co., Ltd., DP422). The operation was carried out in strict accordance with the kit instructions, using 60 μL ddH 2 The extracted DNA was eluted with O and stored at -20°C until use.

1.3

[0070] Primer design and synthesis:

[0071] Primers were designed according to the hexon protein gene sequence of mouse adenovirus K87 strain using Prim...

Embodiment 2

[0083] Example 2. Detection by PCR method

2.1

[0084] specific detection test

[0085] PCR amplification was carried out with the extracted genomic DNA of 10 strains of virus as templates to test the specificity of the PCR method.

2.2

[0086] Sensitivity test

[0087] With MAdV-2 virus plasmid (2.4 × 10 10 copies / reaction) as the template, do a series of gradient dilutions, numbered S4~S10, and the concentrations are 2.4×10 6 copies / reaction, 2.4×10 5 copies / reaction, 2.4×10 4 copies / reaction, 2.4×10 3 copies / reaction, 2.4×10 2 copies / reaction, 2.4×10 1 The positive standard of copies / reaction and 2.4 copies / reaction, the standard product of each gradient is repeated in 3 wells, and the detection method detects the accuracy and stability of the method of the present invention, and NC is ddH 2 O, Sensitivity test for PCR method.

2.3

[0088] Testing of clinical samples

[0089] The 20 DNAs extracted in the above Example 1 were detected by the established PCR m...

Embodiment 3

[0090] Embodiment 3. PCR method detection results

[0091] 3.1 Specific test results

[0092] Taking the genomic DNA of the above 10 strains of viruses as templates, and using the primers for MAdV-2 provided in this application, the above 10 strains of viruses were detected by the fluorescence quantitative PCR method to verify the specificity of the method. The results are shown in Table 1.

[0093] Table 1 Specificity test of PCR method of mouse adenovirus K87 strain

[0094]

[0095] As shown in Table 1, when other strains of viruses were detected with no positive results, the mouse adenovirus K87 strains all detected positive results. From this, it can be seen that the PCR primers provided in this application have good specificity to the mouse adenovirus K87 strain.

[0096] Figure 1A It is the specific detection amplification curve of the primers and methods of the present invention. Figure 1B shows as Figure 1A In the embodiment shown, the melting curve of the fluo...

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Abstract

The present application belongs to the field of detection of pathogenic microorganisms, and relates to PCR primers, PCR methods and kits for detecting mouse adenovirus K87; specifically, the present application provides a pair of mouse adenovirus K87 strains with good specificity and high sensitivity detection primers, fluorescence quantitative PCR method and detection kit. The primers include an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer is the sequence shown in SEQ ID NO.1, and the nucleotide sequence of the downstream primer is the sequence shown in SEQ ID NO.2; the The PCR method includes at least obtaining the upstream primer and the downstream primer through the design of Primer Premier 5 according to the hexon protein gene of the mouse adenovirus K87 strain; the kit provides at least the upstream primer and the downstream primer, or using the PCR method.

Description

technical field [0001] The present application belongs to the field of detection of pathogenic microorganisms, and relates to the detection of mouse adenovirus K87 strain, in particular to PCR primers, a detection method for detecting mouse adenovirus K87 strain, and the application of the detection method. Specifically, the present application provides a pair of detection primers, fluorescent quantitative PCR method and detection kit with good specificity and high sensitivity for mouse adenovirus K87 strain. Background technique [0002] Murine adenovirus (MAdV) belongs to the genus Mammal Adenovirus of the Adenoviridae family, has no envelope, and is a double-stranded DNA virus. Mice are the natural hosts for mouse adenovirus, which is mostly negative for infection in mouse populations and can also cause lethal disease, in which laboratory mice are infected and transmitted mainly through fecal and urine contact. The virus was isolated from mice in the 1960s, and two serot...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11
CPCC12Q1/701C12Q1/6851C12Q2531/113C12Q2563/107Y02A50/30
Inventor 王立鹏朱明星时长军
Owner SUZHOU XISHAN BIOLOGICAL TECH
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