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FAdV-4 PCR detection kit and detection method

A kind of technology of avian adenovirus and kit, which is applied in the detection field of avian adenovirus type 4

Active Publication Date: 2016-04-13
SHIJIAZHUANG SHIMU ANIMAL HUSBANDRY PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no PCR detection kit and detection method for detecting avian adenovirus type 4 in China

Method used

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  • FAdV-4 PCR detection kit and detection method
  • FAdV-4 PCR detection kit and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1 A kind of PCR kit that detects poultry adenovirus type 4 best embodiment

[0028] The kit of this embodiment includes: (1) Avian adenovirus type 4 DNA extraction reagents: proteinase K, lysate, sodium acetate, saturated phenol: chloroform: isoamyl alcohol, absolute ethanol, sterile distilled water.

[0029] (2) PCR reaction reagents: 2×TaqPCRMix, upstream primers, downstream primers; (4) Others: positive control and nuclease-free water. Among them, 2×TaqPCRMix is ​​composed of DNA polymerase, 2×TaqPCR buffer and dNTP Mixture. The positive control is to inoculate chicken embryos of 9-11 days old with avian adenovirus type 4 and collect the allantoic fluid of chicken embryos infected for 96-144 hours. Freeze-dried powder, pure by HPLC. The primer sequences included in the kit of this example are shown in Table 1.

[0030] Table 1 Primer Sequence

[0031]

Embodiment 2

[0032] Embodiment 2 non-diagnostic purpose detects the method for poultry adenovirus type 4 with PCR method

[0033] The detection method of this example uses the kit in Example 1. Tissues such as liver, spleen, lung and kidney were taken from diseased chickens as samples to be tested.

[0034] The detection method of this embodiment comprises the following steps:

[0035] 1. The specific steps of virus DNA extraction are as follows: (1) After cutting the sample to be tested, add physiological saline according to the mass volume ratio of 1:5 and grind evenly, centrifuge at 3000-5000rpm for 5-10 minutes, take the supernatant, clear. (2) Take 300 μL of the above-mentioned treated samples and positive control respectively, add 10 μL of proteinase K, 300 μL of cell lysate, incubate at 50°C for 2 hours, add 610 μL of tris-balanced phenol, fully shake for 5 minutes, and centrifuge at 12000 rpm for 10 minutes. (3) Transfer the supernatant to a new 1.5mL centrifuge tube, add an equ...

Embodiment 3

[0039] The sensitivity test of embodiment 3 poultry adenovirus type 4 PCR detection method

[0040] The sensitivity test was performed on the nucleic acid template with a calculated avian adenovirus type 4 DNA concentration of 129.3 ng / μL measured by an ultra-micro spectrophotometer.

[0041] The operation process of PCR detection of avian adenovirus type 4 was carried out according to Example 2.

[0042] The PCR amplification product was detected by 1% agarose gel electrophoresis, and the results were as follows: figure 2 shown. It can be seen that PCR can detect at least 0.0129ng / μL avian adenovirus type 4 nucleic acid template.

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PUM

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Abstract

The invention discloses an FAdV-4 PCR detection kit and detection method. The kit comprises protease K, a lysing solution, sodium acetate, saturated phenol, chloroform, isoamylol, anhydrous ethanol and sterile distilled water, and is characterized by comprising 2*TaqPCR Mix, a sense primer and a reverse primer, wherein the sequence of the sense primer is shown as SEQ ID No.1, and the sequence of the reverse primer is shown as SEQ ID No.2. The kit can be used for detecting FAdV-4. The method for detecting FAdV-4 by adopting the kit is also provided, and FAdV-4 can be detected quickly and specifically.

Description

technical field [0001] The invention relates to the technical field of detection of avian adenovirus type 4. Background technique [0002] Since June 2015, chicken flocks in Henan, Shandong, Hebei, Liaoning, Anhui, Jilin and other parts of China have developed unexplained diseases characterized by pericardial effusion and hepatomegaly. According to the pathological changes, the disease is called "Chicken pericardial effusion-hepatitis syndrome". The disease mainly occurs in 20-30-day-old broilers, including 817, meat offal, etc. The peak of death is 4-8 days after the onset, the course of disease is 8-15 days, and the mortality rate reaches 20-30%. At the same time, 20-70-day-old and 200-300-day-old laying hens also occurred, but the mortality rate was lower than that of broiler chickens. At present, the incidence area of ​​the disease is constantly expanding, which has caused huge economic losses to the poultry industry in my country. It has been confirmed that the patho...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68
CPCC12Q1/686C12Q1/701C12Q2531/113C12Q2545/113
Inventor 袁万哲孙继国刘聚祥陈萍张姗
Owner SHIJIAZHUANG SHIMU ANIMAL HUSBANDRY PHARMA CO LTD
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