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Primer and kit for detecting duck type-II adenovirus

A technique for detecting adenoviruses and ducks, which is applied in the field of primers and kits for detecting duck type 2 adenoviruses, can solve problems such as the research and reporting of duck type 2 adenoviruses that have not been seen, and achieve shortened detection time, save time and cost, The effect of low false positive

Inactive Publication Date: 2015-11-11
WENS FOOD GRP CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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  • Primer and kit for detecting duck type-II adenovirus
  • Primer and kit for detecting duck type-II adenovirus
  • Primer and kit for detecting duck type-II adenovirus

Examples

Experimental program
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Effect test

Embodiment 1

[0025] Example 1. Detection of duck type 2 adenovirus using Tagman fluorescent quantitative PCR kit for duck type 2 adenovirus

[0026] (1) Extraction of DNA from samples to be tested: 100μL of cell culture medium (infected with the autonomous isolate ZLY, identified as duck type 2 adenovirus, other duck type 2 adenovirus isolates can also be used), 100mg clinical liver tissue (unknown Whether infected with duck adenovirus type 2), clinically-ill duck throat swabs, cloacal swabs, and DMEM (cell culture medium, without DADV2, as a negative control) as samples to be tested, perform the following operations:

[0027] The diseased tissue was fully ground by adding 1 mL of PBS buffer, and then repeatedly frozen and thawed at -20°C for 3 times, and centrifuged at 8000 rpm for 10 minutes to obtain the supernatant for nucleic acid extraction. Add 1mL PBS buffer to the throat swab, shake and mix thoroughly, and take the supernatant to extract nucleic acid. Nucleic acid extraction uses taco...

Embodiment 2

[0044] Example 2. Specificity test

[0045] Taking self-isolated duck type 2 adenovirus infected cell culture virus as a positive control, duck parvovirus, gosling plague virus, Muscovy reovirus, Muscovy duck new reovirus, Duck hepatitis virus, Muscovy C lung Virus and Muscovy Tembusu virus were specifically detected. The method and primer probes of Example 1 were used for fluorescence quantitative PCR amplification. The results showed that there was no amplification curve for other viruses except for the positive control, which shows that the method has good specificity. (see figure 2 ).

Embodiment 3

[0046] Example 3. Sensitivity test

[0047] The standard positive plasmid pMD18-52K containing 52K gene was diluted 10-fold with sterilized double-distilled water, and 2μL of each dilution was used as a template. Perform fluorescence quantitative PCR amplification according to the method in Example 1. Observe the amplification For the curve, the sensitivity is calculated based on the highest dilution of the template amount used for the positive expected curve. image 3 The middle amplification curve from left to right is 10 -5 , 10 -6 , 10 -7 , 10 -8 And 10 -9 The standard positive template amplification curve of the dilution factor indicates that the minimum detection amount is 71 copies (see image 3 ).

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Abstract

The invention discloses a primer and a kit for detecting duck type-II adenovirus, wherein nucleic acid sequences of the primer pair are represented as the SEQ ID No.1-2. The primer and the kit can quickly identify and diagnose infection of the duck type-II adenovirus, are good in repeatability and are accurate in quantitation. The whole sample detection process, from DNA extraction to result, can be completed just in 2-3 h. The method is simple and can save time and cost, is increased in accuracy and allow high-throughput sample detection to be carried out at the same time.

Description

Technical field [0001] The invention relates to the technical field of detection of animal pathogenic microorganisms, in particular to a primer and a kit for detecting duck type 2 adenovirus. Background technique [0002] Duck Adenovirus 2 was first discovered in French Muscovy ducks in 1977. It mainly caused the morbidity and death of Muscovy ducks at 15-35 days. Infected ducks showed listlessness, depression, weight loss, soft feet, yellowish white mucus and thin feces. , Liver enlargement and pale diffuse yellow-white needle-point necrosis, pancreatic white necrosis, spleen enlargement and congestion, kidney enlargement and congestion into yellow strips, mild respiratory symptoms in the early stage of infection, and 1% per day at the peak of the disease- The mortality rate is 1.5%, and lasts for about 10 days. The total mortality rate is as high as 15-25%. In 1982, JFBOUQUET used waterfowl and duck-derived cells to isolate the virus for the first time. At that time, the virus...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
Inventor 招丽婵林丽苗周庆丰覃健萍操胜王占新曾凡桂
Owner WENS FOOD GRP CO LTD
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