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Loop-mediated isothermal amplification kit for detecting BYD viruses, and its application

A ring-mediated isotherm and kit technology, applied in the biological field, can solve the problems of no effective detection means, and achieve the effects of high sensitivity, high specificity, and convenient and simple identification

Active Publication Date: 2013-03-06
MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] There is no effective detection method for this disease, and there is a certain serological cross-reaction with common flaviviruses in my country such as dengue virus and Japanese encephalitis virus in serology. RT-PCR) to detect the pathogen

Method used

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  • Loop-mediated isothermal amplification kit for detecting BYD viruses, and its application
  • Loop-mediated isothermal amplification kit for detecting BYD viruses, and its application
  • Loop-mediated isothermal amplification kit for detecting BYD viruses, and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Embodiment 1, LAMP reaction identification BYD virus

[0071] 1. Design of special primers and preparation of each special primer

[0072] According to the genome sequence of BYD virus, the specific primer sequence is designed as follows, artificially synthesized:

[0073] F3 (sequence 1, primer 1): TGTGGACAAGGCATGTTTGT

[0074] B3 (SEQ ID NO: 2, Primer 2): AGTCAAGTCAATGGCGTTGT

[0075] FIP (SEQ ID NO: 3, primer 3): GGCAAGTCTCCTTGGTGTGGATTTTCGATGTTGAGGCTTGGAAGG

[0076] BIP (SEQ ID NO: 4, Primer 4): TACGATCAGTCAGCAGGCTCGATTTTTCCAAGATCGCGTTCAACTC

[0077] LB (SEQ ID NO: 5, Primer 5): GCACAACATGTGGGTAAGCA

[0078] 2. Preparation of virus samples

[0079] 1. Preparation of virus samples

[0080] The virus liquid of BYD virus Byd1 strain was calculated by plaque test (PFU / ml). Use DMEM medium to carry out gradient dilution of the virus solution to obtain 8 different concentrations of the virus solution (concentrations are 1000PFU / ml, 100PFU / ml, 10PFU / ml, 1PFU / ml, 0.1...

Embodiment 2

[0122] Embodiment 2, clinical sample detection

[0123] The special primers obtained in Step 1 of Example 1 were used to detect the diseased duck liver tissue samples (numbered 1-22) collected from different duck farms in Baiyangdian area, Hebei Province.

[0124] After fully grinding the isolated sick duck liver tissue sample respectively, resuspend with 1ml DMEM medium, centrifuge at 5000g for 10 minutes, get supernatant 210 μ l, extract the RNA of supernatant (extraction method is the same as step 2 of embodiment 1), The ring isothermal amplification method is the same as step three in Example 1, with sample 2 in step two of Example 1 as the positive control (+), and sample 8 as the negative control (-)

[0125] See the test results Figure 5 , #1 to #22 are duck liver samples 1 to 22, "+" is a positive control, and "-" is a negative control. a, c and e are LAMP products under natural light, b, d and f are LAMP products under ultraviolet light. It can be seen that #1, #2...

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Abstract

The invention discloses a loop-mediated isothermal amplification kit for detecting BYD viruses, and its application. Special primers provided by the invention comprise a first primer, a second primer, a third primer and a fourth primer, and the nucleotide sequences of the first primer, the second primer, the third primer and the fourth primer are respectively represented by sequence 1, sequence 2, sequence 3 and sequence 4 in a sequence table. The special primers are suitable for detecting the specificities of the BYD viruses. The loop-mediated isothermal amplification kit has the advantages of performing of an amplification reaction at a constant temperature without special equipment, high specificity, rapidness and high efficiency to realize the completion of the amplification reaction within 35min, high sensitivity, and convenient and simple identification.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a loop-mediated isothermal amplification kit for detecting BYD virus and its application. Background technique [0002] BYD virus, named after its isolated Baiyangdian (Bai Yang Dian), is a member of the flavivirus genus and has a close relationship with Tembusu virus. After the virus infects ducks, it can cause duck egg-drop syndrome (Duck Egg-Drop Syndrome) , DEDS). The disease, first identified in 2010, is highly contagious. The main symptoms of sick ducks are high fever, loss of appetite, decline or even stop of egg production, and ducks with severe disease even lose their ability to move. Necropsy shows serious lesions such as hemorrhage, atrophy, and rupture of the ovaries. my country's coastal duck breeding provinces include Beijing, Anhui, Hebei, Fujian, Guangdong, Guangxi, Jiangsu, Jiangxi, Shandong, and Zhejiang. Both meat ducks and laying ducks can be infected by the vir...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
Inventor 秦成峰姜涛刘娟邓永强秦鄂德
Owner MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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