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Riemerella anatipestifer LAMP detection kit and its detection method

A technique of Riemer's duck disease and detection kit, which is applied in biochemical equipment and methods, determination/inspection of microorganisms, fluorescence/phosphorescence, etc., can solve the problem of low specificity and sensitivity, non-specific appearance, and long detection time and other problems, to achieve the effect of ensuring high specificity, simple operation and low cost

Active Publication Date: 2012-04-11
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Current laboratory diagnostic methods mainly include bacteriological methods (such as isolation and identification of bacteria), serological methods (such as enzyme-linked immunosorbent assay) and molecular biology (such as polymerase chain reaction), but the above methods require The detection time is long, the specificity and sensitivity are low, and special equipment must be equipped, the operation is complicated, and non-specificity often occurs

Method used

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  • Riemerella anatipestifer LAMP detection kit and its detection method
  • Riemerella anatipestifer LAMP detection kit and its detection method
  • Riemerella anatipestifer LAMP detection kit and its detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Detection of Riemerella anatipestifer LAMP

[0030] Prepare Riemerella anatipestifer LAMP detection reaction solution according to the following composition:

[0031] (1) Reaction solution A: Contains 10× reaction buffer, Bst DNA polymerase, dNTP (2.5mM each), Betaine solution (5M), and 3 pairs of primers. The sequence is:

[0032] F3: 3'CTAAAGATGGGGTTTCTGTG 5' (SEQ ID NO.1)

[0033] B3: 3'ATCCATAGGATTAGCTCCAG 5' (SEQ ID NO.2)

[0034] FIP: 3' TCGTTGGTTTTAGATGCTACTTCTTTTTTGCTAAAGAGATAGAGCTAGAGG 5' (SEQ ID NO. 3)

[0035] BIP: 3'TGGCAGGAGATGGTACCACTTTTTCAGCCACATTTTTAAGACCTT5' (SEQ ID NO. 4)

[0036] LF: 3'ACCATTTGAGCTCCCATATT 5' (SEQ ID NO.5)

[0037] LB: 3'GCTACCGTATTAGCGCAAGC 5' (SEQ ID NO.6)

[0038] (2) Reaction solution B: 10000×SYBR green I

[0039] The system composition of 24 μL per tube of reaction solution A is as follows:

[0040] Reaction component volume (μL)

[0041] 10× ThermoPol reaction buffer 10

[0042] Bst DNA (8,000U / mL) 1

[004...

Embodiment 2

[0062] Example 2: Sensitivity of LAMP to detect Riemerella anatipestifer

[0063] (1) Template preparation of Riemerella anatipestifer

[0064] Inoculate Riemerella anatipestifer WJ4 strain into TSB liquid medium, shake the bacteria to OD at 37°C 600 =0.6 or so, take 1mL of the bacterial solution and centrifuge at 12,000r / min for 1min, discard the supernatant, wash 3 times with sterilized PBS, resuspend in 1mL of PBS, and prepare a bacterial solution with a concentration of 5×10 5 CFU / μL, then diluted with PBS: 5×10 4 CFU / μL, 5×10 3 CFU / μL, 5×10 2 CFU / μL, 100CFU / μL, 50CFU / μL, 25CFU / μL, 10CFU / μL, 5CFU / μL.

[0065] (2) Detection comparison of LAMP and PCR sensitivity to Riemerella anatipestifer

[0066] In the reaction solution A, add 1 μL of Riemerella anatipestifer of different concentrations prepared in the above-mentioned Example 1 as a template, bathe in water at 94°C for 5 minutes, and bathe in ice for 20 seconds, add 1 μL of Bst DNA, mix well, and bathe in water at 6...

Embodiment 3

[0072] Embodiment 3: Riemerella anatipestifer rapid LAMP detection kit

[0073] A rapid detection kit for Riemerella anatipestifer, comprising the following reagents:

[0074] Fluorescent dye SYBR green I, 10× reaction buffer, Bst DNA polymerase, dNTP (2.5mMeach), Betaine solution (5M), 3 pairs of primers, the sequence of which is:

[0075] F3: 3'CTAAAGATGGGGTTTCTGTG 5'

[0076] B3: 3'ATCCATAGGATTAGCTCCAG 5'

[0077] FIP: 3'TCGTTGGTTTTAGATGCTACTTCTTTTTTGCTAAAGAGATAGAGCTAGAGG5'

[0078] BIP: 3'TGGCAGGAGATGGTACCACTTTTTTCAGCCACATTTTTTAAGACCTT5'

[0079] LB: 3'GCTACCGTATTAGCGCAAGC 5'

[0080] LF: 3'ACCATTTGAGCTCCCATATT 5'

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Abstract

The invention belongs to biological detection technical field, concretely relates to a riemerella anatipestifer LAMP detection kit and its detection method. The invention discloses the riemerella anatipestifer LAMP detection kit which is characterized in that three primer pairs are contained, the nucleic acid sequences are showed in SEQ ID NO.1-6. The detection kit and its detection method have the advantages of rapidity, sensitivity, specificity, low cost and simple operation, and the defect of current detection method of riemerella anatipestifer LAMP can be compensated, the current detection demand can be satisfied, the riemerella anatipestifer LAMP detection kit and the detection method enable large scope popularization and application, the prevalence of the diseases of ducks and the like can be reduced, and the invention has wide market prospect and large economic benefit.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a Riemerella anatipestifer LAMP detection kit and a detection method thereof. Background technique [0002] Riemerella anatipestifer (RA) disease is a contagious poultry disease that mainly affects ducks, turkeys and other birds, and its serotypes are complex. So far, 21 serotypes of RA have been recognized (types 1 to 21), lack of cross-protection ability between each serotype. The main pathological changes of the disease are fibrinous pericarditis, perihepatitis, air sac inflammation, meningitis, caseous salpingitis, etc. The incidence rate is high and the mortality rate is high. Economic loss is one of the main infectious diseases that endanger the poultry breeding industry at present. [0003] Riemerella anatipestifer is often secondary to other diseases or concurrent with other infectious diseases. Its clinical symptoms and pathological anatomy are...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04G01N21/64
Inventor 于圣青韩先干丁铲胡青海陈鸿军仇旭升宋翠萍
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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