LAMP (loop-mediated isothermal amplification) visual detection kit for chicken mycoplasma synoviae (MS)

The technology of a mycoplasma and a kit is applied in the field of a visual detection kit of Mycoplasma synoviae LAMP, and achieves the effects of good specificity, good application prospect and broad application prospect.

Active Publication Date: 2013-06-05
GUANGXI VETERINARY RES INST
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Loop-mediated isothermal amplification technology (LAMP) has the advantages of simplicity, low cost, and visualized results. It is currently widely used in the detection of some pathogenic microorganisms. At present, there is no research report on the application of LAMP in the detection of Mycoplasma gallisovum in China.

Method used

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  • LAMP (loop-mediated isothermal amplification) visual detection kit for chicken mycoplasma synoviae (MS)
  • LAMP (loop-mediated isothermal amplification) visual detection kit for chicken mycoplasma synoviae (MS)
  • LAMP (loop-mediated isothermal amplification) visual detection kit for chicken mycoplasma synoviae (MS)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1, the design of primer

[0033] Referring to the MS heat shock ATP-dependent protease (heat shock ATP-dependent protease) DNA gene sequence in Genbank, use Primer Exporer V4 online design software to design LAMP primers, including 2 outer primers F3 / B3 and 2 inner primers FIP / BIP , and 2 loop primers Bloop and Floop used to improve the amplification efficiency. The primers were synthesized by Shanghai Invitrogen Company. The specific sequences are shown in Table 1.

[0034] Table 1 is the LAMP primer sequence for amplifying MS

[0035]

[0036] FLoop and BLoop primers can be omitted, and the speed and sensitivity of detection can be improved by adding them.

Embodiment 2

[0037] Embodiment 2, the application of primer in LAMP detection Mycoplasma gallisovoidum (MS)

[0038] 1. Extraction of DNA

[0039] DNA extraction: MS strains were inoculated in MS broth medium, cultured at 37°C until the color turned yellow, and the bacteria were collected by centrifugation; after washing twice with PBS buffer, the precipitate was suspended with appropriate TE buffer, refer to TIANgen DNA extraction kit instructions. The DNA extraction method of other control bacteria (virus) strains is the same.

[0040] 2. Optimizing the LAMP reaction system and reaction conditions

[0041] The establishment of the reaction system: the reaction system of Mycoplasma gallisovii LAMP is 25 μL: Bst DNA polymerase (purchased from New England Biolabs, catalog number: M0275) 8U1μL, 10ⅹBst polymeraMSbuffer2.5uL, F3 and B3 each 0.2uM, FIP and BIP each 1.6uM, FLoop and BLoop each 0.8uM, dNTP1.0mM, betaine0.1M, MgSO 4 4mM, Calcein 5.0uM, MnCl 2 0.5mM, template 1uL.

[0042] In...

Embodiment 3

[0061] Embodiment 3, the detection of clinical sample

[0062] The samples to be tested were 10 chicken disease feed samples (numbered 1-10) collected from chicken farms in Guangxi in 2012, and the DNA of chicken disease feed (synovial fluid and tendon sheath of sick chickens) were extracted respectively.

[0063] The DNA of each sample numbered 1-10 was used as a template, and the LAMP reaction was carried out according to the above-mentioned optimal reaction system and optimal reaction conditions in Example 2.

[0064] The result is as Figure 4 As shown, the samples numbered 1-5 are positive for Mycoplasma gallisovinae (emerald green visible to the naked eye), and the samples numbered 6-10 are negative for Mycoplasma gallisyoviolum (orange-red visible to the naked eye).

[0065] Each sample numbered 1-10 was detected for Mycoplasma gallisovum according to the routine PCR of the control group in Example 2, and the result was consistent with the identification result of the ...

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Abstract

The invention discloses an LAMP (loop-mediated isothermal amplification) visual detection kit for chicken mycoplasma synoviae (MS). The invention provides an LAMP primer group for detecting the chicken mycoplasma synoviae. The LAMP primer group comprises a primer 1, a primer 2, a primer 3 and a primer 4, wherein the nucleotide sequences of the primer 1, the primer 2, the primer 3 and the primer 4 are respectively a sequence 1, a sequence 2, a sequence 3 and a sequence 4 in a sequence table. Experiments in the invention proof that the primer provided by the invention, the prepared MS-LAMP detection reagent and the created method have the characteristics of speediness, flexibility, specificity and simpleness, only one temperature controllable water bath kettle is used, and results can be determined by eyes instead of an instrument, so that the LAMP visual detection kit disclosed by the invention is applicable to rapid detection in a food products factory, a border port, a farm and a primary-level veterinary station, is significant in effective prevention and control of MS and has better application prospect.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a visual detection kit for LAMP of Mycoplasma gallisylinus. Background technique [0002] Mycoplasma synoviae MS (Mycoplasma synoviae MS) is the most important mycoplasma that harms chickens, mainly invading joint synovial fluid, tendon sheath and air sac, causing subclinical upper respiratory tract infection and joint synovial bursitis, and chickens are infected with the disease Significant lameness, stunted growth and carcass degradation can result. Rapid detection of MS is the prerequisite for taking emergency preventive measures and purifying MS. At present, MS-PCR detection technology has been reported. Although MS-PCR detection method is fast and has high sensitivity, it needs to use expensive instruments and reagents. [0003] LAMP technology is a new type of nucleic acid amplification technology. It does not require special equipment. The amplification reaction can be compl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
Inventor 谢芝勋邓显文谢志勤刘加波庞耀珊谢丽基范晴罗思思
Owner GUANGXI VETERINARY RES INST
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