Riemerella anatipestifer detection kit and method based on loop-mediated isothermal amplification technology

A ring-mediated isothermal and Riemerella anatipestifer technology is applied in the direction of microorganism-based methods, biochemical equipment and methods, and microbial measurement/inspection. Achieve the effect of simple and fast operation, strong specificity and low detection cost

Inactive Publication Date: 2010-10-27

CHONGQING ACAD OF ANIMAL SCI +1

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AI-Extracted Technical Summary

Problems solved by technology

But so far there is no report on the detection of Riemerella anatipe...

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Abstract

The invention discloses riemerella anatipestifer detection kit and method based on a loop-mediated isothermal amplification technology. Based on six specific regions of a 16S rRNA conserved region of riemerella anatipestifer, two specific primers and two specific outer primers are designed in the kit and can specifically distinguish the riemerella anatipestifer and other serotype salmonellae so as to ensure the high specificity and the reliability of a detection result of loop-mediated isothermal amplification. The invention detects the riemerella anatipestifer on the basis of the loop-mediated isothermal amplification technology, can amplify a target sequence rapidly, efficiently and specifically under the isothermal condition, has simple and convenient operation and does not use expensive instruments and reagents; an amplification product can be developed directly by using a fluorescent dye and a result can be judged with naked eyes; the detection cost is low; and the invention is particularly suitable for small and medium size units and field tests.

Application Domain

Microbiological testing/measurementMicroorganism based processes

Technology Topic

Field testsFluorescence +5

Examples

Experimental program(5)

Example Embodiment

[0035] Example 1

[0036] 1. A detection kit for Riemerella anatipestifer based on loop-mediated isothermal amplification technology, consisting of the following reagents:

[0037] a. The upstream inner primer FIP aqueous solution and the downstream inner primer BIP aqueous solution each with a concentration of 5 μmol/L, the upstream outer primer F3 aqueous solution and the downstream outer primer B3 aqueous solution with a concentration of 20 μmol/L each: the primer sequence is as follows:

[0038] Upstream inner primer FIP: 5’-caggcttccacccattgtccaa-tgagacacggaccagactc-3’;

[0039] Downstream inner primer BIP: 5’-cgtgaaggacgacggccctat-ctaccctcacgagagtaggt-3’;

[0040] Upstream outer primer F3: 5’-tttagggggcctgagagg-3’;

[0041] Downstream outer primer B3: 5'-ccggtgcttattcgtacagt-3';

[0042] b. A Bst DNA polymerase aqueous solution with a concentration of 8U/μl;

[0043] c. 10× thermal polymerization buffer solution: Tris-HCl with a concentration of 250mmol/L, pH 8.8, potassium chloride with a concentration of 100mmol/L, ammonium sulfate with a concentration of 100mmol/L, and a concentration of 20mmol/L It is composed of magnesium sulfate and Triton X-100 with a mass fraction of 1%;

[0044] d. An aqueous solution of dNTPs with a concentration of 10 mmol/L: consists of dATP, dGTP, dCTP and dTTP with a concentration of 10 mmol/L each;

[0045] e. Magnesium sulfate aqueous solution with a concentration of 100mmol/L;

[0046] f. Betaine aqueous solution with a concentration of 2mol/L;

[0047] g. 10% aqueous solution of fluorescent dye SYBR Green I.

[0048] 2. The method for detecting Riemerella anatipestifer using the ring-mediated isothermal amplification technology-based detection kit for Riemerella anatipestifer includes the following steps:

[0049] a. Extract the bacterial DNA of the sample to be tested as template DNA: In this example, an experimental group and a blank control group are set at the same time. The experimental group is Riemerella anatipestifer, from the Poultry Research Center of Sichuan Agricultural University; DNA extraction reagents are used Kit (Tiangen Biochemical Technology Co., Ltd.) to extract the bacterial DNA of each group, operate according to the kit instructions, the OD of the bacterial DNA aqueous solution obtained in the experimental group 260 /OD 280 The value is 1.8 and the concentration is 20ng/μl.

[0050] b. Loop-mediated isothermal amplification of Riemerella anatipestifer: Prepare a loop-mediated isothermal amplification reaction system with a total volume of 25μl in a reaction tube: add the upstream inner primer FIP aqueous solution and downstream with a concentration of 5μmol/L 1μl each of the inner primer BIP aqueous solution, 1μl each of the upstream outer primer F3 aqueous solution and the downstream outer primer B3 aqueous solution with a concentration of 20μmol/L, 1μl Bst DNA polymerase aqueous solution with a concentration of 8U/μl, 10× thermal polymerization buffer 2.5μl , 2.5μl dNTPs aqueous solution with a concentration of 10mmol/L, 0.75μl magnesium sulfate aqueous solution with a concentration of 100mmol/L, 12.5μl betaine aqueous solution with a concentration of 2mol/L, diluted to 24μl with DNase-free water, and then added template DNA 1μl of the aqueous solution, mix well, and get it; the reaction tube is incubated in a 60-65°C water bath for 60 minutes, and then in a 80°C water bath for 5 minutes to terminate the reaction;

[0051] c. Color detection: Add 1 μl of 10% mass fraction of the fluorescent dye Cyber Green I aqueous solution into the reaction tube, shake it evenly, and observe the color change with naked eyes.

[0052] Results: The color of the blank control group was yellow, indicating that it did not contain Riemerella anatipestifer; the color of the experimental group changed to green, indicating that it contained Riemerella anatipestifer, which was consistent with the expected result.

Example Embodiment

[0053] Example 2

[0054] 1. The Riemerella anatipestifer detection kit based on the loop-mediated isothermal amplification technology is different from the kit described in Example 1 in the specific primer sequence. The primer sequence of this kit is as follows:

[0055] Upstream inner primer FIP: 5’-tattcctcactgctgcctcccg-gtgatcccccacactggt-3’;

[0056] Downstream inner primer BIP: 5’-cgtgaaggacgacggccctat-ctaccctcacgagagtaggt-3’;

[0057] Upstream outer primer F3: 5’-tttagggggcctgagagg-3’;

[0058] Downstream outer primer B3: 5'-ccggtgcttattcgtacagt-3'.

[0059] 2. The method for detecting Riemerella anatipestifer using the ring-mediated isothermal amplification technology-based Riemerella anatipestifer detection kit is the same as the method described in Example 1. As a result, it is found that the color of the blank control group It is yellow, indicating that it does not contain Riemerella anatipestifer; the color of the experimental group changes to green, indicating that it contains Riemerella anatipestifer, which is consistent with the expected result.

Example Embodiment

[0060] Example 3

[0061] 1. The Riemerella anatipestifer detection kit based on the loop-mediated isothermal amplification technology is different from the kit described in Example 1 in the specific primer sequence. The primer sequence of this kit is as follows:

[0062] Upstream inner primer FIP: 5’-caggcttccacccattgtccaa-acactggtactgagacacgg-3’;

[0063] Downstream inner primer BIP: 5’-cgtgaaggacgacggccctat-ctaccctcacgagagtaggt-3’;

[0064] Upstream outer primer F3: 5’-tttagggggcctgagagg-3’;

[0065] Downstream outer primer B3: 5'-ccggtgcttattcgtacagt-3'.

[0066] 2. The method for detecting Riemerella anatipestifer using the ring-mediated isothermal amplification technology-based Riemerella anatipestifer detection kit is the same as the method described in Example 1. As a result, it is found that the color of the blank control group It is yellow, indicating that it does not contain Riemerella anatipestifer; the color of the experimental group changes to green, indicating that it contains Riemerella anatipestifer, which is consistent with the expected result.

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Description & Claims & Application Information

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Classification and recommendation of technical efficacy words

Guaranteed reliability
Strong specificity

Riemerella anatipestifer detection kit and method based on loop-mediated isothermal amplification technology

A ring-mediated isothermal and Riemerella anatipestifer technology is applied in the direction of microorganism-based methods, biochemical equipment and methods, and microbial measurement/inspection. Achieve the effect of simple and fast operation, strong specificity and low detection cost

AI-Extracted Technical Summary

Problems solved by technology

Abstract

Examples

Example Embodiment

Example Embodiment

Example Embodiment

PUM

Description & Claims & Application Information

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