LAMP primer group for detecting broad-spectrum mycoplasma, kit and application of LAMP primer group

Abstract

The invention discloses an LAMP primer group for detecting broad-spectrum mycoplasmas. The LAMP primer group consists of an outer primer F3, an outer primer B3, an inner primer FIP, an inner primer BIP, a loop primer LF and a loop primer LB, wherein the primers are respectively shown as SEQ ID NO.1-6. The invention further discloses a mycoplasma visible detection kit with the primer group, and application of the mycoplasma visible detection kit. The kit disclosed by the invention has advantages of good mycoplasma specificity, sensitivity, rapid and efficient amplification, visible identification and the like. By adoption of the detection system disclosed by the invention, normal mycoplasma contamination can be rapidly and sensitively detected in the cell culture process under an isothermal condition of 60-62 DEG C, no complex instrument is needed, and mycoplasma contamination detection in a cell laboratory at regular time can be relatively well achieved.

Description

technical field

[0001] The invention relates to the field of biotechnology, in particular to a LAMP primer set for detecting broad-spectrum mycoplasma, a kit and an application thereof. Background technique

[0002] In the culture process of mammalian cells, Mycoplasma contamination has always been a troublesome problem. There are three main reasons: 1). It is difficult to see its morphological structure; 2) Mycoplasma is tenacious and can survive under alkaline conditions (pH7.6-8.0), and is resistant to penicillin; 3) In the initial stage of cell contamination by mycoplasma, some sensitive cells can be seen Cell growth and proliferation slowed down, and the shape became round. However, most cells have no obvious changes after contamination, or have slight changes. If they are not treated in time, cross-contamination will also occur. Mycoplasma eventually causes cell death, but more often, mycoplasma coexists with cells. During this process, mycoplasma slowly proliferate...

Examples

Embodiment 1

[0024] Embodiment 1, primer design

[0025] The ITS-23srRNA sequences of 8 kinds of mycoplasma M.hyorhinis, M.fermentans, M.arginini, M.hominis, M.orale, M.salivarium, M.pirum, and AcholeplasmaLaidlawii commonly found in cell culture were retrieved from the American gene database Genbank, Homologous sequence analysis and alignment was performed by clustalX software to find specific conserved target gene fragments. In this example, the 5' end region of the 23srRNA sequence was selected, and primerexplorerV4 was used for online LAMP primer design to obtain F3 / B3 and FIP / BIP primers, and then appropriate loop primers were selected through manual adjustment. The designed primers were analyzed online for their specificity and broad-spectrum through the primer-blast software, and the final optimized primers were as follows:

[0026] Outer primer F3:

[0027] 5'-AAGAGTTTTTGGTGGATGC-3' (as shown in SEQ ID NO.1)

[0028] Outer primer B3:

[0029] 5'-CTGCTGCTACTGAGATGTT-3' (as shown...

Embodiment 2

[0038] Embodiment 2, setting up mycoplasma LAMP reaction system

[0039]Set different final concentrations of magnesium ions (4mM, 6mM, 8mM), dNTP concentrations (1mM, 1.2mM, 1.4mM), betaine (Betaine) concentrations (0.6M, 0.8M, 1M) and Bst2.0 enzyme (BstDNA polymerization enzyme) concentration (4U, 6U, 8U, 10U).

[0040] After the reaction, the color change of the system can be observed with the naked eye, such as figure 1 As shown, the negative is blue-purple (left picture), and the positive is sky blue (right picture).

[0041] The most preferred mycoplasma LAMP reaction system was determined through trial and error, that is, the preparation ratio of 25 μl LAMP reaction solution was: 10×Isothermal Amplification Buffer (isothermal amplification buffer) 2.5 μl, 10×LAMPprimermix (primer mixture) 2.5 μl, 10mMdNTPs 3 μl, 100mM MgSO 4 1.5 μl, 5MBetaine 4μl, 3mM hydroxynaphthol blue (HNB) 1μl, 8U / μl Bst2.0 enzyme (BstDNA polymerase) 1μl, template 1μl, sterilized ultrapure water ...

Embodiment 3

[0047] Broad-spectrum and specificity evaluation of embodiment 3 mycoplasma LAMP detection kit

[0048] The purpose of the present invention is to develop a kit capable of detecting common mycoplasma contamination in the cell culture process, which not only satisfies broad-spectrum mycoplasma detection, but must be specific to non-mycoplasma samples. This embodiment uses ten kinds of mycoplasma samples M.hyorhinis, M.fermentans, M.arginini, M.hominis, M.orale, M.salivarium, M.pirum, M.pulmonis, M.bovis, M.buccale as broad-spectrum Specific detection objects, Escherichia coli, Salmonella enteritidis and Staphylococcus aureus as specific detection objects. The results show that the LAMP reaction system has good broad-spectrum and specificity, and can detect the above ten kinds of mycoplasma samples, and the detection of three non-mycoplasma environmental microorganisms is negative, such as Figure 2a shown. Figure 2b The results of electrophoresis on 2% agarose gel are shown....

technical field

[0001] The invention relates to the field of biotechnology, in particular to a LAMP primer set for detecting broad-spectrum mycoplasma, a kit and an application thereof. Background technique

[0002] In the culture process of mammalian cells, Mycoplasma contamination has always been a troublesome problem. There are three main reasons: 1). It is difficult to see its morphological structure; 2) Mycoplasma is tenacious and can survive under alkaline conditions (pH7.6-8.0), and is resistant to penicillin; 3) In the initial stage of cell contamination by mycoplasma, some sensitive cells can be seen Cell growth and proliferation slowed down, and the shape became round. However, most cells have no obvious changes after contamination, or have slight changes. If they are not treated in time, cross-contamination will also occur. Mycoplasma eventually causes cell death, but more often, mycoplasma coexists with cells. During this process, mycoplasma slowly proliferate...

Examples

Embodiment 1

[0024] Embodiment 1, primer design

[0025] The ITS-23srRNA sequences of 8 kinds of mycoplasma M.hyorhinis, M.fermentans, M.arginini, M.hominis, M.orale, M.salivarium, M.pirum, and AcholeplasmaLaidlawii commonly found in cell culture were retrieved from the American gene database Genbank, Homologous sequence analysis and alignment was performed by clustalX software to find specific conserved target gene fragments. In this example, the 5' end region of the 23srRNA sequence was selected, and primerexplorerV4 was used for online LAMP primer design to obtain F3 / B3 and FIP / BIP primers, and then appropriate loop primers were selected through manual adjustment. The designed primers were analyzed online for their specificity and broad-spectrum through the primer-blast software, and the final optimized primers were as follows:

[0026] Outer primer F3:

[0027] 5'-AAGAGTTTTTGGTGGATGC-3' (as shown in SEQ ID NO.1)

[0028] Outer primer B3:

[0029] 5'-CTGCTGCTACTGAGATGTT-3' (as shown...

Embodiment 2

[0038] Embodiment 2, setting up mycoplasma LAMP reaction system

[0039]Set different final concentrations of magnesium ions (4mM, 6mM, 8mM), dNTP concentrations (1mM, 1.2mM, 1.4mM), betaine (Betaine) concentrations (0.6M, 0.8M, 1M) and Bst2.0 enzyme (BstDNA polymerization enzyme) concentration (4U, 6U, 8U, 10U).

[0040] After the reaction, the color change of the system can be observed with the naked eye, such as figure 1 As shown, the negative is blue-purple (left picture), and the positive is sky blue (right picture).

[0041] The most preferred mycoplasma LAMP reaction system was determined through trial and error, that is, the preparation ratio of 25 μl LAMP reaction solution was: 10×Isothermal Amplification Buffer (isothermal amplification buffer) 2.5 μl, 10×LAMPprimermix (primer mixture) 2.5 μl, 10mMdNTPs 3 μl, 100mM MgSO 4 1.5 μl, 5MBetaine 4μl, 3mM hydroxynaphthol blue (HNB) 1μl, 8U / μl Bst2.0 enzyme (BstDNA polymerase) 1μl, template 1μl, sterilized ultrapure water ...

Embodiment 3

[0047] Broad-spectrum and specificity evaluation of embodiment 3 mycoplasma LAMP detection kit

[0048] The purpose of the present invention is to develop a kit capable of detecting common mycoplasma contamination in the cell culture process, which not only satisfies broad-spectrum mycoplasma detection, but must be specific to non-mycoplasma samples. This embodiment uses ten kinds of mycoplasma samples M.hyorhinis, M.fermentans, M.arginini, M.hominis, M.orale, M.salivarium, M.pirum, M.pulmonis, M.bovis, M.buccale as broad-spectrum Specific detection objects, Escherichia coli, Salmonella enteritidis and Staphylococcus aureus as specific detection objects. The results show that the LAMP reaction system has good broad-spectrum and specificity, and can detect the above ten kinds of mycoplasma samples, and the detection of three non-mycoplasma environmental microorganisms is negative, such as Figure 2a shown. Figure 2b The results of electrophoresis on 2% agarose gel are shown....

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