CN109880896A - A kind of multiple LAMP kit and detection method for the specific parting of quick discriminating bacteria polymyxins drug resistant gene mcr - Google Patents
A kind of multiple LAMP kit and detection method for the specific parting of quick discriminating bacteria polymyxins drug resistant gene mcr Download PDFInfo
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Abstract
The invention discloses a kind of multiple LAMP kits and detection method for the specific parting of quick discriminating bacteria polymyxins drug resistant gene mcr.The present invention devises five pairs of outer primers, five pairs of inner primers and five pairs of ring primers, the specific parting of precise Identification drug resistant gene mcr in being reacted at one, the specific parting of drug resistant gene mcr can be distinguished by Multi-LAMP rapid reaction simultaneously, but also have many advantages, such as rapidly and efficiently, easy to operate, high specific, high sensitivity, detection is easy, is suitble to on-site test, clinically there is high application value.
Description
Technical field
The invention belongs to nucleic acid tests fields, have about one kind for quick discriminating bacteria polymyxins drug resistant gene mcr
The multiple LAMP kit and detection method of body parting.
Background technique
Polymyxins is cationic polypeptide antibiotic family, has broad spectrum antibiotic activity, sterilization machine to gram negative bacilli
There are electrostatic phases between the system residue positively charged dependent on the polymyxins lipid A negatively charged with bacteria wall outer membrane is attached to
The permeability changes of interaction, cell wall lead to bacterial death, to play bactericidal effect, it is multi-drug resistant to be typically considered confrontation
The last line of defense of gram-negative bacterial infections.
Report has found plasmid-mediated polymyxins drug resistance new gene mcr-1, and water can occur between different strain
It flates pass and broadcasts, this causes the extensive concern in the whole world.In life, the strain for carrying drug resistant gene mcr includes escherichia coli, pneumonia gram
The primary bacterium of thunder, clostridium perfringen, bacillus cloacae, salmonella, shigella sonnei, Citrobacter, solution ornithine Raoul bacterium,
Enterobacter sakazakii, Kluyvera, Hosts are also sufficiently complex.The carrying rate of drug resistant gene mcr is also because of sample and area
It is different and different.
In the world altogether report eight mcr gene families (mcr-1, mcr-2, mcr-3, mcr-4, mcr-5, mcr-6,
mcr-7,mcr-8).Mcr-1 and mcr-3 is present in the enterobacteriaceae of different regions in MCR family, in addition the report of six types
It is less.Although mcr gene family is accredited in enterobacteriaceae and non-enterobacteriaceae and comes out, main host is still big
Enterobacteria.Because first five kind gene type it is more common, the amino acid identity with mcr-1 be respectively 81%, 34%, 33% and
31%.China is in the majority with mcr-1, mcr-3 and mcr-4.
Traditional culture of microorganism and drug sensitive test method is required because of the operating process that time-consuming, cumbersome, to instrument and equipment
The reasons such as height fail clinically large-scale promotion use.It is resistance to thus to research and develop a kind of simply and quickly identification bacterium polymyxins
The method of the specific parting of medicine gene mcr is required at present.
Summary of the invention
The purpose of the present invention is to provide a kind of for the specific parting of quick discriminating bacteria polymyxins drug resistant gene mcr
The primer of multiple LAMP.
The purpose of the present invention is to provide a kind of for the specific parting of quick discriminating bacteria polymyxins drug resistant gene mcr
The kit of multiple LAMP.
The purpose of the present invention is to provide a kind of for the specific parting of quick discriminating bacteria polymyxins drug resistant gene mcr
The detection method of multiple LAMP.
The technical solution used in the present invention is:
A kind of multiple LAMP primer group for identifying the specific parting of bacterium polymyxins drug resistant gene mcr, nucleotide sequence
It is as follows:
Mcr-1 outer primer
Mcr-1-F:5'-TGATGCAGCATACTTCTGTG-3'(SEQ ID No.1)；
Mcr-1-R:5'-GACCGTGCCATAAGTGTC-3'(SEQ ID No.2)；
Mcr-1 inner primer
Mcr-1-FIP:5'-GCGATGGGATAGGTTTGGCTAAGCTTGTGTTGCCGTTTTCTTG AC-3'(SEQ ID
No.3)；
Mcr-1-BIP:5'-TGCTGACGATCGCTGTCGAAGCTTGCACATAGCGATACGATGA T-3'(SEQ ID
No.4)；
Mcr-1 ring primer
Mcr-1-LF:5'-AAGGTAAGATTGGCGGTCG-3'(SEQ ID No.5)；
Mcr-1-LB:5'-CTACTGATCACCACGCTGTT-3'(SEQ ID No.6)；
Mcr-2 outer primer
Mcr-2-F:5'-CAAAGACGCCGTGCAGAC-3'(SEQ ID No.7)；
Mcr-2-R:5'-CAGAATACGCCGTCGATGT-3'(SEQ ID No.8)；
Mcr-2 inner primer
Mcr-2-FIP:5'-ACTGCACATGGTCAGCACGCAAGCTTGAGCGTAAGCCACGCCT A-3'(SEQ ID
No.9)；
Mcr-2-BIP:
5'-GGCTATGGCCGTGAGACTTTCCAAGCTTGCCACACGATGTCACTTGG-3'(SEQ ID No.10)；
Mcr-2 ring primer
Mcr-2-LF:5'-ACCGACGACGAACACCAC-3'(SEQ ID No.11)；
Mcr-2-LB:5'-CTTGCCAAAGTTGATGGCTTG-3'(SEQ ID No.12)；
Mcr-3 outer primer
Mcr-3-F:5'-CATTACCAATATTGCTTGTTGC-3'(SEQ ID No.13)；
Mcr-3-R:5'-TTGGCTGGAACAATCTCAC-3'(SEQ ID No.14)；
Mcr-3 inner primer
Mcr-3-FIP:
5'-GCTAACGCCTCATTTTGATTGGAAGCTTGCACTTCTTATCGCACTTAG-3'(SEQ ID No.15)；
Mcr-3-BIP:
5'-TCAAAGGGATTCTAACTCGTGCAAGCTTGCAATAACCGCAATCACTAT-3'(SEQ ID No.16)；
Mcr-3 ring primer
Mcr-3-LF:5'-TCATTGTGTAACTAACGATTGC-3'(SEQ ID No.17)；
Mcr-3-LR:5'-CCTATCGATGTTTGCATCACTT-3'(SEQ ID No.18)；
Mcr-4 outer primer
Mcr-4-F:5'-TGAGTTAAGGCGTTACATTGT-3'(SEQ ID No.19)；
Mcr-4-R:5'-CGCATGAGCTAGTATCGTTAA-3'(SEQ ID No.20)；
Mcr-4 inner primer
Mcr-4-FIP:5'-TTACGACTGGCATTCTTCGCAAAGCTTCTATTTGCAGACGCCC AT-3'(SEQ ID
No.21)；
Mcr-4-BIP:
5'-AGTGGTTGTTGTGGGTGAAACTAAGCTTAGCATTGGTTGGCTTGTTA-3'(SEQ ID No.22)；
Mcr-4 ring primer
Mcr-4-LF:5'-TCTAGGCCAAGTTGTTGGTATT-3'(SEQ ID No.23)；
Mcr-4-LB:5'-CGCGCTCAATGAGCTATCA-3'(SEQ ID No.24)；
Mcr-5 outer primer
Mcr-5-F:5'-CAATGGAGAATGCTGCCCTA-3'(SEQ ID No.25)；
Mcr-5-R:5'-GCGTGGGTATCAGCACATC-3'(SEQ ID No.26)；
Mcr-5 inner primer
Mcr-5-FIP:5'-AGCCCGTTCGTAAAACCCTGACAAGCTTCTTGTTGGTTGCAGC CGT-3'(SEQ ID
No.27)；
Mcr-5-BIP:5'-AGCGGTAATGATGCGCAGCGAAGCTTCATGACTGGCCACAGAC C-3'(SEQ ID
No.28)；
Mcr-5 ring primer
Mcr-5-LF:5'-CTCGCAATCCACCACACGGAT-3'(SEQ ID No.29)；
Mcr-5-BF:5'-TGGCGCTCTCGCCATGA-3'(SEQ ID No.30).
A kind of multiple LAMP kit for identifying the specific parting of bacterium polymyxins drug resistant gene mcr, shown in above-mentioned
Primer sets.
Further, the inner primer in the primer sets: outer primer: the molar ratio of ring primer is (6-8): 1:(3-4).
It further, further include archaeal dna polymerase, LAMP reaction solution, color developing agent, restriction enzyme, enzyme cutting buffering liquid, sun
Property control and negative control；Preferably, color developing agent is SYBR GREEN I.
Further, the archaeal dna polymerase is Bst archaeal dna polymerase；The restriction enzyme is III restriction endonuclease of Hind.
Further, 10mM dNTP, 10 × ThermoPol reaction buffer, 150mM in the LAMP reaction solution
MgSO4The volume ratio of aqueous solution and 5mM glycine betaine is (6-8): (4-5): (2-3): (8-10).
A method of the identification specific parting of bacterium polymyxins drug resistant gene mcr includes the following steps:
(1) measuring samples DNA is extracted；
(2) LAMP amplified reaction is carried out to measuring samples DNA using above-mentioned primer sets and obtains product；
(3) LAMP amplified production is analyzed, determines in sample to be tested whether contain bacterium polymyxins drug resistant gene
Mcr gene；
(4) digestion LAMP product: taking the LAMP product containing bacterium polymyxins drug resistant gene mcr, and DEPC water is added to dilute
After carry out endonuclease reaction；
(5) digestion result judges: the product after endonuclease reaction being carried out gel electrophoresis analysis, judges that bacterium polymyxins is resistance to
The specific parting of medicine gene mcr；
The above method is not suitable for the diagnosing and treating of disease.
Further, step (2) LAMP amplification reaction system are as follows: mcr-1-F 0.2pmol/ μ L, mcr-1-R
0.2pmol/μL、mcr-1-FIP 0.25pmol/μL、mcr-1-BIP 0.25pmol/μL、mcr-1-LF 1pmol/μL、mcr-
1-LB 1pmol/μL、mcr-2-F 0.2pmol/μL、mcr-2-R 0.2pmol/μL、mcr-2-FIP 0.25pmol/μL、
mcr-2-BIP 0.25pmol/μL、mcr-2-LF 1pmol/μL、mcr-2-LB 1pmol/μL、mcr-3-F 0.2pmol/μL、
mcr-3-R 0.2pmol/μL、mcr-3-FIP 0.25pmol/μL、mcr-3-BIP 0.25pmol/μL、mcr-3-LF
1pmol/μL、mcr-3-LB 1pmol/μL、mcr-4-F 0.2pmol/μL、mcr-4-R 0.2pmol/μL、mcr-4-FIP
0.25pmol/μL、mcr-4-BIP 0.25pmol/μL、mcr-4-LF 1pmol/μL、mcr-4-LB 1pmol/μL、mcr-5-F
0.2pmol/μL、mcr-5-R 0.2pmol/μL、mcr-5-FIP 0.25pmol/μL、mcr-5-BIP 0.25pmol/μL、
Mcr-5-LF 1pmol/ μ L, mcr-5-LB 1pmol/ μ L, 12.5 μ L of LAMP reaction solution, archaeal dna polymerase 8U, DNA to be checked
50ng adds the sealing fluid of 20 μ L with sterile deionized water polishing to 25 μ L；The LAMP reaction solution include 10mM dNTP,
10 × Thermo Pol reaction buffer, 150mM MgSO4And 5mM glycine betaine；Preferably, LAMP amplification reaction condition are as follows:
60-63 DEG C of reaction 45-60min.
Further, step (3) is in LAMP amplified production analytic process are as follows: 1-2 μ is added in above-mentioned LAMP amplified production
It is mixed after L color developing agent SYBR Green I, the color change of product after observation mixes contains the more Acarasiales of bacterium if green is presented
Plain drug resistant gene mcr, it is orange not contain bacterium polymyxins drug resistant gene mcr gene then.
Further, step (4) endonuclease reaction system are as follows: the diluted LAMP product of 2~3 μ L, 1 μ L restriction enzyme
Hind III, 2 μ L10 × enzyme cutting buffering liquid, with DEPC water polishing to 20 μ L；The endonuclease reaction condition are as follows: 37 DEG C of reaction 15-
60min。
The multiple LAMP technology (Multi-LAMP technology) of the present invention is also referred to as multiple ring mediated isothermal gene amplification technology.
The beneficial effects of the present invention are:
The specificity of directed toward bacteria polymyxins drug resistant gene mcr-1, mcr-2, mcr-3, mcr-4 and mcr-5 of the present invention,
Primer is separately designed, in conjunction with 6 regions of target gene, specificity with higher can be accurately by the more Acarasiales of bacterium
Plain drug resistant gene mcr is distinguished with other non-bacterial polymyxins drug resistant gene mcr.There is accuracy high, specific good, repeats
Property is good, the advantages of accurately and rapidly analyzing, and is conducive to promote and apply in clinical practice.
Detailed description of the invention
Fig. 1 is embodiment 3 to bacterium polymyxins drug resistant gene mcr-1 (A, F scheme), mcr-2 (B, G figure), mcr-3 (C, H
Figure), mcr-4 (D, I figure) and mcr-5 (E, J scheme) and the specific amplification result to non-bacterial polymyxins drug resistant gene mcr
(1:mcr-1,2:mcr-2,3:mcr-3,4:mcr-4,5:mcr-5,6:KPC-2,7:NDM-1,8:CTX-M-9,9: negative right
According to)；
Fig. 2 is embodiment 4 to bacterium polymyxins drug resistant gene mcr-1 (A, F, K scheme), mcr-2 (B, G, L figure), mcr-3
(C, H, M figure), mcr-4 (D, I, N figure) and mcr-5 (E, J, O figure) progress LAMP and Standard PCR remolding sensitivity compared with experimental result
(A-E: the colour developing result of LAMP amplification of the present invention；F-J: the electrophoresis result of LAMP amplification of the present invention；Wherein, swimming lane 1~7 be containing
There is the pMD19-T plasmid of bacterium polymyxins drug resistant gene mcr, concentration is followed successively by 1.0 × 108、1.0×107、1.0×106、
1.0×105、1.0×104、1.0×103、1.0×102Copy/μ L, swimming lane 8 are negative control DEPC water, M Marker)
Fig. 3 A is multiple LAMP (Multi-LAMP) gel electrophoresis result of mcr-1, mcr-3 and the mcr-4 of embodiment 5
(1:mcr-1,2:mcr-3,3:mcr-4,4:mcr-1/mcr-3/mcr-4,5:DEPC water).
Fig. 3 B is multiple LAMP (Multi-LAMP) gel electrophoresis result (1:mcr- of the mcr-2 and mcr-3 of embodiment 6
2,2:mcr-5,3:mcr-2/mcr-5,4:DEPC water).
Specific embodiment
The present invention is further illustrated combined with specific embodiments below.
The LAMP kit of the detection bacterium polymyxins drug resistant gene mcr of embodiment 1 a kind of
A kind of LAMP kit of detection bacterium polymyxins drug resistant gene mcr, including following component: (1) specificity is drawn
Object group；(2) archaeal dna polymerase；(3) LAMP reaction solution；(4) color developing agent；(5) restriction enzyme Hind III and 10 × digestion buffering
Liquid；(6) positive control and negative control.
(1) design of specific primer:
According to bacterium polymyxins drug resistant gene mcr-1 (GenBank accession number are as follows: NG_050417.1), mcr-2
(GenBank accession number are as follows: NG_051171.1), mcr-3 (GenBank accession number are as follows: KY924928.1), mcr-4 (GenBank
Accession number are as follows: MF543359.1) and mcr-5 (GenBank accession number are as follows: KY807921.1) specificity, separately design 6 spies
Specific primer, nucleotide sequence difference are as follows:
Mcr-1 outer primer
Mcr-1-F:5'-TGATGCAGCATACTTCTGTG-3'(SEQ ID No.1)；
Mcr-1-R:5'-GACCGTGCCATAAGTGTC-3'(SEQ ID No.2)；
Mcr-1 inner primer
Mcr-1-FIP:5'-GCGATGGGATAGGTTTGGCTAAGCTTGTGTTGCCGTTTTCTTG AC-3'(SEQ ID
No.3)；
Mcr-1-BIP:5'-TGCTGACGATCGCTGTCGAAGCTTGCACATAGCGATACGATGA T-3'(SEQ ID
No.4)；
Mcr-1 ring primer
Mcr-1-LF:5'-AAGGTAAGATTGGCGGTCG-3'(SEQ ID No.5)；
Mcr-1-LB:5'-CTACTGATCACCACGCTGTT-3'(SEQ ID No.6)；
Mcr-2 outer primer
Mcr-2-F:5'-CAAAGACGCCGTGCAGAC-3'(SEQ ID No.7)；
Mcr-2-R:5'-CAGAATACGCCGTCGATGT-3'(SEQ ID No.8)；
Mcr-2 inner primer
Mcr-2-FIP:5'-ACTGCACATGGTCAGCACGCAAGCTTGAGCGTAAGCCACGCCT A-3'(SEQ ID
No.9)；
Mcr-2-BIP:5'-GGCTATGGCCGTGAGACTTTCCAAGCTTGCCACACGATGTCAC TTGG-3'(SEQ
ID No.10)；
Mcr-2 ring primer
Mcr-2-LF:5'-ACCGACGACGAACACCAC-3'(SEQ ID No.11)；
Mcr-2-LB:5'-CTTGCCAAAGTTGATGGCTTG-3'(SEQ ID No.12)；
Mcr-3 outer primer
Mcr-3-F:5'-CATTACCAATATTGCTTGTTGC-3'(SEQ ID No.13)；
Mcr-3-R:5'-TTGGCTGGAACAATCTCAC-3'(SEQ ID No.14)；
Mcr-3 inner primer
Mcr-3-FIP:5'-GCTAACGCCTCATTTTGATTGGAAGCTTGCACTTCTTATCGCA CTTAG-3'(SEQ
ID No.15)；
Mcr-3-BIP:
5'-TCAAAGGGATTCTAACTCGTGCAAGCTTGCAATAACCGCAATCACTAT-3'(SEQ ID No.16)；
Mcr-3 ring primer
Mcr-3-LF:5'-TCATTGTGTAACTAACGATTGC-3'(SEQ ID No.17)；
Mcr-3-LR:5'-CCTATCGATGTTTGCATCACTT-3'(SEQ ID No.18)；
Mcr-4 outer primer
Mcr-4-F:5'-TGAGTTAAGGCGTTACATTGT-3'(SEQ ID No.19)；
Mcr-4-R:5'-CGCATGAGCTAGTATCGTTAA-3'(SEQ ID No.20)；
Mcr-4 inner primer
Mcr-4-FIP:5'-TTACGACTGGCATTCTTCGCAAAGCTTCTATTTGCAGACGCCC AT-3'(SEQ ID
No.21)；
Mcr-4-BIP:5'-AGTGGTTGTTGTGGGTGAAACTAAGCTTAGCATTGGTTGGCTT GTTA-3'(SEQ
ID No.22)；
Mcr-4 ring primer
Mcr-4-LF:5'-TCTAGGCCAAGTTGTTGGTATT-3'(SEQ ID No.23)；
Mcr-4-LB:5'-CGCGCTCAATGAGCTATCA-3'(SEQ ID No.24)；
Mcr-5 outer primer
Mcr-5-F:5'-CAATGGAGAATGCTGCCCTA-3'(SEQ ID No.25)；
Mcr-5-R:5'-GCGTGGGTATCAGCACATC-3'(SEQ ID No.26)；
Mcr-5 inner primer
Mcr-5-FIP:5'-AGCCCGTTCGTAAAACCCTGACAAGCTTCTTGTTGGTTGCAGC CGT-3'(SEQ ID
No.27)；
Mcr-5-BIP:5'-AGCGGTAATGATGCGCAGCGAAGCTTCATGACTGGCCACAGAC C-3'(SEQ ID
No.28)；
Mcr-5 ring primer
Mcr-5-LF:5'-CTCGCAATCCACCACACGGAT-3'(SEQ ID No.29)；
Mcr-5-BF:5'-TGGCGCTCTCGCCATGA-3'(SEQ ID No.30).
(2) archaeal dna polymerase is Bst archaeal dna polymerase；
(3) LAMP reaction solution contains: 10mM dNTP, 10 × Thermo Pol reaction buffer, 150mM MgSO4And
5mM glycine betaine, the volume ratio of four substances are 8:5:3:10；
(4) color developing agent is SYBR GREEN I；
(5) restriction enzyme is Hind III；
(6) positive control be respectively contain bacterium polymyxins drug resistant gene mcr-1, mcr-2, mcr-3, mcr-4 and
The pMD19-T plasmid of mcr-5 segment；Negative control is DEPC water.
The LAMP detection method of the bacterium polymyxins drug resistant gene mcr of embodiment 2 a kind of
Utilize the quick detection bacterium polymyxins drug resistant gene mcr of the kit of embodiment 1, the specific steps are as follows:
(1) it extracts template DNA: bacterial genomes DNA is extracted using water-boiling method；
(2) ring mediated isothermal gene amplification reaction (LAMP reaction) system contains: mcr-1-F 0.2pmol/ μ L, mcr-1-
R 0.2pmol/μL、mcr-1-FIP 0.25pmol/μL、mcr-1-BIP 0.25pmol/μL、mcr-1-LF 1pmol/μL、
mcr-1-LB 1pmol/μL、mcr-2-F 0.2pmol/μL、mcr-2-R 0.2pmol/μL、mcr-2-FIP 0.25pmol/μ
L、mcr-2-BIP 0.25pmol/μL、mcr-2-LF 1pmol/μL、mcr-2-LB 1pmol/μL、mcr-3-F 0.2pmol/μ
L、mcr-3-R 0.2pmol/μL、mcr-3-FIP 0.25pmol/μL、mcr-3-BIP 0.25pmol/μL、mcr-3-LF
1pmol/μL、mcr-3-LB 1pmol/μL、mcr-4-F 0.2pmol/μL、mcr-4-R 0.2pmol/μL、mcr-4-FIP
0.25pmol/μL、mcr-4-BIP 0.25pmol/μL、mcr-4-LF 1pmol/μL、mcr-4-LB 1pmol/μL、mcr-5-F
0.2pmol/μL、mcr-5-R 0.2pmol/μL、mcr-5-FIP0.25pmol/μL、mcr-5-BIP 0.25pmol/μL、mcr-
5-LF 1pmol/ μ L, mcr-5-LB 1pmol/ μ L, 12.5 μ L of LAMP reaction solution, archaeal dna polymerase 8U, DNA 50ng to be checked are used
Sterile deionized water polishing adds the sealing fluid of 20 μ L to 25 μ L；By above-mentioned reaction tube in 63 DEG C of reaction 60min；
(3) result judges: 1 μ L 1 × SYBR of color developing agent Green I being added in above-mentioned reaction tube, observes solution after mixing
Color change, if present green if contain bacterium polymyxins drug resistant gene mcr-1, mcr-2, mcr-3, mcr-4 or mcr-
5, bacterium polymyxins drug resistant gene mcr (A~E of such as Fig. 1) is not contained if presentation is orange.
3 specificity experiments of embodiment
Method: the DNA extracted using clinical separation strain is carried out as template using the reaction system and reaction condition of embodiment 2
LAMP amplified reaction, to verify the specificity of the method for designed primer and foundation.
As a result: testing result is shown in A~E of Fig. 1, and only bacterium polymyxins drug resistant gene mcr pipe is green, remaining is non-bacterial
Polymyxins drug resistant gene mcr pipe is orange.The result shows that detection kit specificity of the invention is high, it can be accurately by mcr
And other non-mcr are distinguished.
4 sensitivity experiment of embodiment
With positive reference substance (positive control built be containing bacterium polymyxins drug resistant gene mcr-1, mcr-2,
The pMD19-T plasmid of mcr-3, mcr-4 and mcr-5 segment), it measures its concentration and calculates copy number, according to 10 times of concentration
Gradient dilution chooses 1.0 × 102~1.0 × 108Copy/μ L concentration is tested as sample, then uses this hair respectively
Bright detection method and conventional PCR method is detected.
Primer sequence used in Standard PCR detection method is as follows:
Mcr-1-F1:5 '-AGTCCGTTTGTTCTTGTGGC-3 ' (SEQ ID NO.31)；
Mcr-1-R1:5 '-AGATCCTTGGTCTCGGCTTG-3 ' (SEQ ID NO.32)；
Mcr-2-F1:5 '-CAAGTGTGTTGGTCGCAGTT-3 ' (SEQ ID NO.33)；
Mcr-2-R1:5 '-TCTAGCCCGACAAGCATACC-3 ' (SEQ ID NO.34)；
Mcr-3-F1:5 '-AAATAAAAATTGTTCCGCTTATG-3 ' (SEQ ID NO.35)；
Mcr-3-R1:5 '-AATGGAGATCCCCGTTTTT-3 ' (SEQ ID NO.36)；
Mcr-4-F1:5 '-TCACTTTCATCACTGCGTTG-3 ' (SEQ ID NO.37)；
Mcr-4-R1:5 '-TTGGTCCATGACTACCAATG-3 ' (SEQ ID NO.38)；
Mcr-5-F1:5 '-ATGCGGTTGTCTGCATTTATC-3 ' (SEQ ID NO.39)；
Mcr-5-R1:5 '-TCATTGTGGTTGTCCTTTTCTG-3 ' (SEQ ID NO.40)；
The system of conventional PCR method is identical with the system of existing PCR method.The testing result of kit of the present invention such as Fig. 2
Shown in (A-E is colour developing figure, and F-J is gel electrophoresis figure), the results showed that, this kit is to mcr-1, mcr-2, mcr-4 and mcr-5
The minimum detectability of gene is equal are as follows: 1.0 × 104Copy/μ L, and the minimum detectability of mcr-3 gene are as follows: 1.0 × 105Copy/μ
L。
Shown in the testing result of conventional PCR method such as Fig. 2 (K-O), conventional PCR method to mcr-1, mcr-2, mcr-3,
Mcr-4 and mcr-5 gene minimum detectability is equal are as follows: 1.0 × 105Copy/μ L.
Therefore, LAMP detection method prepared by the present invention is higher than conventional PCR method sensitivity.
5 mcr-1 of embodiment, multiple LAMP (Multi-LAMP) the reaction product detection of mcr-3 and mcr-4 gene
With positive reference substance, (positive control built is to contain bacterium polymyxins drug resistant gene mcr-1, mcr-3
With the pMD19-T plasmid of mcr-4 segment), it is detected with detection method of the invention, LAMP product is taken to be diluted with water 1 times, into
Row endonuclease reaction, takes 5 μ L digestion products to carry out 2% agarose gel electrophoresis, and electrophoresis result is as shown in Figure 3A.
As a result: after digestion, mcr-1 digestion rear electrophoresis band theoretical value is 170bp and 115bp, and mcr-3 theoretical value is
260bp and 155bp；Mcr-4 theoretical value is 270bp and 230bp, 182bp；Mcr-1, mcr-3 and mcr-4 are carried simultaneously, it is theoretical
Value is 260bp, 225bp, 170bp, 120bp and 90bp.
Multiple LAMP (Multi-LAMP) reaction product of 6 mcr-2 and mcr-5 gene of embodiment detects
With positive reference substance, (positive control built is to contain bacterium polymyxins drug resistant gene mcr-2 and mcr-5
The pMD19-T plasmid of segment), it is detected with the detection method of the embodiment of the present invention, LAMP product is taken to be diluted with water 1 times, into
Row endonuclease reaction, takes 5 μ L digestion products to carry out 2% agarose gel electrophoresis analysis, and electrophoresis result is as shown in Figure 3B.
As a result: after digestion, mcr-2 theoretical value is 220bp and 175bp, 140bp；Mcr-5 theoretical value is 120bp and 90bp；
The theoretical value for carrying mcr-2 and mcr-5 simultaneously is 215bp, 170bp, 140bp, 120bp and 90bp.
Above embodiments show that LAMP detection kit of the invention has that accuracy is good, high sensitivity, high specificity
Feature, and the specific parting of the drug resistant gene mcr of energy 100% precise Identification bacterium carrying, are suitble to on-site test.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc. within mind and principle, should all be comprising within protection scope of the present invention.
SEQUENCE LISTING
<110>Zhongshan University
<120>a kind of multiple LAMP kit for the specific parting of quick discriminating bacteria polymyxins drug resistant gene mcr and
Detection method
<130>
<160> 40
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213>artificial primer
<400> 1
tgatgcagca tacttctgtg 20
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<212> DNA
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<400> 2
gaccgtgcca taagtgtc 18
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<211> 45
<212> DNA
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gcgatgggat aggtttggct aagcttgtgt tgccgttttc ttgac 45
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<212> DNA
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<400> 4
tgctgacgat cgctgtcgaa gcttgcacat agcgatacga tgat 44
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<400> 5
aaggtaagat tggcggtcg 19
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ctactgatca ccacgctgtt 20
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caaagacgcc gtgcagac 18
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cagaatacgc cgtcgatgt 19
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actgcacatg gtcagcacgc aagcttgagc gtaagccacg ccta 44
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<212> DNA
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ggctatggcc gtgagacttt ccaagcttgc cacacgatgt cacttgg 47
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accgacgacg aacaccac 18
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cttgccaaag ttgatggctt g 21
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cattaccaat attgcttgtt gc 22
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ttggctggaa caatctcac 19
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gctaacgcct cattttgatt ggaagcttgc acttcttatc gcacttag 48
<210> 16
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<212> DNA
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<400> 16
tcaaagggat tctaactcgt gcaagcttgc aataaccgca atcactat 48
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tcattgtgta actaacgatt gc 22
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cctatcgatg tttgcatcac tt 22
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tgagttaagg cgttacattg t 21
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cgcatgagct agtatcgtta a 21
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ttacgactgg cattcttcgc aaagcttcta tttgcagacg cccat 45
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agtggttgtt gtgggtgaaa ctaagcttag cattggttgg cttgtta 47
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tctaggccaa gttgttggta tt 22
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cgcgctcaat gagctatca 19
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caatggagaa tgctgcccta 20
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gcgtgggtat cagcacatc 19
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agcccgttcg taaaaccctg acaagcttct tgttggttgc agccgt 46
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agcggtaatg atgcgcagcg aagcttcatg actggccaca gacc 44
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ctcgcaatcc accacacgga t 21
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tggcgctctc gccatga 17
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agtccgtttg ttcttgtggc 20
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agatccttgg tctcggcttg 20
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caagtgtgtt ggtcgcagtt 20
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tctagcccga caagcatacc 20
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aaataaaaat tgttccgctt atg 23
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aatggagatc cccgttttt 19
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tcactttcat cactgcgttg 20
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ttggtccatg actaccaatg 20
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atgcggttgt ctgcatttat c 21
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tcattgtggt tgtccttttc tg 22
Claims (10)
1. a kind of multiple LAMP primer group for identifying the specific parting of bacterium polymyxins drug resistant gene mcr, which is characterized in that primer
The nucleotide sequence of group is as follows:
Mcr-1 outer primer
Mcr-1-F:5'-TGATGCAGCATACTTCTGTG-3'(SEQ ID No.1)；
Mcr-1-R:5'-GACCGTGCCATAAGTGTC-3'(SEQ ID No.2)；
Mcr-1 inner primer
Mcr-1-FIP:5'-GCGATGGGATAGGTTTGGCTAAGCTTGTGTTGCCGTTTTCTTG AC-3'(SEQ ID
No.3)；
Mcr-1-BIP:5'-TGCTGACGATCGCTGTCGAAGCTTGCACATAGCGATACGATGA T-3'(SEQ ID
No.4)；
Mcr-1 ring primer
Mcr-1-LF:5'-AAGGTAAGATTGGCGGTCG-3'(SEQ ID No.5)；
Mcr-1-LB:5'-CTACTGATCACCACGCTGTT-3'(SEQ ID No.6)；
Mcr-2 outer primer
Mcr-2-F:5'-CAAAGACGCCGTGCAGAC-3'(SEQ ID No.7)；
Mcr-2-R:5'-CAGAATACGCCGTCGATGT-3'(SEQ ID No.8)；
Mcr-2 inner primer
Mcr-2-FIP:5'-ACTGCACATGGTCAGCACGCAAGCTTGAGCGTAAGCCACGCCT A-3'(SEQ ID
No.9)；
Mcr-2-BIP:5'-GGCTATGGCCGTGAGACTTTCCAAGCTTGCCACACGATGTCAC TTGG-3'(SEQ ID
No.10)；
Mcr-2 ring primer
Mcr-2-LF:5'-ACCGACGACGAACACCAC-3'(SEQ ID No.11)；
Mcr-2-LB:5'-CTTGCCAAAGTTGATGGCTTG-3'(SEQ ID No.12)；
Mcr-3 outer primer
Mcr-3-F:5'-CATTACCAATATTGCTTGTTGC-3'(SEQ ID No.13)；
Mcr-3-R:5'-TTGGCTGGAACAATCTCAC-3'(SEQ ID No.14)；
Mcr-3 inner primer
Mcr-3-FIP:5'-GCTAACGCCTCATTTTGATTGGAAGCTTGCACTTCTTATCGCA CTTAG-3'(SEQ ID
No.15)；
Mcr-3-BIP:
5'-TCAAAGGGATTCTAACTCGTGCAAGCTTGCAATAACCGCAATCACTAT-3'(SEQ ID No.16)；
Mcr-3 ring primer
Mcr-3-LF:5'-TCATTGTGTAACTAACGATTGC-3'(SEQ ID No.17)；
Mcr-3-LR:5'-CCTATCGATGTTTGCATCACTT-3'(SEQ ID No.18)；
Mcr-4 outer primer
Mcr-4-F:5'-TGAGTTAAGGCGTTACATTGT-3'(SEQ ID No.19)；
Mcr-4-R:5'-CGCATGAGCTAGTATCGTTAA-3'(SEQ ID No.20)；
Mcr-4 inner primer
Mcr-4-FIP:5'-TTACGACTGGCATTCTTCGCAAAGCTTCTATTTGCAGACGCCC AT-3'(SEQ ID
No.21)；
Mcr-4-BIP:5'-AGTGGTTGTTGTGGGTGAAACTAAGCTTAGCATTGGTTGGCTT GTTA-3'(SEQ ID
No.22)；
Mcr-4 ring primer
Mcr-4-LF:5'-TCTAGGCCAAGTTGTTGGTATT-3'(SEQ ID No.23)；
Mcr-4-LB:5'-CGCGCTCAATGAGCTATCA-3'(SEQ ID No.24)；
Mcr-5 outer primer
Mcr-5-F:5'-CAATGGAGAATGCTGCCCTA-3'(SEQ ID No.25)；
Mcr-5-R:5'-GCGTGGGTATCAGCACATC-3'(SEQ ID No.26)；
Mcr-5 inner primer
Mcr-5-FIP:5'-AGCCCGTTCGTAAAACCCTGACAAGCTTCTTGTTGGTTGCAGC CGT-3'(SEQ ID
No.27)；
Mcr-5-BIP:5'-AGCGGTAATGATGCGCAGCGAAGCTTCATGACTGGCCACAGAC C-3'(SEQ ID
No.28)；
Mcr-5 ring primer
Mcr-5-LF:5'-CTCGCAATCCACCACACGGAT-3'(SEQ ID No.29)；
Mcr-5-BF:5'-TGGCGCTCTCGCCATGA-3'(SEQ ID No.30).
2. a kind of multiple LAMP kit for identifying the specific parting of bacterium polymyxins drug resistant gene mcr, which is characterized in that the examination
Agent box contains primer sets described in claim 1.
3. multiple LAMP kit according to claim 2, which is characterized in that the inner primer in the primer sets: drawing outside
Object: the molar ratio of ring primer is (6-8): 1:(3-4).
4. the multiple LAMP kit according to shown in claim 2, which is characterized in that further include archaeal dna polymerase, LAMP reaction
Liquid, color developing agent, restriction enzyme Hind III, enzyme cutting buffering liquid, positive control and negative control；Preferably, color developing agent is
SYBR GREENⅠ。
5. multiple LAMP kit according to claim 4, which is characterized in that the archaeal dna polymerase is Bst DNA polymerization
Enzyme；The restriction enzyme is III restriction endonuclease of Hind.
6. multiple LAMP kit according to claim 4, which is characterized in that be 10mM in the LAMP reaction solution
DNTP, 10 × ThermoPol reaction buffer, 150mM MgSO4The volume ratio of aqueous solution and 5mM glycine betaine is (6-8): (4-
5): (2-3): (8-10).
7. a kind of method for identifying the specific parting of bacterium polymyxins drug resistant gene mcr, which comprises the steps of:
(1) measuring samples DNA is extracted；
(2) LAMP amplified reaction is carried out to measuring samples DNA using the primer sets of claim 1 and obtains product；
(3) LAMP amplified production is analyzed, determines in sample to be tested whether contain bacterium polymyxins drug resistant gene mcr base
Cause；
(4) digestion LAMP product: taking the LAMP product containing bacterium polymyxins drug resistant gene mcr, adds the dilution of DEPC water laggard
Row endonuclease reaction；
(5) digestion result judges: the product after endonuclease reaction being carried out gel electrophoresis analysis, judges bacterium polymyxins drug resistance base
Because of the specific parting of mcr；
The above method is not suitable for the diagnosing and treating of disease.
8. the method according to the description of claim 7 is characterized in that step (2) LAMP amplification reaction system are as follows: mcr-1-F
0.2pmol/μL、mcr-1-R 0.2pmol/μL、mcr-1-FIP 0.25pmol/μL、mcr-1-BIP 0.25pmol/μL、
mcr-1-LF 1pmol/μL、mcr-1-LB 1pmol/μL、mcr-2-F 0.2pmol/μL、mcr-2-R 0.2pmol/μL、
mcr-2-FIP 0.25pmol/μL、mcr-2-BIP 0.25pmol/μL、mcr-2-LF 1pmol/μL、mcr-2-LB 1pmol/
μL、mcr-3-F 0.2pmol/μL、mcr-3-R 0.2pmol/μL、mcr-3-FIP 0.25pmol/μL、mcr-3-BIP
0.25pmol/μL、mcr-3-LF 1pmol/μL、mcr-3-LB 1pmol/μL、mcr-4-F 0.2pmol/μL、mcr-4-R
0.2pmol/μL、mcr-4-FIP 0.25pmol/μL、mcr-4-BIP 0.25pmol/μL、mcr-4-LF 1pmol/μL、mcr-
4-LB 1pmol/μL、mcr-5-F 0.2pmol/μL、mcr-5-R 0.2pmol/μL、mcr-5-FIP 0.25pmol/μL、
Mcr-5-BIP 0.25pmol/ μ L, mcr-5-LF 1pmol/ μ L, mcr-5-LB 1pmol/ μ L, 12.5 μ L of LAMP reaction solution,
Archaeal dna polymerase 8U, DNA 50ng to be checked add the sealing fluid of 20 μ L with sterile deionized water polishing to 25 μ L；The LAMP
Reaction solution includes 10mM dNTP, 10 × Thermo Pol reaction buffer, 150mM MgSO4And 5mM glycine betaine；Preferably,
LAMP amplification reaction condition are as follows: 60-63 DEG C of reaction 45-60min.
9. the method according to the description of claim 7 is characterized in that step (3) is in LAMP amplified production analytic process are as follows:
It is mixed after 1-2 μ L color developing agent SYBR Green I is added in LAMP amplified production, the color change of product after observation mixes, if being in
Existing green then contains bacterium polymyxins drug resistant gene mcr, orange not contain bacterium polymyxins drug resistant gene mcr gene then.
10. the method according to the description of claim 7 is characterized in that step (4) endonuclease reaction system are as follows: 2~3 μ L are diluted
LAMP product, 1 μ L restriction enzyme Hind III, 2 μ 10 × enzyme cutting buffering liquids of L, with DEPC water polishing to 20 μ L；Preferably,
The endonuclease reaction condition are as follows: 37 DEG C of reaction 15-60min.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108504723A (en) * | 2018-06-01 | 2018-09-07 | 北京农学院 | A kind of Primer composition and application for detection bacterium MCR-1 genes |
| CN110777212A (en) * | 2019-09-25 | 2020-02-11 | 青岛农业大学 | Primer group for visually detecting drug-resistant gene of MCR-3 polymyxin and detection method thereof |
| CN112522378A (en) * | 2020-11-18 | 2021-03-19 | 深圳市疾病预防控制中心（深圳市卫生检验中心、深圳市预防医学研究所） | Kit for detecting MCR gene, detection method and application thereof |
| CN113817855A (en) * | 2021-10-22 | 2021-12-21 | 上海市计量测试技术研究院 | Digital PCR primer probe composition, kit and method for detecting polymyxin drug resistance gene |
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