CN109680080A - A kind of the nfo-RPA detection method and detection kit of Mycoplasma bovis - Google Patents
A kind of the nfo-RPA detection method and detection kit of Mycoplasma bovis Download PDFInfo
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Abstract
The invention discloses the nfo-RPA detection methods and detection kit of a kind of Mycoplasma bovis.The present invention is by the P80 gene order using Mycoplasma bovis as template, and going out from P80 gene cloning includes the M.b sequence of R1 sequence, using the R1 sequence as target design probe M.b-specific-Probe；Wherein, the M.b sequence is as shown in SEQ ID NO:1.Accordingly, detection kit of the invention, Freeze-dried reaction, 280mM MgAc, Rehydration buffer of its ingredient in addition to commercialization, outside Test device and PCRD Extraction buffer, it mainly include primer, probe and the matched reagent of specially designed Mycoplasma bovis specificity: M.b-specific-Forward, M.b-specific-Reverse, M.b-specific-Probe, Nuclease-free water, positive control template.The present invention is other than for laboratory conventional detection Mycoplasma bovis nucleic acid, on-site test especially suitable for (such as base's Veterinary office, farm) under no laboratory condition, with high specific and sensitivity, it is a kind of fast and accurately Mycoplasma bovis kit for detecting nucleic acid, has a good application prospect.
Description
Technical field
The invention belongs to molecular Biological Detection fields, specifically, being related to a kind of nfo-RP A detection of Mycoplasma bovis
Method and detection kit.
Background technique
Mycoplasma bovis (Mycoplasma bovis, M.bovis) is the important pathogenic pathogen of one kind of infected cattle, energy
Enough cause calf pneumonia, mammitis, otitis, genital tract inflammation, arthritis, keratoconjunctivitis, miscarriage and a variety of diseases such as infertile, this
A little diseases are referred to as Mycoplasma bovis related disease (MbAD).In recent years, MbAD worldwide wide-scale distribution, and ox branch
Substance can also cause secondary infection or mixed infection with other cause of diseases, cause huge economic loss to cattle-raising.
Currently, the detection method to Mycoplasma bovis mainly has Isolation and culture of agent, Serology test, molecular biology
Diagnostic method.Being separately cultured in terms of test operation for Mycoplasma bovis requires strictly, and separation identification difficulty is big, and separation cycle is long,
Only by visually observing when judging result, without enough reliable basis, the accuracy of experiment is influenced；Serologic detection it is not only time-consuming but also
Arduously, and sensitivity is not high；Molecular biology method is convenient and efficient, high sensitivity, mainly has Standard PCR, real-time fluorescence fixed
PCR (qPCR), loop-mediated isothermal amplification technique (LAMP) are measured, although these detection methods avoid lacking for above-mentioned two classes method
It falls into, but needs to operate in the laboratory for having precision instrument, limit the popularization and application on quick checkout and diagnosis at the scene.
Nfo-RPA method relies on specific primer and probe, effectively increases the specificity and sensibility of detection, greatly improves simultaneously
The accuracy rate of detection speed and testing result, has extensive practical, it can be ensured that quickly and accurately detect cause of disease
Body, has broad application prospects and development trend in terms of the fast qualitative detection of pathogen.
Summary of the invention
The purpose of the present invention is to provide the nfo-RPA detection methods and detection kit of a kind of Mycoplasma bovis, it is intended to solve
Certainly prior art sensibility is not high, and testing conditions require excessively high, and accuracy rate is low or reaction system is unstable, is not easy preservation etc. asks
Topic.
The invention is realized in this way a kind of nfo-RPA detection method of Mycoplasma bovis, detects ox by nfo-RPA method
Mycoplasma, this method using the P80 gene order of Mycoplasma bovis as template, design amplimer M.b-specific-Forward and
It includes the M.b sequence of R1 sequence that M.b-specific-Reverse goes out from P80 gene cloning, is visited by target design of the R1 sequence
Needle M.b-specific-Probe；Wherein, the M.b sequence is as shown in SEQ ID NO:1.
Preferably, the amplimer M.b-specific-Forward are as follows: 5 '-CAAAAACAATAAGTCAGTTAAAG
CTCTTTCAG-3 ',
Amplimer M.b-specific-Reverse are as follows: 5 '-Biotin-CTAACATCAGTACCTTTAAGTGTGAAA
ATAAC-3'；
Wherein, Biotin is biotin.
Preferably, the probe M.b-specific-Probe are as follows: 5 '-FAM-TTACAACAAATTCAAAGAATCTCTA
GCTACTA(THF)ATCTCTTACATTACTAG-SpC-3'；
Wherein, FAM is fluorescent reporter group, and SpC is fluorescent quenching group, and THF is tetrahydrofuran.
Preferably, the nfo-RPA method specifically includes the following steps:
(1) using the P80 gene of Mycoplasma bovis as template design primer, the M.b sequence comprising R1 sequence is obtained by amplification；
(2) gel extraction is connected to after purification on pMD18-T carrier, and connection product is then imported into DH-5 α competence
Cell filters out positive clone molecule；
(3) nfo-RPA reaction system is established, includes probe in the system, is separately added into ox toward the nfo-RPA reaction system
Mycoplasma and other genomic DNAs are template, detect positive findings.
Preferably, the nfo-RPA reaction system specifically: reaction system is 50 μ L, wherein Rehyd ration
Buffer 29.5uL, M.b-specific-Forward (10uM) 1.2uL, M.b-specific-Rever se (10uM)
1.2uL, M.b-specific-Probe (10uM) 0.6uL, Nuclease-free water 10uL, Template 5uL,
280mM MgAc 2.5uL。
Invention further discloses a kind of nfo-RPA detection kit of Mycoplasma bovis, which is characterized in that the kit packet
Include Freeze-dried reaction, 280mM MgAc, Rehydration buffer, amplimer M.b-specific-
Forward and M.b-specific-Reverse, probe M.b-specific-Probe, Nucl ease-free water, yin
Property control and positive control, Test device and PCRD Extraction buffer；Wherein,
Amplimer M.b-specific-Forward are as follows: 5 '-CAAAAACAATAAGTCAGTTAAAGCTCTTTCAG-
3 ',
Amplimer M.b-specific-Reverse are as follows: 5 '-Biotin-CTAACATCAGTACCTTTAAGTGTGAAA
ATAAC-3'；
Wherein, Biotin is biotin.
Preferably, the probe M.b-specific-Probe are as follows: 5 '-FAM-TTACAACAAATTCAAAGAATCTCTA
GCTACTA(THF)ATCTCTTACATTACTAG-SpC-3'；
Wherein, FAM is fluorescent reporter group, and SpC is fluorescent quenching group, and THF is tetrahydrofuran.
Preferably, the concentration of the primer is 10uM, and the concentration of probe is 10uM, upstream primer, downstream primer and probe
Volume ratio be 2:2:1.
Preferably, the positive control is the pMD18-T/P80 positive criteria plasmid of building, and the negative control is
Nuclease-free water。
Preferably, the concentration and dosage of each ingredient of the kit are as follows: Rehydration buffer 29.5uL, M.b-
Specific-Forward (10uM) 1.2uL, M.b-specific-Reverse (10uM) 1.2uL, M.b-specific-
Probe (10uM) 0.6uL, ddH2O 10uL, DNA Template 5uL, MgAc (280mM) 2.5uL, Total 50uL.
The present invention overcomes the deficiencies of the prior art and provide the nfo-RPA detection method and detection reagent of a kind of Mycoplasma bovis
Box, the present invention chooses a Duan Xulie of Mycoplasma bovis P80 gene, as shown in SEQ ID NO:1, is based on the gene order, design
Specific primer and probe establish Mycoplasma bovis detection kit, and in order to detect the specificity of the kit, the present invention is with structure
The recombinant plasmid (pMD18-T/P80) built is positive control, using Mycoplasma bovis 08M and other genomic DNAs template,
Nuclease-free water is negative control, carries out nfo-RPA reaction, as a result, it has been found that: remove positive control (pMD18-T/
P80 other than), Mycoplasma bovis 08M genomic DNA testing result is positive (+), remaining testing result is negative (-).
Compared with the prior art the shortcomings that and deficiency, the invention has the following advantages:
(1) the Mycoplasma bovis real-time fluorescence quantitative PCR testing result that the present invention will test that result is established with before is compared
Compared with, find two methods coincidence rate be 100%；Meanwhile the present invention also detects the stability of kit, discovery
Under the conditions of -20 DEG C, the kit established can at least be saved one month, this provides pole for the on-site test of Mycoplasma bovis
Big convenience.Therefore, kit established by the present invention is that one kind has specificity height, conveniently Mycoplasma bovis detection skill
Art means can satisfy the testing requirements of general clinical sample；
(2) present invention is other than for laboratory conventional detection Mycoplasma bovis nucleic acid, especially suitable for no laboratory condition
Under (such as base's Veterinary office, farm) on-site test, there is high specific and sensitivity, be it is a kind of fast and accurately
Mycoplasma bovis kit for detecting nucleic acid, has a good application prospect.
Detailed description of the invention
Fig. 1 is result judgement figure；Wherein C line is nature controlling line, and 1 line and 2 lines are detection lines.If in PCRD test strips simultaneously
There is any one in nature controlling line (C line) and detection line (1 and 2 line), is then determined as the positive；If only gone out in PCRD test strips
Existing nature controlling line (C line), then be determined as feminine gender；
Fig. 2 is specific detection result；As the result is shown: in addition to positive control (pMD18-T/P80), Mycoplasma bovis 08M
Genomic DNA testing result is positive (+), remaining testing result is negative (-)；
Fig. 3 is that Fig. 3 is sensitivity Detection result；As the result is shown: this method can detecte 500fg/uL.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to the accompanying drawings and embodiments, right
The present invention is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, and
It is not used in the restriction present invention.
One, material and method
1, bacterial strain and part mycoplasma, bacterial genomes DNA
Mycoplasma bovis 08M, mycoplasma ovine pneumoniae, mycoplasma agalactiae P2, mycoplasma bovigenitalium PG11, Filamentous mycoplasma
Goat subspecies (Mmc) PG3, mycoplasma capri goat pneumonia subspecies (Mccp) M1601, mycoplasma capri goat subspecies (Mcc)
The mycoplasmas such as D27-1, Pasteurella, ox salmonella, staphylococcus aureus and the genomic DNA of bacterium are by Chinese agriculture
Academy of sciences's Lanzhou veterinary institute provides.
2, key instrument equipment and reagent
Supercentrifuge Eppendorf centrifuge5804R, nucleic acid concentration analyzer NanoDrop-2000, bacterium
Genome DNA extracting reagent kit (TIANamp Bacteria DNA Kit) is purchased from TIANGEN Biotech (Beijing) Co., Ltd.,
Extraction of plasmid DNA kit, pMD18-T Cloning Kit are purchased from precious bioengineering (Dalian) Co., Ltd.
3, the design and synthesis of primer, probe
P80 gene order (the GenBank accession number: NZ_ of Mycoplasma bovis is obtained from ncbi database
LQDV01000064.1), using DNAMAN software (version 5.2.10, Lynnon BioSoft, Vaudreuil,
Canada gene order) is analyzed, it is long compared with abundant region design amplified fragments in polymorphic site according to primer and probe design principle
Degree is the pair of primers of 225bp, and a fluorescence probe is designed in the amplification region of the primer.Primer and probe is raw by giving birth to work
The synthesis of object engineering (Shanghai) limited liability company.Primer and probe sequence is respectively as follows:
M.b-specific-Forward:5 '-CAAAAACAATAAGTCAGTTAAAGCTCTTTCAG-3 ',
M.b-specific-Reverse:5 '-Biotin-CTAACATCAGTACCTTTAAGTGTGAAAATAAC-3 ',
M.b-specific-Probe:5 '-FAM-TTACAACAAATTCAAAGAATCTCTAGCTACTA (THF)
ATCTCTTACATTACTAG-SpC-3'；
Wherein, Biotin is biotin, and FAM is fluorescent reporter group, and SpC is fluorescent quenching group, and THF is tetrahydro furan
It mutters.
4, the building of positive criteria plasmid
Using M.b-specific-Forward and M.b-specific-Reverse as primer, Mycoplasma bovis 08M genome
DNA is template, and PCR amplification obtains the target fragment that size is 225bp.PCR product gel extraction is connected to pMD18-T after purification
On carrier, connection product is then imported into DH-5 α competent cell, filters out positive clone molecule, agarose gel electrophoresis is tested
Plasmid is extracted after card and measures concentration.
5, reaction system
Nfo-RPA reaction system is 50 μ L, wherein Rehydration buffer 29.5uL, M.b-specific-
Forward (10uM) 1.2uL, M.b-specific-Reverse (10uM) 1.2uL, M.b-specific-Probe (10uM)
0.6uL, Nuclease-free water 10uL, Template 5uL, 280mM MgAc2.5uL.
6, specific detection
Using standard plasmid pMD18-T/P80 as positive control, Mycoplasma bovis (08M plants) are separately added by above-mentioned reaction system
It is template, the specificity of detection kit with other genomic DNAs.
7, sensitivity Detection
The DNA for extracting Mycoplasma bovis 08M, measuring concentration is 85ng/uL, and being diluted to concentration is 50ng/uL, then successively 10
× doubling dilution is added to 5ng/uL, 500pg/uL, 50pg/uL, 5pg/uL, 500fg/uL, 50fg/uL by above-mentioned reaction system
Enter DNA profiling, the sensibility of detection method.
8, Detection of Stability
Save in 200 μ L PCR pipes in -20 DEG C several tube reaction premixed liquids (Rehydration buffer 29.5uL,
M.b-specific-Forward (10uM) 1.2uL, M.b-specific-Reverse (10uM) 1.2uL, M.b-specific-
Probe (10uM) 0.6uL, Nuclease-free water 10uL amounts to 42.5uL), and with positive control (pMD18-T/
P80) be template, respectively the 7th day, the 14th day, the 21st day, the 28th day detection premixed liquid preservation effect, i.e., kit is steady
It is qualitative.
Two, result
1, specific detection
Fig. 1 is that result judgement diagram is intended to；If occurring nature controlling line (C line) and detection line (1 He in PCRD test strips simultaneously
2 lines) in any one, then be determined as positive (+)；If only occurring nature controlling line (C line) in PCRD test strips, it is determined as yin
Property (-).
Testing result is as shown in Fig. 2, in Fig. 2,1~12 (number 1~12 of institute's standard above white box) is respectively
PMD18-T/p80, Mycoplasma bovis 08M, sheep mycoplasma, mycoplasma agalactiae, mycoplasma bovigenitalium, Filamentous mycoplasma goat are sub-
Kind, mycoplasma capri goat pneumonia subspecies, mycoplasma capri goat subspecies, Pasteurella, ox salmonella, Staphylococcus aureus
Bacterium, Nuclease-free water.As shown in Fig. 2, being shown according to nfo-RPA testing result, positive control (pMD18-T/
P80) and Mycoplasma bovis (08M plants) be positive (+), remaining testing result is negative (-).
2, sensitivity Detection
Nfo-RPA testing result is as shown in figure 3, in Fig. 3, and 1~6 (number 1~6 of institute's standard above white box) is respectively
It is the DNA (5ng/uL~50fg/uL) of the Mycoplasma bovis 08M of 10 × doubling dilution, the 7 (numbers of white box top institute standard
It 7) is Nuclease-free water.From figure 3, it can be seen that the method for the present invention sensibility can reach 500fg/uL.
3, Detection of Stability
The p- 20 DEG C stability for saving the 7th day, the 14th day, the 21st day and the 28th day premixed liquid have carried out nfo-RPA respectively
Detection, discovery positive control (pMD18-T/P80) testing result are positive (+).Illustrate above-mentioned premixed liquid or kit -20
Long-term preservation can be carried out under DEG C preservation condition, at least can be reserved for 1 month.
The present invention chooses a Duan Xulie of Mycoplasma bovis P80 gene, is based on the gene order, designs specific primer and spy
Needle establishes Mycoplasma bovis nfo-RPA detection kit.The present invention is positive with the recombinant plasmid (pMD18-T/P80) constructed
Control, using Mycoplasma bovis 08M and other genomic DNAs negative control, detects the specificity of this kit.
As a result, it has been found that: in addition to positive control (pMD18-T/P80), the genomic DNA testing result of Mycoplasma bovis 08M is
Positive (+), the testing result of remaining genomic DNA are negative (-).The present invention will test the ox branch that result is established with before
Substance real-time fluorescence quantitative PCR testing result is compared, it is found that the coincidence rate of two methods is 100%.Show institute of the present invention
This has preferable specific box sensibility to the kit of foundation, can satisfy the testing requirements of general clinical sample.Meanwhile this
Invention also detects the stability of kit, finds under the conditions of -20 DEG C, the kit established can at least save
One month, this was provided a great convenience for the on-site test of Mycoplasma bovis.Therefore, the Mycoplasma bovis nfo- that the present invention establishes
RPA detection kit is a kind of detection kit of special, convenient and fast Mycoplasma bovis.
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., should all be included in the protection scope of the present invention within mind and principle.
Sequence table
<110>Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences
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Claims (10)
1. a kind of nfo-RPA detection method of Mycoplasma bovis detects Mycoplasma bovis by nfo-RPA method, which is characterized in that the party
Method designs amplimer M.b-specific-Forward and M.b- using the P80 gene order of Mycoplasma bovis as template
It includes the M.b sequence of R1 sequence that specific-Reverse goes out from P80 gene cloning, using the R1 sequence as target design probe
M.b-specific-Probe；Wherein, the M.b sequence is as shown in SEQ ID NO:1.
2. the nfo-RPA detection method of Mycoplasma bovis as described in claim 1, which is characterized in that the amplimer M.b-
Specific-Forward are as follows: 5 '-CAAAAACAATAAGTCAGTTAAAGCTCTTTCAG-3 ',
Amplimer M.b-specific-Reverse are as follows: 5 '-Biotin-CTAACATCAGTACCTTTAAGTGTGAAAATA
AC-3'；
Wherein, Biotin is biotin.
3. the nfo-RPA detection method of Mycoplasma bovis as described in claim 1, which is characterized in that the probe M.b-
Specific-Probe are as follows: 5 '-FAM-TTACAACAAATTCAAAGAATCTCTAGCTACTA (THF)
ATCTCTTACATTACTAG-SpC-3'；
Wherein, FAM is fluorescent reporter group, and SpC is fluorescent quenching group, and THF is tetrahydrofuran.
4. the nfo-RPA detection method of Mycoplasma bovis as claimed in claim 2, which is characterized in that the nfo-RPA method is specific
The following steps are included:
(1) using the P80 gene of Mycoplasma bovis as template design primer, the M.b sequence comprising R1 sequence is obtained by amplification；
(2) gel extraction is connected to after purification on pMD18-T carrier, and connection product is then imported into DH-5 α competent cell,
Filter out positive clone molecule；
(3) nfo-RPA reaction system is established, includes probe in the system, is separately added into Niu Zhiyuan toward the nfo-RPA reaction system
Body and other genomic DNAs are template, detect positive findings.
5. the nfo-RPA detection method of Mycoplasma bovis as claimed in claim 4, which is characterized in that the nfo-RPA reactant
System specifically: reaction system is 50 μ L, wherein Rehydration buffer 29.5uL, M.b-specific-Forward
(10uM) 1.2uL, M.b-specific-Reverse (10uM) 1.2uL, M.b-specific-Probe (10uM) 0.6uL,
Nuclease-free water 10uL, Template 5uL, 280mM MgAc 2.5uL.
6. a kind of nfo-RPA detection kit of Mycoplasma bovis, which is characterized in that the kit includes Freeze-dried
Reaction, 280mM MgAc, Rehydration buffer, amplimer M.b-specific-Forward and M.b-
Specific-Reverse, probe M.b-specific-Probe, Nuclease-free water, negative control and the positive are right
According to Test device and PCRD Extraction buffer；Wherein,
Amplimer M.b-specific-Forward are as follows: 5 '-CAAAAACAATAAGTCAGTTAAAGCTCTTTCAG-3 ',
Amplimer M.b-specific-Reverse are as follows: 5 '-Biotin-CTAACATCAGTACCTTTAAGTGTGAAAATA
AC-3'；
Wherein, Biotin is biotin.
7. the nfo-RPA detection kit of Mycoplasma bovis as claimed in claim 6, which is characterized in that the probe M.b-
Specific-Probe are as follows: 5 '-FAM-TTACAACAAATTCAAAGAATCTCTAGCTACTA (THF)
ATCTCTTACATTACTAG-SpC-3'；
Wherein, FAM is fluorescent reporter group, and SpC is fluorescent quenching group, and THF is tetrahydrofuran.
8. the nfo-RPA detection kit of Mycoplasma bovis as claimed in claim 6, which is characterized in that the concentration of the primer
For 10uM, the concentration of probe is 10uM, and the volume ratio of upstream primer, downstream primer and probe is 2:2:1.
9. the nfo-RPA detection kit of Mycoplasma bovis as claimed in claim 6, which is characterized in that the positive control is
The pMD18-T/P80 positive criteria plasmid of building, the negative control are Nuclease-free water.
10. the nfo-RPA detection kit of Mycoplasma bovis as claimed in claim 6, which is characterized in that the kit respectively at
The concentration and dosage divided are as follows: Rehydration buffer 29.5uL, M.b-specific-Forward (10uM) 1.2uL,
M.b-specific-Reverse (10uM) 1.2uL, M.b-specific-Probe (10uM) 0.6uL, ddH2O 10uL, DNA
Template 5uL, MgAc (280mM) 2.5uL, Total 50uL.
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Cited By (1)
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| CN110746504A (en) * | 2019-11-06 | 2020-02-04 | 华中农业大学 | Monoclonal antibody of anti-mycoplasma bovis Mbovp579 protein and application thereof |
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| CN110746504B (en) * | 2019-11-06 | 2021-01-15 | 华中农业大学 | Monoclonal antibody of anti-mycoplasma bovis Mbovp579 protein and application thereof |
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