CN106198968A - The detection method of one mycoplasma species and detection kit - Google Patents
The detection method of one mycoplasma species and detection kit Download PDFInfo
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- CN106198968A CN106198968A CN201610478333.4A CN201610478333A CN106198968A CN 106198968 A CN106198968 A CN 106198968A CN 201610478333 A CN201610478333 A CN 201610478333A CN 106198968 A CN106198968 A CN 106198968A
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/56933—Mycoplasma
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Abstract
The invention provides detection method and the detection kit of a mycoplasma species.Round pcr is combined by described detection method with elisa technique, it it is a kind of sxemiquantitative nucleic acid detection technique, the feature that existing round pcr is quick, sensitive, PCR method has been surmounted again in terms of specificity identification and quantization, make in the enhancing of experimental result specificity, sensitivity raising, objective degree more credible, it is to avoid the drawback of false positive and false negative result easily occurs in PCR method.Described detection kit, simple in construction, easy to operate, it is possible to the reagent that detection method described in all promising policy needs, improve the work efficiency of experimenter.
Description
Technical field
The invention belongs to medical science detection analysis technical field, be specifically related to detection method and the detectable of a mycoplasma species
Box.
Background technology
Mycoplasma (Mycoplasma) is the prokaryotic cell microorganism of the acellular wall of class between antibacterial and virus,
Be current known can the minimum microorganism of growth and breeding in without life culture medium.The harm that mycoplasma infection causes is the widest
General, the multiple diseases such as people, animal, plant and insecticide can be caused, attract widespread attention.But based on mycoplasma, there is bacterium
Volume morphing is little, various, the biological characteristics such as the existence of cross-reacting antigen between poor growth and mycoplasma kind, limits mycoplasma inspection
The method of some simple, intuitive surveyed.
At present, state-of-the-art in prior art is to use PCR method detection mycoplasma, and PCR method detection mycoplasma mainly depends on
The most conservative according to procaryotic rRNA base sequence, and encode in rRNA operon between each biological species in DNA spacer of rRNA
Base sequence difference very big, set with the DNA of the interior transcriptional domain between mycoplasma 16S rRNA and 16S-23S and 23S rRNA
The primer of meter carries out sleeve type PCR amplification, runs glue by ethidium bromide agarose and detects.PCR method is to utilize round pcr, analogue body
Some are difficult to, by culture method and the mycoplasma of serological method detection, more to show it by the amplification in vitro method of interior DNA replication dna
Efficiently, sensitive specificity.
But in PCR method detection mycoplasma, because the defect of round pcr itself, there is also some problems, such as: easily occur
Pollute, cross reaction easily occurs, produce false positive false negative result etc..
Summary of the invention
In order to solve the problems referred to above that prior art exists, the invention provides detection method and the detection of a mycoplasma species
Test kit.Round pcr is combined by described detection method with elisa technique, is a kind of sxemiquantitative nucleic acid detection technique, existing
The feature that round pcr is quick, sensitive, has surmounted again PCR method in terms of specificity identification and quantization, has made experimental result specificity
In enhancing, sensitivity raising, objective degree more credible, it is to avoid easily there is the drawback of false positive and false negative result in PCR method.
The technical solution adopted in the present invention is:
The detection method of one mycoplasma species, comprises the following steps:
A, take testing sample: take cell supernatant to be measured as sample；
B, PCR react: with the supernatant in step A as template, carry out PCR reaction, obtain PCR product；
C, ELISA react: the standard substance of the PCR product obtained in step B and variable concentrations are carried out ELISA respectively
Reaction, the OD450 value of described PCR product and variable concentrations standard substance is measured in ELISA reaction respectively after terminating；
D, determine sample concentration: according to the OD450 value of the standard substance of variable concentrations, draw OD450 value and normal concentration
Relation curve；According to described relation curve, according to the OD450 value of described PCR product, check in the concentration of PCR product；
According to the concentration of described PCR product, it is calculated the concentration of described testing sample.
Round pcr is combined by described detection method with elisa technique, the feature that existing round pcr is quick, sensitive, again
In terms of specificity identification and quantization, surmount PCR method, made in the enhancing of experimental result specificity, sensitivity raising, objective degree
More credible, it is to avoid the drawback of false positive and false negative result easily occurs in PCR method.
In step A, take the supernatant of the cell culture fluid that incubation time is 3-7 days as sample.
In step B, using the primer being fluorescently labeled, the fluorescence labeling method of the primer used is: with 5 ' end labellings
The primer of biotin expands for PCR, and a chain of PCR reaction double-stranded products is just with biotin, and described biotin is as mark
Note thing.
It should be noted that in step C in the present invention, described biotin can with in ELISA Plate coated Avidin
In conjunction with, PCR product after denaturing, is removed another unmarked chain, is added the probe of digoxigenin labeled, and is combined in enzyme
Strand specific hybrid on target, then adds enzyme mark anti digoxin antibody, and the digoxin on probe is combined, and then shows
Color, measures OD value, has completed whole experiment process.
In the present invention, use improved primer that PCR primer is marked, and in the follow-up convenient survey of ELISA reaction
Determine OD value, PCR reaction and ELISA reaction are organically combined.
It addition, in step B, PCR reaction system include primer, dNTP mixture, Buffer, Takara Ex Taq and
Sample, the cumulative volume of described PCR reaction system is that 25 μ L, the content of the most each component or concentration are: primer concentration is 10pmol/
The concentration of ul, dNTP mixture is 2.5mmol/L, sample content 2 μ L, and surplus is water.
In step B, the PCR amplification program used in PCR reaction is: denaturation 1min at a temperature of 94 DEG C；Then carry out
35 circulations, the most each circulation comprises the following steps successively: denaturation 1min at a temperature of 94 DEG C, anneals at a temperature of 55 DEG C
2min.1min is extended at a temperature of 72 DEG C；After completing 35 circulations, i.e. complete PCR amplification program.
Certainly, for those skilled in the art, it is possible to according to prior art, other the joining of PCR reaction system is made
Ratio and response procedures, do not repeating at this.
In step C, described ELISA reaction is: to the PCR product obtained in step B and the standard substance of variable concentrations
Being added separately to in the ELISA Plate of Avidin, offset plate covers, and hatches successively and cleans；Then in described enzyme mark version
Adding one to resist, offset plate covers, and hatches successively and cleans；Then adding two in enzyme mark version to resist, offset plate covers, and carries out successively
Hatch and clean；After having hatched, add 3,3', 5,5'-tetramethyl benzidines, lucifuge；It is eventually adding stop solution, terminates reaction
The rear OD450 value measuring described PCR product and variable concentrations standard substance respectively.
Preferably, in step C, clean every time and use 1XWashing Buffer to wash 3 times.
Further, in step B, increase positive controls: described positive controls add positive template, remaining arrange with
Described PCR reaction in step B arranges identical.
In the present invention, whether positive control mainly confirmatory sample there is obstruction thing, do not contain the inside sequence of mycoplasma
Row, for synthetic.When using positive control, amplified fragments size is 800bp, if not having fragment to expand when using positive control
Increasing, illustrating to detect in sample has obstruction thing, detection sample need to be carried out DNA extraction, then expand for template with it.
Present invention also offers the detection kit of a kind of detection method for mycoplasma, including box body, reagent bottle, eight
Connecting leg and eight connecting leg framves, the bottom of described box body is provided with fixed layer, and described fixed layer offers multiple fixing hole, described examination
Agent bottle is arranged in fixing hole, described reagent bottle include PCR reactant liquor reagent bottle, positive template reagent bottle, standard substance reagent bottle,
Diluent reagent bottle, cleanout fluid reagent bottle, an anti-reagent bottle, two anti-reagent bottle, nitrite ion reagent bottle and stop buffer reagent bottle；Institute
State eight connecting legs and eight connecting leg framves are arranged on the inside of described box body.
Equipped with PCR reactant liquor reagent in described PCR reactant liquor reagent bottle, positive template reagent bottle tries equipped with positive template
Agent, equipped with standard substance reagent in standard substance reagent bottle, equipped with diluent reagent in diluent reagent bottle, fills in cleanout fluid reagent bottle
There is cleanout fluid reagent, equipped with an anti-reagent in an anti-reagent bottle, equipped with two anti-reagent in two anti-reagent bottle, in nitrite ion reagent bottle
Equipped with nitrite ion reagent, equipped with stop buffer reagent in stop buffer reagent bottle.
Described PCR reactant liquor tries, extremely in addition to PCR reaction template, and mixing of other common reagents in PCR reaction system
Close liquid.
In the present invention, the principle of ELISA reaction is: enzyme-linked immunosorbent assay (ELISA) is by known antigen or antibody
Absorption, at surface of solid phase carriers, makes the technology that the antigen antibody reaction of enzyme labelling is carried out at solid phase surface.This technology can be used for examining
Survey macromole antigen and specific antibody etc., there is quick, sensitive, easy, carrier and be prone to the advantages such as standardization.During measurement, anti-
Former (antibody) is first combined on solid phase carrier, but still retains its immunocompetence, then adds a kind of antibody (antigen) and is combined into enzyme
Conjugate (label), this conjugate still retains its former immunocompetence and enzymatic activity, anti-when on conjugate and solid phase carrier
After former (antibody) reaction bonded, add the corresponding substrate of enzyme, i.e. chemically reactive and present color.Its color generated is deep
Shallow it is directly proportional to the antigen to be surveyed (antibody) content, detects light absorption value by microplate reader, draw standard substance light absorption value-concentration bent
Line, just can calculate the content of institute's test sample product according to the light absorption value of sample.
The invention have the benefit that
1, the invention provides the detection method of a mycoplasma species, described detection method is by round pcr and elisa technique phase
In conjunction with, it is a kind of sxemiquantitative nucleic acid detection technique, the feature that existing round pcr is quick, sensitive, again in specificity identification and amount
Change aspect has surmounted PCR method, make in the enhancing of experimental result specificity, sensitivity raising, objective degree more credible, it is to avoid PCR method
The drawback of false positive and false negative result easily occurs.
2, described detection kit, simple in construction, easy to operate, it is possible to the examination that detection method described in all promising policy needs
Agent, improves the work efficiency of experimenter.
Accompanying drawing explanation
Fig. 1 is the structural representation concentrating detection kit tray interior in the present invention.
In figure: 1, PCR reactant liquor reagent bottle；2, positive template reagent bottle；3, standard substance reagent bottle；4, diluent reagent
Bottle；5, cleanout fluid reagent bottle；6, an anti-reagent bottle；7, two anti-reagent bottle；8, nitrite ion reagent bottle；9, stop buffer reagent bottle；10、
Eight connecting legs；11, eight connecting leg frame.
Detailed description of the invention
Below in conjunction with embodiment, further illustrate present disclosure.Should be appreciated that the enforcement of the present invention is not limited to
In the following examples, any pro forma accommodation and/or the change of being made the present invention fall within scope.
In the present invention, if not refering in particular to, all of part, percentage ratio are unit of weight, and all of equipment and raw material etc. are equal
It is commercially available or the industry is conventional.Method in following embodiment, if no special instructions, is the routine of this area
Method.
Embodiment 1
The detection method of one mycoplasma species, comprises the following steps:
A, take testing sample: take the supernatant of the cell culture fluid that incubation time is 7 days as sample；
B, PCR react: with the supernatant in step A as template, carry out PCR reaction, obtain PCR product；Use quilt
Fluorescently-labeled primer, the fluorescence labeling method of the primer used is: expand for PCR with the primer of 5 ' end labelling biotin
Increasing, a chain of PCR reaction double-stranded products is just with biotin, and described biotin is as label；
PCR reaction system includes primer, dNTP mixture, Buffer, Takara Ex Taq and sample, and described PCR is anti-
The cumulative volume answering system is that 25 μ L, the content of the most each component or concentration are: primer concentration is 10pmol/ul, dNTP
The concentration of mixture is 2.5mmol/L, sample content 2 μ L, and surplus is water.
The PCR amplification program used in PCR reaction is: denaturation 1min at a temperature of 94 DEG C；Then 35 circulations are carried out,
The most each circulation comprises the following steps successively: denaturation 1min at a temperature of 94 DEG C, and anneal at a temperature of 55 DEG C 2min.72
1min is extended at a temperature of DEG C；After completing 35 circulations, i.e. complete PCR amplification program.
C, ELISA react: described ELISA reaction is: to the PCR product obtained in step B and the mark of variable concentrations
Quasi-product are added separately to in the ELISA Plate of Avidin, and offset plate covers, and hatches successively and cleans；Then at described enzyme mark
Adding one in version to resist, offset plate covers, and hatches successively and cleans；Then adding two in enzyme mark version to resist, offset plate covers, successively
Hatch and clean；After having hatched, add 3,3', 5,5'-tetramethyl benzidines, lucifuge；It is eventually adding stop solution, terminates
The OD450 value of described PCR product and variable concentrations standard substance is measured respectively after reaction；
It should be noted that in step C, described biotin can be combined by coated Avidin in ELISA Plate, PCR reacts
Product after denaturing, is removed another unmarked chain, is added the probe of digoxigenin labeled, with the strand being combined in ELISA Plate
Specific hybrid, then adds enzyme mark anti digoxin antibody, and the digoxin on probe is combined, and then develops the color, and measures OD value,
To complete whole experiment process.
D, determine sample concentration: according to the OD450 value of the standard substance of variable concentrations, draw OD450 value and normal concentration
Relation curve；According to described relation curve, according to the OD450 value of described PCR product, check in the concentration of PCR product；
According to the concentration of described PCR product, it is calculated the concentration of described testing sample.
Embodiment 2
The detection method of one mycoplasma species, comprises the following steps:
A, take testing sample: take the supernatant of the cell culture fluid that incubation time is 3 days as sample；
B, PCR react: with the supernatant in step A as template, carry out PCR reaction, obtain PCR product；Use quilt
Fluorescently-labeled primer, the fluorescence labeling method of the primer used is: expand for PCR with the primer of 5 ' end labelling biotin
Increasing, a chain of PCR reaction double-stranded products is just with biotin, and described biotin is as label；
In step B, increasing positive controls: described positive controls adds positive template, remaining is arranged and in step B
Described PCR reaction arranges identical.Whether positive control mainly confirmatory sample there is obstruction thing, does not contains the inside sequence of mycoplasma
Row, for synthetic.When using positive control, amplified fragments size is 800bp, if not having fragment to expand when using positive control
Increasing, illustrating to detect in sample has obstruction thing, detection sample need to be carried out DNA extraction, then expand for template with it.
PCR reaction system includes primer, dNTP mixture, Buffer, Takara Ex Taq and sample, and described PCR is anti-
The cumulative volume answering system is that 25 μ L, the content of the most each component or concentration are: primer concentration is 10pmol/ul, dNTP
The concentration of mixture is 2.5mmol/L, sample content 2 μ L, and surplus is water.
The PCR amplification program used in PCR reaction is: denaturation 1min at a temperature of 94 DEG C；Then 35 circulations are carried out,
The most each circulation comprises the following steps successively: denaturation 1min at a temperature of 94 DEG C, and anneal at a temperature of 55 DEG C 2min.72
1min is extended at a temperature of DEG C；After completing 35 circulations, i.e. complete PCR amplification program.
C, ELISA react: described ELISA reaction is: to the PCR product obtained in step B and the mark of variable concentrations
Quasi-product are added separately to in the ELISA Plate of Avidin, and offset plate covers, and hatches successively and cleans；Then at described enzyme mark
Adding one in version to resist, offset plate covers, and hatches successively and cleans；Then adding two in enzyme mark version to resist, offset plate covers, successively
Hatch and clean；After having hatched, add 3,3', 5,5'-tetramethyl benzidines, lucifuge；It is eventually adding stop solution, terminates
The OD450 value of described PCR product and variable concentrations standard substance is measured respectively after reaction；
It should be noted that in step C, described biotin can be combined by coated Avidin in ELISA Plate, PCR reacts
Product after denaturing, is removed another unmarked chain, is added the probe of digoxigenin labeled, with the strand being combined in ELISA Plate
Specific hybrid, then adds enzyme mark anti digoxin antibody, and the digoxin on probe is combined, and then develops the color, and measures OD value,
Complete whole experiment process.
D, determine sample concentration: according to the OD450 value of the standard substance of variable concentrations, draw OD450 value and normal concentration
Relation curve；According to described relation curve, according to the OD450 value of described PCR product, check in the concentration of PCR product；
According to the concentration of described PCR product, it is calculated the concentration of described testing sample.
Embodiment 3
As it is shown in figure 1, the invention provides the detection kit of a kind of detection method for mycoplasma, including box body,
Reagent bottle, eight connecting legs and eight connecting leg framves 11, the bottom of described box body is provided with fixed layer, described fixed layer offers multiple solid
Determining hole, described reagent bottle is arranged in fixing hole, described reagent bottle include PCR reactant liquor reagent bottle 1, positive template reagent bottle 2,
Standard substance reagent bottle 3, diluent reagent bottle 4, cleanout fluid reagent bottle 5, the anti-reagent bottle 7 of anti-reagent bottle 6, two, nitrite ion reagent
Bottle 8 and stop buffer reagent bottle 9；Described eight connecting legs and eight connecting leg framves 11 are arranged on the inside of described box body.In the present embodiment, made
Reagent bottle be the reagent bottle of industry universal.
Equipped with PCR reactant liquor reagent in described PCR reactant liquor reagent bottle 1, equipped with positive template in positive template reagent bottle 2
Reagent, equipped with standard substance reagent in standard substance reagent bottle 3, equipped with diluent reagent in diluent reagent bottle 4, cleanout fluid reagent bottle
Equipped with cleanout fluid reagent in 5, equipped with an anti-reagent in an anti-reagent bottle 6, equipped with two anti-reagent, nitrite ion in two anti-reagent bottle 7
Equipped with nitrite ion reagent (TMB) in reagent bottle 8, equipped with stop buffer reagent (Stop solution) in stop buffer reagent bottle 9.
Described PCR reactant liquor tries, extremely in addition to PCR reaction template, and mixing of other common reagents in PCR reaction system
Close liquid.Described detection kit, simple in construction, easy to operate, it is possible to the reagent that detection method described in all promising policy needs, carry
The high work efficiency of experimenter.
It should be noted last that, above example is only in order to illustrate technical scheme and unrestricted, although ginseng
According to preferred embodiment, the present invention is described in detail, it should be understood that and the foregoing is only being embodied as of the present invention
Mode, the protection domain being not intended to limit the present invention, all within the spirit and principles in the present invention, that is done any repaiies
Change, equivalent, improvement etc., should be included within the scope of the present invention.
Claims (10)
1. the detection method of a mycoplasma species, it is characterised in that comprise the following steps:
A, take testing sample: take cell supernatant to be measured as sample；
B, PCR react: with the supernatant in step A as template, carry out PCR reaction, obtain PCR product；
C, ELISA react: the standard substance of the PCR product obtained in step B and variable concentrations are carried out ELISA respectively anti-
Should, the OD450 value of described PCR product and variable concentrations standard substance is measured in ELISA reaction respectively after terminating；
D, determine sample concentration: according to the OD450 value of the standard substance of variable concentrations, draw OD450 value and the relation of normal concentration
Curve；According to described relation curve, according to the OD450 value of described PCR product, check in the concentration of PCR product；According to
The concentration of described PCR product, is calculated the concentration of described testing sample.
Detection method the most according to claim 1, it is characterised in that in step A, takes the cell that incubation time is 3-7 days
The supernatant of culture fluid is as sample.
Detection method the most according to claim 1, it is characterised in that in step B, uses the primer being fluorescently labeled, institute
The fluorescence labeling method of the primer used is: expanding for PCR with the primer of 5 ' end labelling biotin, PCR reacts double-stranded products
A chain just with biotin, described biotin is as label.
Detection method the most according to claim 1, it is characterised in that in step B, PCR reaction system includes primer, dNTP
Mixture, Buffer, Takara Ex Taq and sample, the cumulative volume of described PCR reaction system is 25 μ L, the most each component
Content or concentration is: primer concentration is that the concentration of 10pmol/ul, dNTP mixture is 2.5mmol/L, sample content 2 μ L,
Surplus is water.
Detection method the most according to claim 1, it is characterised in that in step B, the PCR used in PCR reaction expands journey
Sequence is: denaturation 1min at a temperature of 94 DEG C；Then carrying out 35 circulations, the most each circulation comprises the following steps successively:
Denaturation 1min at a temperature of 94 DEG C, anneal at a temperature of 55 DEG C 2min；1min is extended at a temperature of 72 DEG C；Complete 35 circulations
Afterwards, PCR amplification program is i.e. completed.
Detection method the most according to claim 1, it is characterised in that in step C, described ELISA reaction is: to step B
In the standard substance of the PCR product that obtains and variable concentrations be added separately to in the ELISA Plate of Avidin, offset plate covers,
Hatch successively and clean；Then adding one in described enzyme mark version to resist, offset plate covers, and hatches successively and cleans；So
After in enzyme mark version add two resist, offset plate cover, hatch successively and clean；After having hatched, add 3,3', 5,5'-tetra-
Methyl biphenyl amine, lucifuge；It is eventually adding stop solution, after terminating reaction, measures described PCR product and variable concentrations mark respectively
The OD450 value of quasi-product.
Detection method the most according to claim 6, it is characterised in that clean every time and use 1XWashing Buffer to wash 3
Secondary.
8. according to the arbitrary described detection method of claim 1-7, it is characterised in that in step B, increase positive controls: institute
State positive controls and add positive template, remaining arrange react with the described PCR in step B arrange identical.
9. the detection kit for the detection method of mycoplasma, it is characterised in that include box body, reagent bottle, eight connecting legs
(10) and eight connecting leg framves (11), the bottom of described box body is provided with fixed layer, and described fixed layer offers multiple fixing hole, institute
Stating reagent bottle to be arranged in fixing hole, described reagent bottle includes PCR reactant liquor reagent bottle (1), positive template reagent bottle (2), mark
Quasi-product reagent bottle (3), diluent reagent bottle (4), cleanout fluid reagent bottle (5), an anti-reagent bottle (6), two anti-reagent bottle (7), aobvious
Color liquid reagent bottle (8) and stop buffer reagent bottle (9)；Described eight connecting legs (10) and eight connecting leg framves (11) are arranged on the interior of described box body
Portion.
Test kit the most according to claim 9, it is characterised in that anti-equipped with PCR in described PCR reactant liquor reagent bottle (1)
Answer liquid reagent, equipped with positive template reagent in positive template reagent bottle (2), equipped with standard substance reagent in standard substance reagent bottle (3),
Equipped with diluent reagent in diluent reagent bottle (4), equipped with cleanout fluid reagent in cleanout fluid reagent bottle (5), an anti-reagent bottle (6)
In equipped with an anti-reagent, equipped with two anti-reagent in two anti-reagent bottle (7), equipped with nitrite ion reagent in nitrite ion reagent bottle (8), eventually
Only equipped with stop buffer reagent in liquid reagent bottle (9).
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| CN201610478333.4A CN106198968A (en) | 2016-06-27 | 2016-06-27 | The detection method of one mycoplasma species and detection kit |
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| CN201610478333.4A CN106198968A (en) | 2016-06-27 | 2016-06-27 | The detection method of one mycoplasma species and detection kit |
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| CN108165607A (en) * | 2018-03-14 | 2018-06-15 | 安徽农业大学 | The new method of mycoplasma infection in a kind of detection cell culture |
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Application publication date: 20161207 |