CN108165607A - The new method of mycoplasma infection in a kind of detection cell culture - Google Patents
The new method of mycoplasma infection in a kind of detection cell culture Download PDFInfo
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- CN108165607A CN108165607A CN201810208306.4A CN201810208306A CN108165607A CN 108165607 A CN108165607 A CN 108165607A CN 201810208306 A CN201810208306 A CN 201810208306A CN 108165607 A CN108165607 A CN 108165607A
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- C12Q1/6804—Nucleic acid analysis using immunogens
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Abstract
The invention belongs to technical field of biological, disclose a kind of new method for detecting mycoplasma infection in cell culture, take the supernatant of cell culture fluid as sample；PCR system is added in by template of the DNA of extraction, amplified reaction is carried out, obtains PCR reaction products；PCR system is added in as template to be enucleated sour water, amplified reaction is carried out, as negative control；After all amplified reactions, it is carried out at the same time agarose gel electrophoresis；Observe agarose gel electrophoresis detection figure；Then ELISA reactions are carried out；Determine sample concentration.This invention ensures that the reliability and stability of experiment, than positive control is separately provided.Round pcr is combined with elisa technique, the characteristics of existing round pcr is quick, sensitive, PCR methods are surmounted in terms of specificity identification and quantization again, have enhanced experimental result specificity, is more credible in objective degree, avoiding PCR methods and the drawbacks of false positive and false negative result easily occur.
Description
Technical field
The invention belongs to technical field of biological more particularly to a kind of new sides for detecting mycoplasma infection in cell culture
Method.
Background technology
Mycoplasma (Mycoplasma) is the prokaryotic cell microorganism of the acellular wall of one kind between bacterium and virus,
Be it is current known can in no life culture medium growth and breeding minimum microorganism.It is endangered caused by mycoplasma infection very wide
It is general, a variety of diseases such as people, animal, plant and insect can be caused, attracted widespread attention.But bacterium is had based on mycoplasma
Volume morphing is small, the biological characteristics such as presence of cross-reacting antigen between various, slow-growing and mycoplasma kind, limits mycoplasma inspection
The method for some simple, intuitives surveyed.In PCR methods detect mycoplasma, because the defects of round pcr itself, there is also some to ask
Topic, such as：Pollution easily occurs, cross reaction easily occurs, generates false positive false negative result etc..The PCR of general mycoplasma contamination
In detection method mostly to be enucleated sour water as negative control template, positive control template is mycoplasma genomic DNA, is judged with this
Testing result.Positive control is separately provided in detecting in this way, not only wastes reagent, but also PCR processes and electrophoresis detection process
It will be more cumbersome.Mycoplasma genomic DNA is also prepared separately simultaneously, more increases the fussy degree of detection；It is most important
The problem of be detection architecture from positive control system office in different PCR systems, even if positive control is normal, it is also possible to go out
Situation about now misjudging.
To sum up, problem of the existing technology is：Existing detection of mycoplasma method easily occurs pollution, easily occurs to intersect instead
Should, it generate false positive false negative result etc.；Reagent is wasted, PCR processes and electrophoresis detection process all can be more cumbersome, it is possible to go out
Situation about now misjudging.
Invention content
In view of the problems of the existing technology, the present invention provides a kind of new sides for detecting mycoplasma infection in cell culture
Method.
The invention is realized in this way a kind of new method for detecting mycoplasma infection in cell culture
Further, include the following steps：
Step 1 takes the supernatant of cell culture fluid that incubation time is 3-7 days as sample；
Step 2 extracts the DNA of cell to be checked；
Step 3 adds in PCR system by template of the DNA of extraction, carries out amplified reaction, obtain PCR reaction products；
Step 4 adds in PCR system as template to be enucleated sour water, amplified reaction is carried out, as negative control；All amplifications
After reaction, it is carried out at the same time agarose gel electrophoresis；
Step 5, observation agarose gel electrophoresis detection figure；
Step 6, ELISA reactions；
ELISA reactions are carried out respectively to the standard items of reaction product and various concentration, ELISA is measured respectively after reaction
The OD450 values of PCR reaction products and various concentration standard items；
Step 7 determines sample concentration；
According to the OD450 values of the standard items of various concentration, the relation curve of OD450 values and normal concentration is drawn；According to pass
It is curve, according to the OD450 values of PCR reaction products, checks in the concentration of PCR reaction products；According to the concentration of PCR reaction products,
The concentration of sample to be tested is calculated.
Further, the agarose gel electrophoresis detection figure, the method for judging testing result are as follows：
1) when the corresponding amplified production of primer pair of specific amplification Chinese hamster ovary celI glyceraldehyde-3-phosphate dehydrogenase DNA sequence dna
Occur, when amplified production does not occur in negative control, illustrate that PCR system works normally, PCR testing results at this time accurately may be used
It leans on；
If 1.1) at this point, there is not the primer pair phase of specific amplification mycoplasma 16srRNA conservative region DNA sequence dnas
The amplified production answered illustrates mycoplasma infection do not occur in CHO culture cells；
1.2) it is at this point, corresponding in the event of the primer pair of specific amplification mycoplasma 16srRNA conservative region DNA sequence dnas
Amplified production, illustrate CHO culture cell in there is mycoplasma infection；
2) if the primer pair of specific amplification Chinese hamster ovary celI glyceraldehyde-3-phosphate dehydrogenase DNA sequence dna expands production accordingly
Object does not occur, and illustrates that PCR system does not work normally, testing result is invalid at this time；
If 3) amplified production occurs in negative control, illustrate that PCR system is infected, testing result is invalid at this time.
Advantages of the present invention and good effect are：When whether detecting culture cell by mycoplasma infection with PCR method, to carry
The DNA of the cell of culture is taken as the reaction template in PCR system, and has a large amount of cell genomic dna in template, is being detected
House keeper's gene cell glyceraldehyde-3-phosphate dehydrogenase (GADPH) DNA in specific amplification cellular genome is added in PCR system
The primer pair of sequence, you can to treat as the positive control whether detection PCR reaction systems work normally, it is possible to save and individually set
The step of putting positive control, and positive control and detection carry out in same PCR system, and then ensure that the reliability of experiment
And stability, than positive control is separately provided, result is relatively reliable, intuitive.Round pcr with elisa technique is combined, is
A kind of the characteristics of sxemiquantitative nucleic acid detection technique, existing round pcr is quick, sensitive, and in terms of specificity identification and quantization
PCR methods are surmounted, have made the enhancing of experimental result specificity, sensitivity raising, more credible in objective degree, avoid PCR methods and easily go out
The drawbacks of existing false positive and false negative result.
Description of the drawings
Fig. 1 is the new method flow chart that the present invention implements mycoplasma infection in the detection cell culture provided.
Specific embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
The application principle of the present invention is further described below in conjunction with the accompanying drawings.
Include the following steps as shown in Figure 1, the present invention provides a kind of new method for detecting mycoplasma infection in cell culture：
S101 takes the supernatant of cell culture fluid that incubation time is 3-7 days as sample；
S102 extracts the DNA of cell to be checked；
S103 adds in PCR system by template of the DNA of extraction, carries out amplified reaction, obtain PCR reaction products；
S104 adds in PCR system as template to be enucleated sour water, amplified reaction is carried out, as negative control；All amplifications are anti-
After answering, it is carried out at the same time agarose gel electrophoresis；
S105, observation agarose gel electrophoresis detection figure；
S106, ELISA react；ELISA reactions are carried out respectively to the standard items of above-mentioned reaction product and various concentration,
ELISA measures PCR reaction products and the OD450 values of various concentration standard items respectively after reaction；
S107 determines sample concentration；
According to the OD450 values of the standard items of various concentration, the relation curve of OD450 values and normal concentration is drawn；According to pass
It is curve, according to the OD450 values of PCR reaction products, checks in the concentration of PCR reaction products；According to the concentration of PCR reaction products,
The concentration of sample to be tested is calculated.
It is as follows that the present invention provides agarose gel electrophoresis detection figure, the method for judging testing result in step S105：
1) when the corresponding amplified production of primer pair of specific amplification Chinese hamster ovary celI glyceraldehyde-3-phosphate dehydrogenase DNA sequence dna
Occur, when amplified production does not occur in negative control, illustrate that PCR system works normally, PCR testing results at this time accurately may be used
It leans on；
If 1.1) at this point, there is not the primer pair phase of specific amplification mycoplasma 16srRNA conservative region DNA sequence dnas
The amplified production answered illustrates mycoplasma infection do not occur in CHO culture cells；
1.2) it is at this point, corresponding in the event of the primer pair of specific amplification mycoplasma 16srRNA conservative region DNA sequence dnas
Amplified production, illustrate CHO culture cell in there is mycoplasma infection；
2) if the primer pair of specific amplification Chinese hamster ovary celI glyceraldehyde-3-phosphate dehydrogenase DNA sequence dna expands production accordingly
Object does not occur, and illustrates that PCR system does not work normally, testing result is invalid at this time；
If 3) amplified production occurs in negative control, illustrate that PCR system is infected, testing result is invalid at this time.
The foregoing is merely a prefered embodiment of the invention, is not intended to limit the invention, it is all the present invention spirit and
All any modification, equivalent and improvement made within principle etc., should all be included in the protection scope of the present invention.
Claims (3)
1. a kind of new method for detecting mycoplasma infection in cell culture, which is characterized in that branch is former in the detection cell culture
The new method of body-sensing dye is using cell suspension and dot blot, by genomic samples point on cellulose nitrate film, upper primary antibody and
Secondary antibody, primary antibody are anti-m6A antibody, and secondary antibody is the antibody of anti-m6A antibody, and secondary antibody has color developing effect, according to the difference of colour developing, sentences
It is disconnected whether to have mycoplasma infection；If signal is especially high, there is mycoplasma infection.
2. the new method of mycoplasma infection in cell culture is detected as claimed in claim 1, which is characterized in that the detection cell training
The new method of mycoplasma infection includes the following steps in supporting：
Step 1 takes the supernatant of cell culture fluid that incubation time is 3-7 days as sample；
Step 2 extracts the DNA of cell to be checked；
Step 3 adds in PCR system by template of the DNA of extraction, carries out amplified reaction, obtain PCR reaction products；
Step 4 adds in PCR system as template to be enucleated sour water, amplified reaction is carried out, as negative control；All amplified reactions
After, it is carried out at the same time agarose gel electrophoresis；
Step 5, observation agarose gel electrophoresis detection figure；
Step 6, ELISA reactions；The standard items of reaction product and various concentration are carried out with ELISA reactions, ELISA reactions respectively
After measure PCR reaction products and the OD450 values of various concentration standard items respectively；
Step 7 determines sample concentration；
According to the OD450 values of the standard items of various concentration, the relation curve of OD450 values and normal concentration is drawn；According to relationship song
Line according to the OD450 values of PCR reaction products, checks in the concentration of PCR reaction products；According to the concentration of PCR reaction products, calculate
Obtain the concentration of sample to be tested.
3. the new method of mycoplasma infection in cell culture is detected as claimed in claim 1, which is characterized in that the Ago-Gel
Electrophoresis detection figure, the method for judging testing result are as follows：
1) when the corresponding amplified production of primer pair of specific amplification Chinese hamster ovary celI glyceraldehyde-3-phosphate dehydrogenase DNA sequence dna goes out
It is existing, when amplified production does not occur in negative control, illustrate that PCR system works normally, PCR testing results at this time are accurately and reliably；
1.1) at this point, if the primer pair for specific amplification mycoplasma 16srRNA conservative region DNA sequence dnas do not occur is corresponding
Amplified production illustrates mycoplasma infection do not occur in CHO culture cells；
1.2) at this point, expanding accordingly in the event of the primer pair of specific amplification mycoplasma 16srRNA conservative region DNA sequence dnas
Increase production object, illustrate mycoplasma infection occur in CHO culture cells；
2) if the corresponding amplified production of primer pair of specific amplification Chinese hamster ovary celI glyceraldehyde-3-phosphate dehydrogenase DNA sequence dna does not have
It occurs, illustrates that PCR system does not work normally, testing result is invalid at this time；
If 3) amplified production occurs in negative control, illustrate that PCR system is infected, testing result is invalid at this time.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201810208306.4A CN108165607A (en) | 2018-03-14 | 2018-03-14 | The new method of mycoplasma infection in a kind of detection cell culture |
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| CN201810208306.4A CN108165607A (en) | 2018-03-14 | 2018-03-14 | The new method of mycoplasma infection in a kind of detection cell culture |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005098045A1 (en) * | 2004-03-31 | 2005-10-20 | Sphingomonas Research Partners, Lp | Diagnosis of systemic lupus erythematosus |
| CN103627781A (en) * | 2012-08-24 | 2014-03-12 | 华北制药集团新药研究开发有限责任公司 | Kit and method for detecting mycoplasma pollution in CHO cultured cells |
| CN106198968A (en) * | 2016-06-27 | 2016-12-07 | 武汉思安医疗技术有限公司 | The detection method of one mycoplasma species and detection kit |
-
2018
- 2018-03-14 CN CN201810208306.4A patent/CN108165607A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005098045A1 (en) * | 2004-03-31 | 2005-10-20 | Sphingomonas Research Partners, Lp | Diagnosis of systemic lupus erythematosus |
| CN103627781A (en) * | 2012-08-24 | 2014-03-12 | 华北制药集团新药研究开发有限责任公司 | Kit and method for detecting mycoplasma pollution in CHO cultured cells |
| CN106198968A (en) * | 2016-06-27 | 2016-12-07 | 武汉思安医疗技术有限公司 | The detection method of one mycoplasma species and detection kit |
Non-Patent Citations (2)
| Title |
|---|
| GAMAL A.M.YOUNIS等: "Molecular Identification and Sequencing of Mycoplasma gallisepticum Recovered from Broilers in Egypt", 《PAKISTAN JOURNAL OF BIOLOGICAL SCIENCES》 * |
| 刘吉荣等: "原核生物16SrDNA的PCR-ELISA检测方法的研究", 《中华儿科杂志》 * |
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Application publication date: 20180615 |
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