CN115976238B - qPCR rapid detection kit containing 15 mycoplasma, and use method and application thereof - Google Patents
qPCR rapid detection kit containing 15 mycoplasma, and use method and application thereof Download PDFInfo
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Abstract
The invention provides a qPCR rapid detection kit for simultaneously amplifying 15 mycoplasma, which comprises a PCR amplification reaction reagent, a multiplex amplification primer mixture, a positive control and a negative control, and can multiplex and amplify mycoplasma leydig, mycoplasma argininosum, mycoplasma fermentum, mycoplasma gallisepticum, mycoplasma genitalium, mycoplasma hominis, mycoplasma hyopneumoniae, mycoplasma stomatae, mycoplasma pyriform, mycoplasma pneumoniae, mycoplasma salivarius, mycoplasma synoviae, mycoplasma pharyngis and mycoplasma otolytium in single tube reaction. The invention utilizes mycoplasma PCR detection to simulate in-vivo DNA replication in vitro, controls DNA renaturation and denaturation through temperature change, uses a primer as a promoter, can amplify a specific nucleic acid sequence of mycoplasma under the action of Taq enzyme and dNTP, and is used for qualitative detection of mycoplasma and detection of cellular gene therapy products, such as cell mycoplasma detection, lentiviral vector mycoplasma detection and the like.
Description
Technical Field
The invention relates to a detection kit, a using method and application thereof, in particular to a qPCR rapid detection kit, a using method and application thereof.
Background
Mycoplasma, also known as mycoplasmal, was found in 1898 to be the smallest and simplest prokaryote found today, with a size of 0.1-0.3 microns, able to pass through filters, in a highly polymorphic form, mycoplasma genome is a circular double stranded DNA, lacking cell walls, small molecular weight, the only visible organelle being ribosomes, most mycoplasma being predominantly spherical, having great variability in cell membranes with three-layer structure, having a certain pathogenicity to both humans and animals and able to grow and reproduce on inanimate artificial medium. The genome of mycoplasma is mostly double-stranded DNA, interspersed throughout the cell in pseudonuclear or non-formed nuclear regions. The sterol contained in the cell membrane of mycoplasma plays a great role in maintaining the integrity of the cell membrane, and gram staining is not easy to stain, and various characteristics of mycoplasma are enough to indicate the characteristics that the mycoplasma are not easy to detect and clear.
Foreign surveys have found that about twenty or more mycoplasma can contaminate cells, and that some cell lines can be contaminated with even more than two mycoplasma simultaneously. Studies have shown that more than 95% of mycoplasma contaminating cells are four of the following: mycoplasma stomatitis (M.orale), mycoplasma arginini (M.arginini), mycoplasma hyorhinis (M.hyorhinis), lei's non-cholesterol mycoplasma (A.laidlawii). In addition, mycoplasma fermentans (M.fermentans), mycoplasma hominus (M.hominis), mycoplasma salivarius (M.salivarium), mycoplasma pulmonary (M.pulmonis) and Mycoplasma pyriformis (M.pirum) are also common. The sources of mycoplasma pollution mainly comprise pollution of working environment, pollution of operators (some mycoplasma is normal flora in human body), pollution of culture medium, cross pollution caused by polluted cells, pollution of experimental equipment, pollution of original tissues and organs of prepared cells, and the like.
The contamination rate of mycoplasma to cultured cells is high, and the average rate is estimated to be about 60%. Meanwhile, the method is quite secret, the cells are not easy to find in the early stage, after the cells are polluted, the growth state and the morphology of the cells are affected, the nutrition substances supporting the growth of the cells in the culture medium are reduced due to the existence of mycoplasma, the growth of the cells is slow or even stopped, and cell microtubules are depolymerized to cause a series of lesions of the cells; also can affect the normal metabolism and function of cells, the synthesis of proteins, DNA and RNA is blocked, the integrity of cell membranes is destroyed, the signal transmission of cells is affected, and the chromosome is abnormal; in addition, mycoplasma contamination can also affect the yield of some viruses in cells, decrease the oncogenic potential of malignant cells, affect lymphocyte differentiation, etc.
The manufacturing process of viral vectors includes various techniques based on adherent or suspension cell production culture. Production of viral vectors mostly relies on adherent cells cultured in a two-dimensional (2D) static system. These systems may include petri dishes and T-masks, but in order to maintain a larger scale, cell factories and roller bottles may be used, which if mycoplasma contaminate cells, can have a great impact on the production of viral vectors, while lentiviral vectors as raw materials for CAR-T cell preparation pose a great risk to the final drug safety.
At present, the common detection methods for detecting mycoplasma in biological products include a traditional separation culture method, an indicator cell culture method (DNA staining method), an ELISA method, a PCR method and the like. The isolated culture method does not have false negative results basically, and is therefore considered as a gold standard for mycoplasma detection. However, there are two disadvantages, firstly, the detection time is too long, and the mycoplasma takes at least 4 weeks to grow obvious clones; secondly, although most mycoplasma species can be detected, the isolation culture method is also very powerful, for example mycoplasma hyorhinis (m.hyorhinis). The sensitivity is lower when the cell culture method (DNA staining method) is indicated, and the light mycoplasma pollution of the cells is not easy to detect due to the existence of background fluorescence; cell lysis death produces debris that can also be stained by fluorescent dyes and mistakenly considered mycoplasma staining to cause false positives; in addition, fluorescent staining generally requires culturing of contaminant-free indicator cells as a control, increasing the detection effort.
The PCR method is the mainstream mycoplasma detection means. The mycoplasma is used as a prokaryote, the rRNA gene is formed by arranging conserved and multiple variable sequences at intervals, primers are designed by using a highly conserved sequence in a 16SrRNA nucleic acid sequence, and the mycoplasma pollution in cells is detected by adopting a PCR amplification and agarose electrophoresis method, and the detection time of the PCR method is greatly shortened compared with that of the traditional separation culture method, but the existing PCR mycoplasma detection also has some problems, such as poor primer design, and insufficient detection sensitivity and specificity; moreover, the current mainstream detection kit can only be aimed at one or a few mycoplasma, and has poor broad spectrum. ZL 202210318135.7 discloses a composition for detecting mycoplasma gallisepticum by adopting droplet-type digital PCR and application thereof, wherein the composition can only detect one mycoplasma, the flux is low, ZL 202210646129.4 detects mycoplasma gallisepticum by adopting the LAMP-Taqman technology, the flux is low, the application range is narrow, ZL202111573467.1 discloses a PCR kit for detecting mycoplasma pollution in cell culture and application thereof, and the primer can amplify a plurality of mycoplasma, but the detection process is complex, time-consuming and labor-consuming.
Disclosure of Invention
The invention aims to provide a kit for quickly detecting mycoplasma qPCR and a use method thereof, and the kit can quickly detect whether mycoplasma pollution exists in a cell bank sample and a virus vector sample.
In order to solve the technical problems, the invention adopts the following technical scheme:
a qPCR rapid detection kit for simultaneously amplifying 15 mycoplasma comprises the following components: 15 primer pairs for specifically amplifying 15 mycoplasma; the 15 mycoplasma are respectively: mycoplasma hyopneumoniae, mycoplasma stomatae, mycoplasma pyriform, mycoplasma hyopneumoniae, mycoplasma salivarius, mycoplasma synoviae, mycoplasma pharyngeal, and mycoplasma otozoae; the primer pairs and primer concentrations for the 15 mycoplasma are shown in table 1:
TABLE 1
PCR is a complex molecular kinetic process. With the increase of the number of primers in a multiplex amplification system, the mutual interference among different primers is more and more serious, the dynamics of a reaction system is more and more complex, so that a large number of specific primers are required to be designed and modified for complex testing, the optimal concentration ratio among the primers in the multiplex amplification system is searched, and finally, more mycoplasma are compositely detected on the premise of not reducing the specificity and sensitivity of the kit.
Further, the above detection kit further comprises the following components: as shown in table 2 below:
TABLE 2
The kit of the invention needs to test different PCR amplification procedures because the adaptability of different samples and the tolerance of different annealing temperatures are required to be satisfied, and the final amplification procedure is as follows:
step 1, 10min at 95 ℃;
step 2, 15s at 95 ℃;
step 3, 60 ℃ for 60s;
step 2 to step 3 are repeated for 40 times, and fluorescence signals are received;
step 4, 15s at 95 ℃;
step 5, 60 ℃ for 60s; a fluorescent signal is received.
Step 6 15s at 95 ℃.
Preferably, the amplification products of the kit of the invention are detected using qPCR methods.
The PCR kit is used for detecting cell gene therapy products, such as cell bank mycoplasma detection, lentivirus (LVV) carrier mycoplasma detection and the like.
The invention has the advantages that:
the invention creatively obtains 15 mycoplasma real-time fluorescence PCR detection primer combinations through design, combines the characteristic of rapid real-time fluorescence quantitative PCR, and greatly shortens the mycoplasma detection time compared with the traditional separation culture method.
Based on a real-time fluorescent quantitative PCR technical platform, the invention carries out qualitative detection on Zhi Yuanti specific target genes, can detect a plurality of samples by one experiment, has the characteristics of rapidness, specificity, economy and the like, and greatly reduces the mycoplasma detection cost of cell gene therapy samples.
And 3, positive control which is designed independently is used in the invention, and target sequences of 15 mycoplasma are respectively constructed on 3T carrier plasmids, so that the pollution of positive strains to a kit and the environment in the extraction process is effectively avoided.
The technical scheme provided by the invention is suitable for industrialization, is efficient and simple, and provides a higher-level detection method for preventing and controlling mycoplasma pollution in cellular gene therapy products.
And 5, through a large number of experiments and repeated test verification, the invention finally obtains the optimal primer and concentration ratio and avoids false positive amplification.
Drawings
FIG. 1 is a map of the amplification of a synthetic plasmid of the target sequence of Mycoplasma hyopneumoniae in a positive control;
FIG. 2 is a graph of the results of detection of Chlamydia in a specificity assay; wherein A is a Chlamydia amplification map; b is an amplification profile after mixing chlamydia with positive control; c is a negative control amplification profile; d is a positive control amplification profile.
FIG. 3 is a DMEM medium amplification map; wherein A is a DMEM medium amplification map; b is an amplification map after the DMEM and the positive control are mixed; c is a negative control amplification profile; d is a positive control amplification profile.
FIG. 4 is an amplification map of Mycoplasma doubtful medium; wherein A is a mycoplasma culture medium amplification map; b is an amplification map after mixing mycoplasma culture medium and positive control; c is a negative control amplification profile; d is a positive control amplification profile.
Description of the embodiments
The invention will be described in further detail below with reference to the drawings by means of specific embodiments. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The specific techniques or conditions are not identified in the examples and are performed according to techniques or conditions described in the literature in this field or according to the product specifications. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1: determining a mycoplasma detection; kit primer design, amplification system and determination of amplification method.
1. Determination of detection of mycoplasma
Studies have shown that more than 95% of mycoplasma contaminating cells are four of the following: mycoplasma stomatitis (M.orale), mycoplasma arginini (M.arginini), mycoplasma hyorhinis (M.hyorhinis), lei's non-cholesterol mycoplasma (A.laidlawii). In addition, mycoplasma fermentans (M.fermentans), mycoplasma hominus (M.hominis), mycoplasma salivarius (M.salivarium), mycoplasma pulmonary (M.pulmonis) and Mycoplasma pyriformis (M.pirum) are also common. According to the literature, 15 kinds of mycoplasma which are easy to pollute in cells and gene therapy products are selected, the gene sequences in GenBank are referenced, and specific primer design is carried out by selecting target sequences through nucleic acid sequence comparison, and the information is shown in the following table 3 (mycoplasma target information).
TABLE 3 Table 3
2. Optimization and establishment of composite amplification system
1. Primer design
After determining a mycoplasma target sequence to be detected, adopting oligo 6.0 software to carry out primer design, wherein all primers are synthesized by a biological engineering (Shanghai) limited company, and when the primers are designed, the moderate length of the primers is ensured, and the primers have similar physical characteristics and reaction dynamics characteristics, the Tm value and the GC content are similar, and a dimer cannot be formed among the primers; primer design should take into account the specificity of primer amplification without generating non-specific and false positive amplifications. Primer pairs and primer concentrations for 15 mycoplasma are shown in table 1.
2. Multiplex amplification
After a large number of experiments and modification and optimization, the kit for simultaneously analyzing 15 mycoplasma composite amplifications is finally determined:
a mixture was prepared, power SYBR Green PCR Master Mix (2×) purchased from Thermo Fisher company, 10× Primer Mix (10×) self-prepared, and the theoretical total volume of individual test wells was 18 μl, the volume of the mixture being as shown in table 4 below:
TABLE 4 Table 4
| Component (A) | Volume (mul) |
| Power SYBR Green PCR Master Mix(2×) | 15 |
| 10×Primer Mix(10×) | 3 |
| Total volume of | 18 |
The detection pore reaction system comprises: test wells, test interference wells (test + positive control), positive control wells, and negative control wells. The formulation of the reaction system is shown in Table 5 below:
TABLE 5
| Component (A) | Test sample hole | Test article interference hole | Negative control well | Positive control well |
| Test article (mul) | 10 | 10 | NA | NA |
| Mixed solution (mul) | 18 | 18 | 18 | 18 |
| Water (mu l) | 2 | NA | 12 | 10 |
| Positive control (μl) | NA | 2 | NA | 2 |
| Total volume (μl) | 30 | 30 | 30 | 30 |
3. On-machine detection
3.1, starting the instrument, and sequentially performing program setting: 96-well (0.2 ml), melt Curve, SYBR Green Reagents, standard.
3.2 defines the report type for all detection wells. Meanwhile, specific names are defined for the test wells.
Target Name：MycoSEQ
Reporter：SYBR
Quencher：None
3.3 define PCR procedure, total reaction volume: 30 μl.
Hold stage：95℃，10min；
PCR stage：95℃，15s；60℃，1min；40cycles；
Melt Curve stage：95℃，15s；60℃，1min；95℃，15s；
3.4 click "Start Run" to Start the procedure, which takes approximately 1.6 hours.
4. Data analysis and recording:
4.1 "Manual CT" was selected and set to "Threshold" of 0.2.
4.2, the Ct, tm and DV of the positive control, the negative control and the test sample are recorded respectively.
4.3 recording Ct, tm and DV of the test article interference hole and calculating delta Ct=Ct Interference of test sample -Ct Positive control
5. The results are shown in Table 6
TABLE 6
| Control | Ct | Tm(℃) | DV | |ΔCt| |
| Positive control well | ≤36 | 84.5±1 | ＞0.05 | NA |
| Negative control well | ＞36 | NA | NA | NA |
| Test article interference hole | ≤36 | 84.5±1 | ＞0.05 | ＜2 |
5.1 positive control wells: ct is less than or equal to 36, DV is more than 0.05, tm (DEG C) =84.5+/-1, and the three judgment standards can be judged positive when being simultaneously established.
5.2 negative control wells: ct > 36, negative.
5.3 test article interference hole: when Ct is less than or equal to 36, DV is more than 0.05, tm (DEG C) =84.5+/-1 and |delta Ct| < 2 and the four judging standards are met, judging that the sample does not interfere with the analysis method, and judging the detection result of the sample; if the four judgment standards are not met at the same time, the test sample is proved to have interference on the analysis method, and judgment is carried out after the interference is eliminated.
5.4 test sample Ct > 36, it is determined that Mycoplasma is not detected.
5.5 when the Ct of the sample is less than or equal to 36, judging according to the following different conditions:
(1) when DV is less than 0.05, the Tm value is not taken as a reference, and the test sample is judged to be that mycoplasma is not detected.
(2) If DV is not less than 0.05, but Tm (DEG C) is not within the range of 75-81 ℃, judging that mycoplasma is not detected.
(3) If DV is more than or equal to 0.05 and Tm (DEG C) is between 75 ℃ and 81 ℃, the test sample mycoplasma is determined to be detected positive.
Example 2: construction and Synthesis of kit Positive control plasmid
In order to evaluate the specificity, sensitivity and method stability of the kit primer, commercial company (Jin Weizhi, su zhou) is entrusted to synthesizing plasmids according to the primer targeting nucleotide sequence, and the synthesized plasmid standard is respectively used as a positive control, and 15 plasmids can be well amplified by adopting the amplification analysis system in the embodiment 1, and the representative map is shown in fig. 1, so that the kit composite amplification primer has good specificity and can be effectively amplified on target genes.
Example 3: kit specificity performance verification
The PCR detection kit used in this example was the same as in example 1.
Directly using the existing Escherichia coli, staphylococcus aureus, staphylococcus epidermidis, bacillus tetanus, diplococcus pneumoniae and Chlamydia in laboratory, and extracting DNA by using a nucleic acid extraction kit (ABI procurement) according to the following steps:
1, preparation of a test article:
the sample was the above microorganism sample, and 100. Mu.l was directly aspirated into the EP tube.
2. Sample cracking:
2.1 preheating the metal bath to 56 ℃ and preheating the water bath kettle to 37 ℃ for standby.
2.2 into the EP tube containing the sample, 200. Mu.l of Lysis Buffer (reagent in kit) was added, and mixed by vortexing.
2.3 to this EP tube were added 2. Mu.l of 0.5 mol/L EDTA solution, 18. Mu.l of RNase Cocktail (reagent in kit) in sequence, and mixed with gentle shaking.
2.4 the EP tube was incubated in a metal bath at 56℃for 15min.
2.5 the EP tube was removed, 2. Mu.l of Proteinase K (reagent in kit) was added to the tube, mixed with gentle shaking and incubated in a metal bath at 56℃for 10min.
2.6 the EP tube was removed, left at room temperature for 5min, 700. Mu.l of Lysis Buffer was added to the EP tube and mixed well on a vortex shaker.
DNA adsorption:
3.1 Advance incubation of Magnetic Particles (kit reagents) in a 37℃water bath for about 10min.
3.2 into the EP tube was added 30 μ l Magnetic Particles, vortexing.
3.3 to the EP tube 525 μ l Binding Solution (reagent in kit) was added and mixed upside down.
3.4 after mixing the EP tube was placed on a vortex shaker and shaken vertically at about 1500rpm for 5min.
3.5, after removal, the instantaneous maximum speed is centrifuged on a centrifuge for about 15s.
3.6 the EP tube was placed on a magnet holder for about 5min.
3.7 the supernatant was aspirated with a 1000. Mu.l pipette and discarded, avoiding touching Magnetic Particles during aspiration.
Dna washing:
4.1 into an EP tube, 300. Mu.l Wash Buffer (reagent in kit) was added and mixed well on a vortex shaker.
4.2 the EP tube was centrifuged at the instantaneous maximum speed on the centrifuge for about 15s.
4.3 after removal, the EP tube was placed on a magnet rack for about 1min.
4.4 the supernatant was aspirated with a 200. Mu.l pipette and discarded, avoiding touching Magnetic Particles during aspiration.
4.5 repeating the steps 5.6.1 to 5.6.4 once. The remaining supernatant at the bottom of the tube was aspirated with a 200. Mu.l pipette.
4.6, keeping the tube cover in an open state, standing for 5min at room temperature, and drying Magnetic Particles to precipitate.
Dna elution:
5.1 preheating the metal bath to 70℃for further use.
5.2 to the EP tube in step 5.6.6, 100. Mu.l of Elutation Buffer (in-kit reagent) was added.
5.3 the EP tube was oscillated on a vortex shaker for about 10s.
5.4 incubating the EP tube in a metal bath at 70℃for 7min, and taking out the centrifuge tube during incubation and shaking the tube on a vortex shaker for 2-3 times to fully suspend Magnetic Particle.
5.5 the EP tube was then centrifuged at 13000rpm for 5min.
5.6 after removal the EP tube was placed on a magnetic rack for 3min.
5.7 transfer of the elution supernatant to a new 1.5ml EP tube.
The results of the PCR amplification of the 6 genomes obtained in this example are shown in table 7 (the results of the specificity verification of the PCR detection kit) respectively, which indicates that the kit does not obtain effective amplification of 6 microorganisms, and does not interfere with the detection of mycoplasma by the kit, and the results of false positives are shown in fig. 2.
TABLE 7
| Genome (genome) | Coli bacterium | Staphylococcus aureus | Staphylococcus epidermidis | Tetanus bacillus | Pneumococci | Chlamydia (Chlamydia) | Positive control group |
| Results | — | — | — | — | — | — | + |
Example 4: application verification of PCR detection kit
Various test cells being cultured in the laboratory: vero cells, CHO cells, 293T cells, HEK293 cells, HT1080 cells were extracted with 100. Mu.l of culture supernatant, and DNA extraction was performed according to the DNA extraction procedure described in example 3; DMEM cell culture medium, a bottle of a multi-time used suspected mycoplasma culture medium was sampled for 100. Mu.l each, and DNA extraction was performed as in the DNA extraction step of example 3; the positive standard mycoplasma, mycoplasma pneumoniae and mycoplasma stomatitis strains (purchased from ATCC) used in the laboratory mycoplasma culture method were taken for DNA extraction according to the DNA extraction procedure in example 3, a total of 9 genomic DNA samples were amplified using the kit of example 1, the results are summarized in table 8 (sample detection results of the PCR detection kit), one bottle of used mycoplasma medium had been contaminated with mycoplasma, and neither the test cells nor the culture medium had been contaminated. This result helps the laboratory to find the contamination problem of the medium in time, avoiding significant deviations in the detection. This also demonstrates that the PCR detection kit has very high detection sensitivity in practical applications. FIG. 3 shows a representative pattern of negative amplification of the samples used for verification (DMEM medium amplification pattern), and FIG. 4 shows a representative pattern of positive amplification of the samples used for verification (Mycoplasma doubtful medium amplification pattern).
TABLE 8
| Genome (genome) | Vero cells | CHO cells | 293T cells | HEK293 cells | HT1080 cells |
| Results | — | — | — | — | — |
| Genome (genome) | DMEM | Mycoplasma doubtful culture medium | Mycoplasma pneumoniae DNA | Mycoplasma stomatitis DNA | NA |
| Results | — | + | + | + | NA |
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations and modifications of the present invention will be apparent to those of ordinary skill in the art in light of the foregoing description. It is not necessary here nor is it exhaustive of all embodiments. And obvious variations or modifications thereof are contemplated as falling within the scope of the present invention.
Claims (4)
1. A kit for qPCR detection of amplified mycoplasma is characterized in that: the kit comprises 15 groups of primer sequences, which are respectively: SEQ ID NO. 1-2, SEQ ID NO. 3-4, SEQ ID NO. 5-6, SEQ ID NO. 7-8, SEQ ID NO. 9-10, SEQ ID NO. 11-12, SEQ ID NO. 13-14, SEQ ID NO. 15-16, SEQ ID NO. 17-18, SEQ ID NO. 19-20, SEQ ID NO. 21-22, SEQ ID NO. 23-24, SEQ ID NO. 25-26, SEQ ID NO. 27-28 and SEQ ID NO. 29-30.
2. The kit for qPCR detection of mycoplasma amplification according to claim 1, wherein the final concentration of the primer sequence in the amplification system is: SEQ ID NO. 1 and SEQ ID NO. 2 are 0.15. Mu.M; SEQ ID NO. 3 and SEQ ID NO. 4 are 0.12. Mu.M; SEQ ID NO. 5 and SEQ ID NO. 6 are 0.14. Mu.M; SEQ ID NO. 7 and SEQ ID NO. 8 are 0.16. Mu.M; SEQ ID NO. 9 and SEQ ID NO. 10 are 0.19. Mu.M; SEQ ID NO. 11 and SEQ ID NO. 12 are 0.2. Mu.M; SEQ ID NO. 13 and SEQ ID NO. 14 are 0.24. Mu.M; SEQ ID NO. 15 and SEQ ID NO. 16 are 0.11. Mu.M; SEQ ID NO. 17 and SEQ ID NO. 18 are 0.09. Mu.M; SEQ ID NO. 19 and SEQ ID NO. 20 are 0.1. Mu.M; SEQ ID NO. 21 and SEQ ID NO. 22 are 0.1. Mu.M; SEQ ID NO. 23 and SEQ ID NO. 24 are 0.06. Mu.M; SEQ ID NO. 25 and SEQ ID NO. 26 are 0.1. Mu.M; SEQ ID NO. 27 and SEQ ID NO. 28 are 0.14. Mu.M; SEQ ID NO. 29 and SEQ ID NO. 30 are 0.21. Mu.M.
3. A method of using an amplified mycoplasma qPCR assay kit according to claim 1 or 2, said method being for non-disease diagnostic and therapeutic purposes, comprising the steps of:
1)95℃，10min；
2)95℃，15s；
3)60℃，60s；
4) Repeating 1) to 3) for 40 times and receiving fluorescent signals;
5)95℃，15s；
6) 60 ℃ for 60s; receiving a fluorescent signal;
7)95℃，15s。
4. use of an amplified mycoplasma qPCR detection kit according to claim 1 or 2 for detecting cellular gene therapy products for non-disease diagnosis and treatment purposes, said cellular gene therapy products being mycoplasma cell wall and/or mycoplasma lentivirus vectors.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20150007049A (en) * | 2013-07-10 | 2015-01-20 | 솔젠트 (주) | Method for Detecting Mycoplasma Contaminated in Therapeutic Cells or Biological Medicine By Using Polymerase Chain Reaction and Kit for the Same Method |
| KR20150012128A (en) * | 2013-07-24 | 2015-02-03 | 솔젠트 (주) | Method for Detecting Mycoplasma Contaminated in Therapeutic Cells or Biological Medicine By Using Real-time Polymerase Chain Reaction and Kit for the Same Method |
| CN106319080A (en) * | 2016-10-26 | 2017-01-11 | 武汉科前生物股份有限公司 | PCR detection kit for rapidly identifying mycoplasma hyopeumoniae, mycoplasma synoviae and mycoplasma hyorhinis, and applications of PCR detection kit |
| CN107805668A (en) * | 2017-12-11 | 2018-03-16 | 杭州华安生物技术有限公司 | Detect mycoplasma primer and application and apply its product and method |
| CN109468395A (en) * | 2018-12-10 | 2019-03-15 | 湖南中岸生物药业有限公司 | A kind of primer, kit, detection method and application detecting mycoplasma |
| CN114350825A (en) * | 2021-12-21 | 2022-04-15 | 苏州鉴达生物科技有限公司 | PCR kit for detecting mycoplasma pollution in cell culture and application thereof |
-
2022
- 2022-11-15 CN CN202211422763.6A patent/CN115976238B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20150007049A (en) * | 2013-07-10 | 2015-01-20 | 솔젠트 (주) | Method for Detecting Mycoplasma Contaminated in Therapeutic Cells or Biological Medicine By Using Polymerase Chain Reaction and Kit for the Same Method |
| KR20150012128A (en) * | 2013-07-24 | 2015-02-03 | 솔젠트 (주) | Method for Detecting Mycoplasma Contaminated in Therapeutic Cells or Biological Medicine By Using Real-time Polymerase Chain Reaction and Kit for the Same Method |
| CN106319080A (en) * | 2016-10-26 | 2017-01-11 | 武汉科前生物股份有限公司 | PCR detection kit for rapidly identifying mycoplasma hyopeumoniae, mycoplasma synoviae and mycoplasma hyorhinis, and applications of PCR detection kit |
| CN107805668A (en) * | 2017-12-11 | 2018-03-16 | 杭州华安生物技术有限公司 | Detect mycoplasma primer and application and apply its product and method |
| CN109468395A (en) * | 2018-12-10 | 2019-03-15 | 湖南中岸生物药业有限公司 | A kind of primer, kit, detection method and application detecting mycoplasma |
| CN114350825A (en) * | 2021-12-21 | 2022-04-15 | 苏州鉴达生物科技有限公司 | PCR kit for detecting mycoplasma pollution in cell culture and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| Detection of multiple mycoplasma infection in cell cultures by PCR;J Timenetsky等;Braz J Med Biol Res;第39卷(第7期);第907-914页 * |
| 支原体检测多重定量PCR方法的建立及应用;霍欣洋等;现代生物医学进展;第20卷(第7期);第1259-1262页 * |
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