CN118064616A - Method for rapidly detecting bacterial, fungal and mycoplasma pollution in mesenchymal stem cell culture process based on fluorescence quantitative PCR - Google Patents
Method for rapidly detecting bacterial, fungal and mycoplasma pollution in mesenchymal stem cell culture process based on fluorescence quantitative PCR Download PDFInfo
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Abstract
The invention provides a method for rapidly detecting bacterial, fungal and mycoplasma pollution in a mesenchymal stem cell culture process based on fluorescence quantitative PCR, belonging to the technical field of microorganism detection. A DNA specific fragment for detecting bacteria, fungi and mycoplasma comprises a DNA fragment with a nucleotide sequence shown as SEQ ID NO. 1 or a DNA fragment with a length of more than 50% which is intercepted on the basis of SEQ ID NO. 1. Based on the DNA specific fragment, the invention designs a universal primer for detecting bacteria, fungi and mycoplasma. The qPCR detection by using the universal primer can rapidly and specifically detect whether bacterial, fungal and mycoplasma pollution exists in the mesenchymal stem cell preparation, and the method is simple, quick and accurate, takes shorter time than the conventional culture method, and can perform qualitative analysis on the result.
Description
Technical Field
The invention belongs to the technical field of microorganism detection, and particularly relates to a method for rapidly detecting bacterial, fungal and mycoplasma pollution in a mesenchymal stem cell culture process based on fluorescence quantitative PCR.
Background
The mesenchymal stem cells and the exosomes thereof have wide application prospects in clinical medicine, so that pollution-free culture of the mesenchymal stem cells is increasingly receiving attention. The mesenchymal stem cell culture process needs to meet strict quality control detection. Quality control tests include sterility, tumorigenicity, cell purity, cell activity. Wherein sterility includes detection of mycoplasma, bacteria, fungi, and endotoxins.
The traditional mycoplasma detection method mainly comprises a culture method and an indicator cell method, and detection is generally required to be carried out simultaneously. Both methods generally require a longer time, such as at least 28 days of culture. In recent years, cell therapeutic drugs are rapidly developed, and the cell therapeutic drugs have the advantages of rapid marketing period, short shelf life, and incapability of meeting the time requirement of drug release by both culture methods and cell indication methods. Real-time fluorescent quantitative PCR is a method by which a specific DNA sequence in a sample to be measured is quantitatively analyzed. The real-time fluorescent quantitative PCR is to detect the PCR process in real time through fluorescent signals in the PCR amplification process. Since the CT value of the template and the initial copy number of the template have a linear relationship in the exponential phase of PCR amplification, the quantitative basis is established. Single peak plot of melting profile of PCR product shows the singleness of PCR amplification product. Therefore, detection of mycoplasma based on real-time fluorescent quantitative PCR technology is expected to achieve good detection results. However, mesenchymal stem cells are also susceptible to bacterial and fungal contamination during culture, and although specific fragments and universal primers for detecting bacteria and/or fungi have been reported, there are no universal primers and methods for detecting bacterial, fungal and mycoplasma contamination during mesenchymal stem cell culture.
Disclosure of Invention
In view of the above, the present invention aims to provide a universal primer for detecting mesenchymal stem cell contamination, which can realize simultaneous detection of bacteria, fungi and mycoplasma, and provides a basis for simplifying detection operations.
The invention also aims to provide a universal primer for detecting bacteria, fungi and mycoplasma, which not only can detect a plurality of typical mycoplasma related to Chinese pharmacopoeia, but also can detect a plurality of bacteria and fungi, and can rapidly and specifically detect whether the mesenchymal stem cell preparation has pollution of cells, fungi and mycoplasma, thereby simplifying experimental operation and reducing detection cost.
The invention provides a DNA specific fragment for detecting bacteria, fungi and mycoplasma, which comprises a DNA fragment with a nucleotide sequence shown as SEQ ID NO.1 or a DNA fragment with a length of more than 50% intercepted on the basis of SEQ ID NO. 1.
Preferably, the DNA fragment with the nucleotide sequence shown in SEQ ID NO. 2-SEQ ID NO. 5 is included.
The present invention provides a universal primer for detecting bacteria, fungi and mycoplasma, comprising
At least one of the following primer pairs for amplifying the DNA-specific fragment: the nucleotide sequence is shown in SEQ ID NO:6 and the nucleotide sequence of the forward primer F1 is shown as SEQ ID NO:7, the nucleotide sequence of the reverse primer R1 is shown as SEQ ID NO:8 and the nucleotide sequence of the forward primer F2 is shown as SEQ ID NO:7, the nucleotide sequence of the reverse primer R1 is shown as SEQ ID NO:9 and the nucleotide sequence of the forward primer F3 is shown as SEQ ID NO:7 and the nucleotide sequence of the reverse primer R1 is shown as SEQ ID NO:6 and the nucleotide sequence of the forward primer F1 is shown as SEQ ID NO:10, and a reverse primer R2 shown in FIG. 10.
The invention provides application of the universal primer in preparation of a kit for simultaneously detecting bacteria, fungi and mycoplasma.
The invention provides a kit for detecting microbial contamination of mesenchymal stem cells based on a fluorescent quantitative PCR technology, which comprises the universal primer and a premix solution for fluorescent quantitative PCR amplification.
The invention provides application of the universal primer in preparation of a kit for detecting microbial contamination of mesenchymal stem cells.
The invention provides a method for detecting bacterial, fungal and mycoplasma pollution in a mesenchymal stem cell culture system, which comprises the following steps:
Extracting DNA from a mesenchymal stem cell culture system to be detected;
preparing a qPCR reaction system by using the extracted DNA as a template and utilizing the universal primer to perform qPCR amplification;
Judging whether bacterial, fungal and mycoplasma pollution exists in a mesenchymal stem cell culture system to be detected according to the shape of an amplification curve and a CT value of a detection channel:
positive: the CT value of the detection channel is less than 29, and the amplification curve has obvious exponential growth trend;
negative: an amplification curve with a sample detection result CT more than or equal to 30 or no CT value and no obvious exponential growth trend;
suspicious: the CT value is more than or equal to 29 and less than 30, and the sample with the typical amplification curve suggests repeated tests, and the repeated test results show that the CT value is more than or equal to 29 and the typical amplification curve is positive, otherwise, the sample with the typical amplification curve is negative.
Preferably, the qPCR reaction system comprises 10 μlMASTERQPCR MIX-SYBR (+UDG) 5. Mu.L, 10. Mu.M forward primer 0.5. Mu.L, 10. Mu.M reverse primer 0.5. Mu.L and DNA 4. Mu.L.
Preferably, the reaction system of qPCR amplification is at 50 ℃ for 2min;95 ℃ for 2min; fluorescence signals were collected at 95℃for 15S,55℃for 30S,72℃for 30S, and 32 cycles at 72 ℃.
Preferably, the bacteria include at least one of: staphylococcus aureus, pseudomonas aeruginosa, escherichia coli, bacillus subtilis and clostridium sporogenes;
the fungi include candida albicans and/or aspergillus niger;
The mycoplasma includes mycoplasma stomatitis and/or mycoplasma pneumoniae.
The invention provides a DNA specific fragment for detecting bacteria, fungi and mycoplasma, which comprises a DNA fragment with a nucleotide sequence shown as SEQ ID NO. 1 or a DNA fragment with a length of more than 50% intercepted on the basis of SEQ ID NO. 1. The invention firstly carries out sequence comparison on the complete genomes of 7 bacterial fungi (staphylococcus aureus, pseudomonas aeruginosa, escherichia coli, bacillus subtilis, clostridium sporogenes, candida albicans and aspergillus niger) and 2 mycoplasma (mycoplasma stomatitis and mycoplasma pneumoniae), finds a section of homologous conserved sequence, designs an amplification primer according to the homologous conserved sequence, and utilizes the primer to carry out fluorescent quantitative PCR detection verification, thereby realizing the detection of bacteria, fungi and mycoplasma, greatly simplifying the detection operation and providing a basis for simultaneously detecting the bacteria, the fungi and the mycoplasma.
The invention provides a universal primer for detecting mesenchymal stem cell pollution based on a fluorescent quantitative PCR technology, which comprises the following nucleotide sequences as shown in SEQ ID NO:3 and the nucleotide sequence of the forward primer is shown as SEQ ID NO: 4. The universal primer provided by the invention can be used for rapidly and specifically detecting whether bacterial fungus and mycoplasma pollution exists in the mesenchymal stem cell preparation. According to the embodiment of the invention, 7 bacteria fungi (staphylococcus aureus, pseudomonas aeruginosa, escherichia coli, bacillus subtilis, clostridium sporogenes, candida albicans and aspergillus niger) and 2 mycoplasma (mycoplasma stomatitis and mycoplasma pneumoniae) recorded in pharmacopoeia are used as representatives, and inactivated DNA (deoxyribonucleic acid) is used as a template to perform a real-time fluorescence quantitative PCR reaction, so that the universal primer can detect bacteria, fungi and mycoplasma simultaneously, has good detection specificity and has detection sensitivity higher than that of the traditional culture method recorded in Chinese pharmacopoeia. The development of the universal primer provides great convenience for the application environment for rapidly and efficiently detecting bacteria, fungi and mycoplasma, and can greatly reduce the detection cost and labor input.
The invention provides a method for detecting bacterial, fungal and mycoplasma pollution in a mesenchymal stem cell culture system, which is characterized in that whether bacterial, fungal and mycoplasma pollution exists in the mesenchymal stem cell culture system to be detected is judged by utilizing the shape of an amplification curve and a CT value of a detection channel through a real-time fluorescence quantitative PCR method. The method provided by the invention meets the requirements of rapid and efficient detection, greatly simplifies the detection steps of the mesenchymal stem cell culture system polluted microorganisms, and has great industrial popularization and application values.
Drawings
FIG. 1 shows the result of a conserved sequence alignment of bacteria, fungi and mycoplasma (nine total);
FIG. 2 is a melting curve of Staphylococcus aureus-F1/R1 primers;
FIG. 3 shows melting curves of the B.subtilis-F1/R1 primers;
FIG. 4 shows melting curves of Clostridium sporogenes-F1/R1 primers;
FIG. 5 is a melting curve of E.coli-F1/R1 primer;
FIG. 6 is a melting curve of Candida albicans-F1/R1 primer;
FIG. 7 shows melting curves of Aspergillus niger-F1/R1 primers;
FIG. 8 is a melting curve of the Pseudomonas aeruginosa-F1/R1 primer;
FIG. 9 shows melting curves of Mycoplasma stomatitis-F1/R1 primers;
FIG. 10 shows melting curves of Mycoplasma pneumoniae-F1/R1 primers;
FIG. 11 is a melting curve of Staphylococcus aureus-F2/R1 primers;
FIG. 12 is a melting curve of the B.subtilis-F2/R1 primer;
FIG. 13 is a melting curve of Clostridium sporogenes-F2/R1 primer;
FIG. 14 is a melting curve of E.coli-F2/R1 primer;
FIG. 15 is a melting curve of Candida albicans-F2/R1 primer;
FIG. 16 shows melting curves of Aspergillus niger-F2/R1 primers;
FIG. 17 is a melting curve of the Pseudomonas aeruginosa-F2/R1 primer;
FIG. 18 shows melting curves of Mycoplasma stomatitis-F2/R1 primers;
FIG. 19 shows melting curves of Mycoplasma pneumoniae-F2/R1 primers;
FIG. 20 is a melting curve of Staphylococcus aureus-F3/R1 primers;
FIG. 21 is a melting curve of the B.subtilis-F3/R1 primer;
FIG. 22 shows melting curves of Clostridium sporogenes-F3/R1 primers;
FIG. 23 is a melting curve of E.coli-F3/R1 primer;
FIG. 24 is a melting curve of Candida albicans-F3/R1 primer;
FIG. 25 shows melting curves of Aspergillus niger-F3/R1 primers;
FIG. 26 is a melting curve of the Pseudomonas aeruginosa-F3/R1 primer;
FIG. 27 is a melting curve of Mycoplasma stomatitis-F3/R1 primer;
FIG. 28 shows melting curves of Mycoplasma pneumoniae-F3/R1 primers;
FIG. 29 is a melting curve of Staphylococcus aureus-F1/R2 primers;
FIG. 30 is a melting curve of the B.subtilis-F1/R2 primer;
FIG. 31 is a melting curve of Clostridium sporogenes-F1/R2 primers;
FIG. 32 is a melting curve of E.coli-F1/R2 primer;
FIG. 33 is a melting curve of Candida albicans-F1/R21 primer;
FIG. 34 is a melting curve of the A.niger-F1/R2 primer;
FIG. 35 is a melting curve of the Pseudomonas aeruginosa-F1/R2 primer;
FIG. 36 shows melting curves of Mycoplasma stomatitis-F1/R2 primers;
FIG. 37 shows melting curves of Mycoplasma pneumoniae-F1/R2 primers.
Detailed Description
The invention provides a DNA specific fragment for detecting bacteria, fungi and mycoplasma, which comprises a DNA fragment with a nucleotide sequence shown as SEQ ID NO. 1 (GCCACACTGGAACTGAGACACGGTC CAGACTCCTACGGGAGGCAGCAGTGGGGAATATT) or a DNA fragment with a length of more than 50% which is intercepted on the basis of SEQ ID NO. 1.
In the invention, the method for obtaining the DNA specific fragment preferably comprises the step of comparing the sequences of the whole genomes of 7 bacterial fungi (staphylococcus aureus, pseudomonas aeruginosa, escherichia coli, bacillus subtilis, clostridium sporogenes, candida albicans and aspergillus niger) and 2 mycoplasma (mycoplasma stomatitis and mycoplasma pneumoniae) to obtain a section of homologous conserved sequence.
In the present invention, the DNA specific fragment is preferably a DNA fragment having a length of 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% cut on the basis of SEQ ID NO. 1. In the present invention, a DNA fragment having a nucleotide sequence shown as SEQ ID NO:2(CGGTCCAGACTCCTA CGGGAGGCAGCAGTGGGGAAT)、SEQ ID NO:3(GACACGGTCCAGA CTCCTACGGGAGGCAGCAGTGGGGAAT)、SEQ ID NO:4(CACGGTCCA GACTCCTACGGGAGGCAGCAGTGGGGAAT)、SEQ ID NO:5(CGGTCCA GACTCCTACGGGAGGCAGCAGTGGGGAA) is exemplified, and detection of bacteria, fungi and mycoplasma can be simultaneously achieved based on the DNA-specific fragment, but it is not construed that the scope of the present invention is limited. The experimental result shows that based on the DNA specific fragment, the existence of the polluted microorganisms of bacteria, fungi and mycoplasma can be detected at the same time, and the specific detection experimental result shows that the DNA fragment exists only in the bacteria, the fungi and the mycoplasma, is not conserved in the cultured target cells and viruses, and cannot obtain a positive amplification result, so that the DNA fragment has good specificity.
The invention provides a universal primer for detecting bacteria, fungi and mycoplasma, comprising at least one of the following primer pairs for amplifying the DNA specific fragment: the nucleotide sequence is shown in SEQ ID NO:6 (CGGTCCAGACTCCTACGG) and the forward primer F1 with the nucleotide sequence shown in SEQ ID NO:7 (ATTCCCCACTGCTGCCTC), the nucleotide sequence of which is shown in SEQ ID NO:8 (GACACGGTCCAGACTCCT) and the nucleotide sequence of the forward primer F2 is shown as SEQ ID NO:7 (ATTCCCCACTGCTGCCTC), the nucleotide sequence of which is shown in SEQ ID NO:9 (CACGGTCCAGACTCCTAC) and the forward primer F3 with the nucleotide sequence shown in SEQ ID NO:7 (ATTCCCCACTGCTGCCTC), the nucleotide sequence of which is shown in SEQ ID NO:6 (CGGTCCAGACTCCTACGG) and the forward primer F1 with the nucleotide sequence shown in SEQ ID NO:10 (TTCCCCACTGCTGCCTCCC) A reverse primer R2.
In the invention, 4 pairs of primers are designed aiming at specific fragments shown in SEQ ID NO. 1. The source of the universal primer is preferably designated as the gene synthesis company.
In the invention, sensitivity detection experiments show that the detection sensitivity is higher than the sensitivity (10 CFU/ml) of the traditional culture method recorded in Chinese pharmacopoeia, wherein the detection sensitivity of mycoplasma stomatitis is 5CFU/ml, and the detection sensitivity of mycoplasma pneumoniae is 2.5CFU/ml.
In view of the fact that the universal primer can realize specific detection on bacteria, fungi and mycoplasma at the same time, the invention provides application of the universal primer in preparation of a kit for detecting bacteria, fungi and mycoplasma at the same time.
In the present invention, the kit is suitable for detection of all kinds of bacteria, fungi and mycoplasma. In the examples of the present invention, in order to describe the effects obtained by the kit in detail, staphylococcus aureus, pseudomonas aeruginosa, escherichia coli, bacillus subtilis and clostridium sporogenes are represented as bacteria, candida albicans and/or aspergillus niger are represented as fungi, and mycoplasma stomatitis and mycoplasma pneumoniae are represented as mycoplasma. The result shows that the kit not only can detect bacteria, fungi and mycoplasma at the same time, but also can distinguish cultured cells and viruses, and has good detection specificity. In addition, the detection sensitivity of the kit meets the detection requirement.
The invention provides a kit for detecting microbial contamination of mesenchymal stem cells based on a fluorescent quantitative PCR technology, which comprises the universal primer and a premix solution for fluorescent quantitative PCR amplification.
The kind and source of the premix for fluorescent quantitative PCR amplification are not particularly limited, and those known in the art may be used. In the embodiment of the invention, the premix for fluorescent quantitative PCR amplification preferably comprisesMASTER QPCR Mix (+UDG) from Beijing Optimago Biotechnology Inc., lot OFC19901.
In the present invention, the mesenchymal stem cell-contaminating microorganisms preferably include bacteria, fungi and mycoplasma. In the embodiment of the invention, staphylococcus aureus, pseudomonas aeruginosa, escherichia coli, bacillus subtilis and clostridium sporogenes are used as bacteria, candida albicans and/or aspergillus niger are used as fungi, and mycoplasma stomatitis and mycoplasma pneumoniae are used as mycoplasma. The result shows that the kit not only can detect bacteria, fungi and mycoplasma at the same time, but also can distinguish cultured cells and viruses, and has good detection specificity. In addition, the detection sensitivity of the kit meets the detection requirement.
The invention provides application of the universal primer in preparation of a kit for detecting microbial contamination of mesenchymal stem cells.
In the present invention, the kit is suitable for detection of all kinds of bacteria, fungi and mycoplasma. In the examples of the present invention, in order to describe the effects obtained by the kit in detail, staphylococcus aureus, pseudomonas aeruginosa, escherichia coli, bacillus subtilis and clostridium sporogenes are represented as bacteria, candida albicans and/or aspergillus niger are represented as fungi, and mycoplasma stomatitis and mycoplasma pneumoniae are represented as mycoplasma. The result shows that the kit not only can detect bacteria, fungi and mycoplasma at the same time, but also can distinguish cultured cells and viruses, and has good detection specificity. In addition, the detection sensitivity of the kit meets the detection requirement.
The invention provides a method for detecting bacterial, fungal and mycoplasma pollution in a mesenchymal stem cell culture system, which comprises the following steps:
Extracting DNA from a mesenchymal stem cell culture system to be detected;
preparing a qPCR reaction system by using the extracted DNA as a template and utilizing the universal primer to perform qPCR amplification;
Judging whether bacterial, fungal and mycoplasma pollution exists in a mesenchymal stem cell culture system to be detected according to the shape of an amplification curve and a CT value of a detection channel:
positive: the CT value of the detection channel is less than 29, and the amplification curve has obvious exponential growth trend;
negative: an amplification curve with a sample detection result CT more than or equal to 30 or no CT value and no obvious exponential growth trend;
suspicious: the CT value is more than or equal to 29 and less than 30, and the sample with the typical amplification curve suggests repeated tests, and the repeated test results show that the CT value is more than or equal to 29 and the typical amplification curve is positive, otherwise, the sample with the typical amplification curve is negative.
The invention extracts DNA from the mesenchymal stem cell culture system to be tested.
The method for extracting DNA of the present invention is not particularly limited, and methods for extracting DNA known in the art, such as a kit method, may be employed. After extraction of the DNA, the DNA is preferably subjected to quality tests, including concentration and integrity tests. Diluting the DNA which is qualified in detection, and refrigerating for standby.
The invention prepares a qPCR reaction system by using the universal primer and carries out qPCR amplification by taking the extracted DNA as a template.
In the present invention, the qPCR reaction system preferably comprises 10. Mu.l ofMASTER QPCR Mix-SYBR (+UDG) 5. Mu.L, 10. Mu.M forward primer 0.5. Mu.L, 10. Mu.M reverse primer 0.5. Mu.L and DNA 4. Mu.L. The reaction system of qPCR amplification is preferably at 50 ℃ for 2min;95 ℃ for 2min; fluorescence signals were collected at 95℃for 15S,55℃for 30S,72℃for 30S, and 32 cycles at 72 ℃. The apparatus used for qPCR amplification is not particularly limited, and qPCR apparatuses known in the art may be used.
In the present invention, the bacterium preferably includes at least one of: staphylococcus aureus, pseudomonas aeruginosa, escherichia coli, bacillus subtilis and clostridium sporogenes. The fungus preferably comprises candida albicans and/or aspergillus niger. The mycoplasma preferably includes mycoplasma stomatitis and mycoplasma pneumoniae.
In the embodiment of the invention, the CT values of the primer detection concentration of the mycoplasma stomatitis DNA of 5CFU/ml and the primer detection concentration of the mycoplasma pneumoniae DNA of 2.5CFU/ml are lower than 29, which indicates that the primer can detect the mycoplasma stomatitis DNA and the mycoplasma pneumoniae DNA. Meanwhile, the primers can detect DNA corresponding to 7 bacteria and fungi, which indicates that the primers can detect bacteria and fungi. The invention also develops a specificity experiment, which takes the cultured mesenchymal stem cells and the human polyomavirus JCV as the contrast, and the detection result is negative. It is described that the universal primers can detect only bacteria, fungi and mycoplasma, but not mesenchymal stem cells and conventional viruses in a culture system, and the primers have good detection specificity.
The following is a detailed description of a method for rapid detection of bacterial, fungal and mycoplasma contamination during mesenchymal stem cell culture based on fluorescent quantitative PCR provided by the present invention, but they should not be construed as limiting the scope of the present invention.
Example 1
Screening method of bacterial, fungal and mycoplasma DNA specific fragments
And searching corresponding gene sequences on NCBI and GenBank according to the English names of nine bacterial fungi and mycoplasma, and then carrying out homologous sequence comparison analysis, wherein the comparison result is shown in figure 1, so that a section of conserved sequence is obtained, and the nucleotide sequence is shown as SEQ ID NO.1 (GCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATT).
TABLE 1 summary of strain information
Example 2
Design and verification of universal primers for bacteria, fungi and mycoplasma
1. Primer design
The universal primers were designed using PRIMER PREMIER software using the conserved sequences obtained in example 1 as templates, and the results are shown in Table 2.
TABLE 2 primer sequence information
2. Verification method
2.1 Strain information is shown in Table 3.
TABLE 3 strains used in experiments
| Bacterial strain | Source(s) |
| Bacillus subtilis | Wenzhou micro dome microbiological technologies Co |
| Clostridium sporogenes | Wenzhou micro dome microbiological technologies Co |
| Escherichia coli | Wenzhou micro dome microbiological technologies Co |
| Staphylococcus aureus | Wenzhou micro dome microbiological technologies Co |
| Candida albicans | Wenzhou micro dome microbiological technologies Co |
| Aspergillus niger | Wenzhou micro dome microbiological technologies Co |
| Pseudomonas aeruginosa | Wenzhou micro dome microbiological technologies Co |
| Oral cavity mycoplasma inactivated bacterial liquid | Huzhou Shen Ke Biotechnology Co Ltd |
| Mycoplasma pneumoniae inactivated bacterial liquid | Huzhou Shen Ke Biotechnology Co Ltd |
3. Extraction of Strain/bacterial liquid DNA
Extracting the genome DNA of the nine standard strains/bacterial solutions according to the instruction of a root genome DNA extraction kit, wherein the specific method is as follows:
1. 200 μl of buffer GA was added and shaken until thoroughly mixed.
2. Mu.l of protease K solution was added and mixed well.
3. 200 Μl of buffer GB was added, mixed well by inversion, left at 70deg.C for 10min, the solution was clear and centrifuged briefly to remove water droplets from the inner wall of the tube cap.
4. 200 Μl of absolute ethanol was added, and the mixture was thoroughly shaken and mixed for 15sec, at which time flocculent precipitate was likely to appear, and centrifuged briefly to remove water droplets on the inner wall of the tube cap.
5. The solution obtained in the previous step and the flocculent precipitate were both added to an adsorption column CB3, centrifuged at 12000rpm for 30sec, and the waste liquid was discarded, and the adsorption column CB3 was returned to the collection tube.
6. To the adsorption column CB3, 500. Mu.l of the buffer solution GD was added, and the mixture was centrifuged at 12000rpm for 30sec, and the waste liquid was poured off, whereby the adsorption column CB3 was placed in a collection tube.
7. 600. Mu.l of the rinse PW was added to the adsorption column CB3, centrifuged at 12000rpm for 30sec, and the waste liquid was poured off, and the adsorption column CB3 was placed in a collection tube.
8. And repeating the operation step 7.
9. The adsorption column CB3 was put back into the collection tube, centrifuged at 12000rpm for 2min, and the waste liquid was discarded. The adsorption column CB3 was left at room temperature for several minutes to thoroughly dry the residual rinse solution in the adsorption material.
10. Transferring the adsorption column CB3 into a clean centrifuge tube, suspending and dripping 50 μl of elution buffer TE into the middle part of the adsorption film, standing for 5min at room temperature, centrifuging at 12000rpm for 2min, and collecting the solution into the centrifuge tube.
TABLE 4 concentration of bacterial DNA
| Sample name | DNA concentration (ng/. Mu.l) |
| Clostridium sporogenes | 4.407 |
| Escherichia coli | 4.727 |
| Candida albicans | 4.342 |
| Aspergillus niger | 4.393 |
| Bacillus subtilis | 4.044 |
| Staphylococcus aureus | 3.873 |
| Pseudomonas aeruginosa | 4.479 |
| Mycoplasma stomatitis | 10.462 |
| Mycoplasma pneumoniae | 23.119 |
4. Fluorescent quantitative PCR reaction system and program
10 Μl of the total reaction system comprising the following reagents: 2X TSINGKE MASTER QPCR Mix-SYBR (+UDG) 5. Mu.l, upstream primer (10. Mu.M) 0.5. Mu.l, downstream primer (10. Mu.M) 0.5. Mu.l, sample DNA 4. Mu.l. The final concentration of the upstream and downstream primers was 0.4. Mu.M each. The same template was subjected to a multiplex reaction, all reactions were performed on a LightCycler480 fluorescent quantitative PCR instrument. Reaction procedure for real-time fluorescent quantitative PCR: pre-denaturation at 50 ℃ for 2min; pre-denaturation at 95℃for 2min; denaturation at 95℃for 15s, annealing at 55℃for 30s, extension at 72℃for 30s,32 cycles, and fluorescent signal collection at 72℃ (LightCycler 480, SYBR Green I channel).
Result judgment (qualitative analysis):
And judging whether the sample to be tested contains mycoplasma or not according to CT values of the positive control and the negative control (BET water).
Positive: the CT value of the detection channel is less than 29, and the amplification curve has obvious exponential growth trend;
negative: an amplification curve with a sample detection result CT more than or equal to 30 or no CT value and no obvious exponential growth trend;
suspicious: the CT value is more than or equal to 29 and less than 30, and the sample with the typical amplification curve suggests repeated tests, and the repeated test results show that the CT value is more than or equal to 29 and the typical amplification curve is positive, otherwise, the sample with the typical amplification curve is negative.
5. The experimental results are shown in tables 5 to 8 and FIGS. 2 to 37. As shown by the detection results, the detection CT values of the universal primer pair bacteria, fungi and mycoplasma are all lower than 30, and the amplification curves are all obviously exponentially increased, so that the detection results are positive.
TABLE 5 CT value for detection of F1/R1 Universal primer
| Melting curve | Bacterial strain | CT value |
| Unimodal peak | Staphylococcus aureus | 23.55 |
| Unimodal peak | Bacillus subtilis | 23.97 |
| Unimodal peak | Clostridium sporogenes | 22.78 |
| Unimodal peak | Escherichia coli | 23.66 |
| Unimodal peak | Candida albicans | 25.08 |
| Unimodal peak | Aspergillus niger | 22.74 |
| Unimodal peak | Pseudomonas aeruginosa | 21.83 |
| Unimodal peak | Mycoplasma stomatitis | 24.87 |
| Unimodal peak | Mycoplasma pneumoniae | 28.35 |
TABLE 6 CT number for detection of F2/R1 Universal primer
TABLE 7 CT value for detection of F3/R1 Universal primer
| Melting curve | Bacterial strain | CT value |
| Unimodal peak | Staphylococcus aureus | 26.35 |
| Unimodal peak | Bacillus subtilis | 25.74 |
| Unimodal peak | Clostridium sporogenes | 24.46 |
| Unimodal peak | Escherichia coli | 24.36 |
| Unimodal peak | Candida albicans | 25.52 |
| Unimodal peak | Aspergillus niger | 25.52 |
| Unimodal peak | Pseudomonas aeruginosa | 21.55 |
| Unimodal peak | Mycoplasma stomatitis | 25.29 |
| Unimodal peak | Mycoplasma pneumoniae | 27.30 |
Table 8 CT values for detection of F1/R2 Universal primers
| Melting curve | Bacterial strain | CT value |
| Unimodal peak | Staphylococcus aureus | 25.06 |
| Unimodal peak | Bacillus subtilis | 24.24 |
| Unimodal peak | Clostridium sporogenes | 24.33 |
| Unimodal peak | Escherichia coli | 24.14 |
| Unimodal peak | Candida albicans | 23.99 |
| Unimodal peak | Aspergillus niger | 24.43 |
| Unimodal peak | Pseudomonas aeruginosa | 24.89 |
| Unimodal peak | Mycoplasma stomatitis | 25.48 |
| Unimodal peak | Mycoplasma pneumoniae | 27.18 |
Example 3
CT values of 7 bacterial fungi (Staphylococcus aureus, pseudomonas aeruginosa, escherichia coli, bacillus subtilis, clostridium sporogenes, candida albicans and Aspergillus niger) DNA were examined using universal primers
1. Strain information 7 bacterial fungi (staphylococcus aureus, pseudomonas aeruginosa, escherichia coli, bacillus subtilis, clostridium sporogenes, candida albicans and aspergillus niger) are standard strains recommended to be subjected to aseptic routine detection by the chinese pharmacopoeia, and are representative.
Standard strain genomic DNA of 7 bacterial fungi (Staphylococcus aureus, pseudomonas aeruginosa, escherichia coli, bacillus subtilis, clostridium sporogenes, candida albicans and Aspergillus niger, 100 CFU/ml) was extracted according to the instructions of the Tiangen genomic DNA extraction kit.
2. Fluorescent quantitative PCR reaction system and program
10. Mu.l of the total reaction system, 5. Mu.l of 2X TSINGKE MasterqPCRMix-SYBR (+UDG), 0.5. Mu.l of the upstream primer (10. Mu.M), 0.5. Mu.l of the downstream primer (10. Mu.M) and 4. Mu.l of the sample DNA. The final concentration of the upstream and downstream primers was 0.5. Mu.M each. The same template was subjected to a multiplex reaction, all reactions were performed on a LightCycler480 fluorescent quantitative PCR instrument. Reaction procedure for real-time fluorescent quantitative PCR: pre-denaturation at 50 ℃ for 2min; pre-denaturation at 95℃for 2min; denaturation at 95℃for 15s, annealing at 55℃for 30s, extension at 72℃for 30s,32 cycles, and fluorescence signal acquisition at 72 ℃.
4. The experimental results are shown in Table 9.
Table 9 CT values obtained by detecting DNA of 7 bacteria and fungi with the universal primers
Conclusion: the universal primers can detect DNA corresponding to 7 bacteria and fungi, and the sensitivity reaches the sensitivity (100 CFU/ml) of the traditional culture method recorded in Chinese pharmacopoeia. Therefore, the sensitivity of the detection method provided by the invention is more superior than that of the traditional method, the time is short, the result can be obtained after 3 hours, and the traditional method is used for culturing for 14 days.
Example 4
Universal primer for detecting CT value of oral cavity mycoplasma and pneumonia mycoplasma DNA
1. Strain information, mycoplasma stomatitis and mycoplasma pneumoniae are standard strains recommended by the Chinese pharmacopoeia for routine detection of mycoplasma, and are representative.
Extracting genome DNA of two mycoplasma inactivated bacterial liquids according to the description of a root genome DNA extraction kit.
2. Dilution of DNA from two Mycoplasma species
Diluting the DNA extracted from the two mycoplasma inactivated bacteria solutions by using sterile water, wherein the concentration of the diluted oral mycoplasma DNA corresponding to the original bacteria solution is 5CFU/ml, and the concentration of the mycoplasma pneumoniae corresponding to the original bacteria solution is 2.5CFU/ml. 2. Mu.l of diluted DNA was used for qPCR detection.
3. Fluorescent quantitative PCR reaction system and program
10 Μl of the total reaction system: 2X TSINGKE MasterqPCRMix-SYBR (+UDG) 5. Mu.l, upstream primer (10. Mu.M) 0.5. Mu.l, downstream primer (10. Mu.M) 0.5. Mu.l, sample DNA 4. Mu.l. The final concentration of the upstream and downstream primers was 0.5. Mu.M each. The same template was subjected to a multiplex reaction, all reactions were performed on a LightCycler480 fluorescent quantitative PCR instrument. Reaction procedure for real-time fluorescent quantitative PCR: pre-denaturation at 50 ℃ for 2min; pre-denaturation at 95℃for 2min; denaturation at 95℃for 15s, annealing at 55℃for 30s, extension at 72℃for 30s,32 cycles, and fluorescence signal acquisition at 72 ℃.
4. The experimental results are shown in Table 10.
Table 10 CT values obtained by detecting two Mycoplasma species with the primers used in general
| Primer(s) | CT value of oral mycoplasma of 5CFU/ml | 2.5CFU/ml Mycoplasma pneumoniae CT value |
| F1/R1 | 24.87 | 28.35 |
| F2/R1 | 25.28 | 28.36 |
| F3/R1 | 25.29 | 27.30 |
| F1/R2 | 25.48 | 27.18 |
Conclusion: the universal primers can detect DNA corresponding to mycoplasma stomatitis and mycoplasma pneumoniae, and can be regarded as the detection sensitivity of the method for mycoplasma stomatitis is 5CFU/ml, the detection sensitivity for mycoplasma pneumoniae is 2.5CFU/ml, and the sensitivity is higher than the sensitivity of the traditional culture method described in Chinese pharmacopoeia by 10CFU/ml. As can be seen, the sensitivity of the method is more superior than that of the traditional method, the time is short, the result can be obtained after 3 hours, and the traditional method is used for culturing for 28 days.
Example 5
Specificity verification of Universal primers
QPCR was performed on human amniotic mesenchymal stem cell DNA and human polyomavirus JCV DNA using the method of example 2, and the results were negative (see Table 11), which indicated that the two pairs of universal primers were specific and were able to detect whether there was bacterial fungus and mycoplasma (9 microorganisms verified, see examples 3 and 4) contamination in the samples.
Table 11 general primers for detecting DNA of human amniotic mesenchymal stem cells and human polyomavirus JCV
| Primer number | Human amniotic mesenchymal stem cell DNA | DNA of human polyomavirus JCV |
| F1/R1 | Negative of | Negative of |
| F2/R1 | Negative of | Negative of |
| F3/R1 | Negative of | Negative of |
| F1/R2 | Negative of | Negative of |
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. A DNA specific fragment for detecting bacteria, fungi and mycoplasma is characterized by comprising a DNA fragment with a nucleotide sequence shown as SEQ ID NO. 1 or a DNA fragment with a length of more than 50% which is intercepted on the basis of SEQ ID NO. 1.
2. The DNA specific fragment for detecting bacteria, fungi and mycoplasma according to claim 1, wherein the DNA specific fragment comprises a DNA fragment having a nucleotide sequence shown in SEQ ID NO. 2 to SEQ ID NO. 5.
3. A universal primer for detecting bacteria, fungi and mycoplasma comprising at least one of the following primer pairs for amplifying the DNA-specific fragment of claim 1 or 2: the nucleotide sequence is shown in SEQ ID NO:6 and the nucleotide sequence of the forward primer F1 is shown as SEQ ID NO:7, the nucleotide sequence of the reverse primer R1 is shown as SEQ ID NO:8 and the nucleotide sequence of the forward primer F2 is shown as SEQ ID NO:7, the nucleotide sequence of the reverse primer R1 is shown as SEQ ID NO:9 and the nucleotide sequence of the forward primer F3 is shown as SEQ ID NO:7 and the nucleotide sequence of the reverse primer R1 is shown as SEQ ID NO:6 and the nucleotide sequence of the forward primer F1 is shown as SEQ ID NO:10, and a reverse primer R2 shown in FIG. 10.
4. Use of the universal primer according to claim 3 for the preparation of a kit for simultaneous detection of bacteria, fungi and mycoplasma.
5. A kit for detecting microbial contamination of mesenchymal stem cells based on a fluorescent quantitative PCR technology is characterized by comprising the universal primer as set forth in claim 3 and a premix for fluorescent quantitative PCR amplification.
6. Use of the universal primer according to claim 3 for preparing a kit for detecting microbial contamination of mesenchymal stem cells.
7. A method for detecting bacterial, fungal and mycoplasma contamination in a mesenchymal stem cell culture system, comprising the steps of:
Extracting DNA from a mesenchymal stem cell culture system to be detected;
Preparing a qPCR reaction system by using the universal primer of claim 3 and taking the extracted DNA as a template for qPCR amplification;
Judging whether bacterial, fungal and mycoplasma pollution exists in a mesenchymal stem cell culture system to be detected according to the shape of an amplification curve and a CT value of a detection channel:
positive: the CT value of the detection channel is less than 29, and the amplification curve has obvious exponential growth trend;
negative: an amplification curve with a sample detection result CT more than or equal to 30 or no CT value and no obvious exponential growth trend;
suspicious: the CT value is more than or equal to 29 and less than 30, and the sample with the typical amplification curve suggests repeated tests, and the repeated test results show that the CT value is more than or equal to 29 and the typical amplification curve is positive, otherwise, the sample with the typical amplification curve is negative.
8. The method of claim 7, wherein the qPCR reaction system comprises, in 10. Mu.lMASTER QPCR Mix-SYBR (+UDG) 5. Mu.L, 10. Mu.M forward primer 0.5. Mu.L, 10. Mu.M reverse primer 0.5. Mu.L and DNA 4. Mu.L.
9. The method of claim 7, wherein the qPCR amplification reaction system is at 50 ℃ for 2min;95 ℃ for 2min; fluorescence signals were collected at 95℃for 15S,55℃for 30S,72℃for 30S, and 32 cycles at 72 ℃.
10. The method according to any one of claims 7 to 9, wherein the bacteria comprise at least one of: staphylococcus aureus, pseudomonas aeruginosa, escherichia coli, bacillus subtilis and clostridium sporogenes;
the fungi include candida albicans and/or aspergillus niger;
The mycoplasma includes mycoplasma stomatitis and/or mycoplasma pneumoniae.
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