Composition and kit for detecting mycoplasma

Abstract

The present invention belongs to the technical field of biology. Provided are a composition and kit for detecting mycoplasma. The composition for detecting mycoplasma is characterized in that the composition is an aqueous solution containing primer M-F, primer M-R and probe M-P; the sequence of M-F is as shown in SEQ ID NO: 1; the sequence of M-R is as shown in SEQ ID NO: 2; and the nucleotide sequence of probe M-P is as shown in SEQ ID NO: 3, the 5' end thereof is linked to fluorescent group FAM, and the 3' end thereof is linked to quenching group BHQ1. The composition is used for detecting mycoplasma, has high sensitivity, high specificity, and a wide range of detection.

Description

Compositions and kits for detecting mycoplasma Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a composition and a kit for detecting mycoplasma. Background Art

[0002] A major challenge in cell culture is mycoplasma contamination. In 1956, researchers at Johns Hopkins reported mycoplasma contamination of HeLa cells used in their laboratory. This was the first time mycoplasma was detected in cell culture. Mycoplasma contamination of cells can cause their metabolism to weaken and their proliferation to slow. However, since mycoplasma infection is non-lethal to cells, it can often coexist with cells for a long time and generally does not cause obvious morphological changes in the cells. In the early stages of contamination, the culture medium will not become turbid, making it difficult to determine with the naked eye whether the cell culture is contaminated with mycoplasma. However, contaminated cells can undergo a series of biological changes, such as changes in cell membrane composition, chromosomal abnormalities, changes in enzyme systems, and changes in viral load, which can greatly mislead scientific research and seriously interfere with experimental results.

[0003] Mycoplasma as a cell culture contaminant primarily originates from animal serum, trypsin, and aerosols. Besides cholesteroplasma (one of the most common contaminants), soil and other inanimate sources must also be considered. Since commercially available trypsin is obtained from commercially available porcine pancreas, Mycoplasma hyorhinis can also enter cell cultures through this reagent. As early as 1960, Pollock et al. found that 57% of 166 mammalian cell lines and sublines were contaminated with mycoplasmas. Furthermore, their study found that mycoplasma-contaminated cell cultures exhibited slower growth and a shortened logarithmic growth phase in mammalian cell cultures grown in vitro.

[0004] The Veterinary Pharmacopoeia of the People's Republic of China stipulates two methods for detecting mycoplasmas: culture and DNA fluorescence staining. However, the use of conventional culture methods for mycoplasma detection has the disadvantages of heavy workload and long cycle time. For some mycoplasmas with more stringent nutritional requirements, there may be cases of missed detection. In addition, due to the long time span during the culture process, contamination may also occur, leading to false positives. The DNA fluorescence staining method is highly sensitive, but the results are difficult to judge and are easily affected by the subjective judgment of the tester. The time required is about one week, which is slightly shorter than the culture method, but there are still many limitations for its application in scientific research. The existing technology lacks a mycoplasma detection method with high sensitivity, strong specificity, and a wide detection range.

[0005] Summary of the Invention

[0006] The purpose of the present invention is to provide a composition for detecting mycoplasma with high sensitivity, strong specificity and a wide detection range.

[0007] The purpose of the present invention is achieved by adopting the following technical solutions:

[0008] A composition for detecting mycoplasma, characterized by comprising an aqueous solution containing a primer MF, a primer MR, and a probe MP; the sequence of MF is shown in SEQ ID NO: 1; the sequence of MR is shown in SEQ ID NO: 2; the nucleotide sequence of the probe MP is shown in SEQ ID NO: 3, the 5' end is connected to a fluorescent group FAM, and the 3' end is connected to a quenching group BHQ1.

[0009] In the present invention, the molar concentration ratio of the primer MF, the primer MR and the probe MP is 1:0.8-1.2:0.8-1.2.

[0010] The present invention also provides a kit for detecting mycoplasma, comprising the composition.

[0011] In the present invention, the molar concentration ratio of the primer MF, the primer MR and the probe MP in the kit is 1:0.8-1.2:0.8-1.2.

[0012] In the present invention, the kit further comprises a positive plasmid, which is obtained by inserting a fragment with a sequence as shown in SEQ ID NO: 4 into a pUC57 plasmid vector.

[0013] The present invention also provides a method for detecting mycoplasma using the composition for non-diagnostic purposes, which is characterized by comprising the following steps:

[0014] (1) Extracting sample DNA;

[0015] (2) Using sample DNA as a template, qPCR detection was performed using primers MF, primer MR, and probe MP;

[0016] (3) When the Ct value of the qPCR test of the sample DNA is less than 38 and there is a typical S-shaped amplification curve, it is judged as positive, indicating that mycoplasma is present in the sample; when the Ct value of the qPCR test of the sample DNA is ≥38 or there is no Ct value or no typical S-shaped amplification curve, it is judged as negative, indicating that there is no mycoplasma in the sample.

[0017] In the present invention, the reaction system in the qPCR detection includes: 12.5 μL of fluorescent PCR reaction solution, 1 μL of sample DNA, 3 μL of the composition according to claim 1, and 8.5 μL of ddH2O.

[0018] In the present invention, the qPCR reaction procedure is: 95°C for 3 min; 95°C for 15 sec, 60°C for 30 sec, for a total of 40 amplification cycles.

[0019] The composition of the present invention demonstrates high sensitivity, strong specificity, and a wide detection range for mycoplasma detection. 106 random cell samples from different laboratories in different regions were collected for testing. The positive samples detected using the composition of the present invention had a 100% concordance rate with the culture method, significantly shortening detection time. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 shows the detection results of the qPCR method in Example 1, where (1) is the qPCR detection result of 15 types of mycoplasma; (2) is the qPCR detection result of cells, bacteria, and viruses.

[0021] Figure 2 Electrophoresis of the detection results of mycoplasma using a commercial nested mycoplasma detection PCR kit, where M: DL2000 DNA Marker; 1. MG; 2: Mhs; 3. Mp; 4. M. orale; 5. M. hyorhinis; 6. A. laidlawii; 7. M. fermentans; 8. MS; 9. S. citri; 10. Mf; 11. MO; 12. Mh; 13. negative control; 14. positive control; 15. Mb; 16. M. arginini; 17. M. pirum.

[0022] Figure 3. Results of mycoplasma detection using commercial qPCR kits, where (1) is the amplification curve of 15 mycoplasma samples, (2) is the amplification curve of Mycoplasma pirum, and (3) is the amplification curve of A. laidlawii.

[0023] FIG4 shows the amplification curves of 106 cell samples detected by the qPCR method in Example 1.

[0024] Figure 5 shows the amplification curves of 106 cell samples detected using a commercial qPCR kit. DETAILED DESCRIPTION

[0025] Example 1 Composition, kit, and detection method for detecting mycoplasma

[0026] 1. Composition for detecting mycoplasma

[0027] To find a highly sensitive and universal qPCR method for detecting mycoplasmas, the applicant conducted a whole-genome comparison analysis of 143 mycoplasma sequences published in the NCBI database and designed dozens of pairs of primers and probes. It was found that only one pair of primers (MF and MR) and probe MP could detect all 15 tested mycoplasmas with high sensitivity.

[0028] Among them, the sequence of MF (SEQ ID NO: 1) is as follows: 5'-ATCCATCCCCACGTTCTCGT-3'; the sequence of MR (SEQ ID NO: 2) is as follows: 5'-TGCGGTGAATACGTTCTCGGG-3'; the nucleotide sequence of probe MP (SEQ ID NO: 3) is as follows: 5'-ACGGGCGGTGTGTACA-3', with the fluorescent group FAM (carboxyfluorescein) connected to the 5' end and the quencher group BHQ1 (succinimidyl ester) connected to the 3' end.

[0029] The composition for detecting mycoplasma is an aqueous solution containing 10 μM MF, 10 μM MR and 10 μM probe MP.

[0030] 2. qPCR method for detecting mycoplasma

[0031] The qPCR method for detecting mycoplasma includes the following steps:

[0032] (1) Extracting sample DNA;

[0033] (2) qPCR detection;

[0034] A qPCR reaction was performed using the sample DNA as a template. The total qPCR reaction system was 25 μL: 12.5 μL of fluorescent PCR reaction solution (Novozymes, product number Q112-AA), 1 μL of sample DNA, 3 μL of the composition for mycoplasma detection, and 8.5 μL of ddH2O. The reaction system is shown in Table 1. The PCR reaction tube containing the qPCR total reaction system was placed into the detection well of an ABI fluorescent PCR instrument. The FAM channel (quencher: BHQ-1) was selected for detection. The reaction system was set to 25 μL. The cycling parameters were: 95°C for 3 min; 95°C for 15 sec; 60°C for 30 sec, for a total of 40 amplification cycles. The FAM fluorescence signal was collected at the end of each cycle.

[0035] In addition, negative and positive controls were set up, wherein the negative control used ddH2O instead of sample DNA, and the positive control used positive plasmid DNA instead of sample DNA. The other qPCR detection methods were the same as those for the samples. The positive plasmid DNA used in the positive control was a positive plasmid obtained by ligating a gene fragment from Spiroplasma (sequence shown in SEQ ID NO: 4) into a pUC57 plasmid vector via two restriction sites, BamHI and XhoI. The positive plasmid was chemically transformed into the competent Escherichia coli XL10 strain for proliferation.

[0036] Table 1 qPCR reaction system

[0037] (3) Result determination.

[0038] When the Ct value of the qPCR test of the sample DNA is less than 38 and there is a typical S-shaped amplification curve, it is judged as positive, indicating that mycoplasma is present in the sample; when the Ct value of the qPCR test of the sample DNA is ≥38 or there is no Ct value or no typical S-shaped amplification curve, it is judged as negative, indicating that there is no mycoplasma in the sample.

[0039] Example 2 Specificity and sensitivity of qPCR

[0040] 1. Specificity

[0041] (1) The qPCR method in Example 1 was used to detect 15 types of mycoplasmas, multiple bacteria, viral nucleic acids and different cells. The 15 types of mycoplasmas are: A.laidlawii, M.fermentans, M.hyorhinis, M.orale, M.arginini, M.pneumoniae (Mp), M.gallisepticum (MG), M.synoviae (MS), S.citri, M.hyoviae (Mhs), M.hominis (Mh), M.pirum, M.flocculus (Mf), M.bovis (Mb) and Mianyang M.pneumoniae (MO). A variety of bacteria, viral nucleic acids and different cells include: Salmonella (S.pullorum), Escherichia coli (E.coli), Staphylococcus aureus (S.aureus), Pseudomonas fragi (P.fragi), Yeast, PCV-2, Pseudorabies virus (PRV), Porcine reproductive and respiratory syndrome virus (PRRSV), African green monkey kidney cells (Vero), porcine kidney cells (PK-15), canine kidney cells (MDCK), human laryngeal epidermoid carcinoma cells (Hep-2), mouse monocytic macrophage leukemia cells (RAW264.7), etc.

[0042] The qPCR method described in Example 1 detected no peaks for the common cells, viruses, and bacteria mentioned above. The qPCR method described in Example 1 also detected DNA from the 15 mycoplasma species, yielding Ct values ​​< 38 (Table 2) and typical S-shaped amplification curves (Figure 1). These results demonstrate that the qPCR method described in Example 1 has good broad spectrum and specificity for detecting mycoplasmas.

[0043] Table 2 CT values ​​of 15 mycoplasma species detected by qPCR Note: In Table 2, Undet means no CT value was detected, Pos means positive control, and Neg means negative control, the same below.

[0044] (2) Commercial nested PCR method

[0045] The commercial nested mycoplasma detection PCR kit GMyc-PCR Mycoplasma Test Kit (Yisheng Biological) was used to detect the 15 mycoplasma samples (1) in the title 1 of this example.

[0046] Procedure: The first-round PCR reaction system is shown in Table 3, and the reaction procedure is shown in Table 4. After the first-round PCR amplification reaction, the amplified product was diluted 1000-fold and used as a template for the second-round PCR reaction. The second-round PCR reaction system is shown in Table 5, and the reaction procedure is the same as the first-round.

[0047] Table 3 First round PCR reaction system

[0048] Table 4 First round PCR reaction conditions

[0049] Table 5 Second round PCR reaction system

[0050] The commercial nested mycoplasma detection PCR kit was used to detect 15 mycoplasma strains. As shown in Figure 2, only 12 mycoplasma strains were detected. This method requires two rounds of PCR reactions and agarose gel electrophoresis, which is cumbersome to operate. The detection rate is 20% lower than that of the qPCR method of the present invention.

[0051] (3) Commercial qPCR method

[0052] The commercial qPCR kit MycAway TM Mycoplasma Real-time qPCR Detection Kit (Yisheng Bio) was used to detect the 15 mycoplasma samples listed in (1) in Title 1 of this Example.

[0053] Commercial qPCR reaction components include 4× qPCR Reaction Buffer, Primer & Probe MIX, positive / negative controls, and sterile nuclease-free water. See Table 6 for the qPCR reaction system.

[0054] Select FAM as the reporter fluorophore and MGB as the quencher fluorophore. Set the reaction volume to 40 μL and the cycling parameters to: 95°C for 5 minutes, 95°C for 15 seconds, 62°C for 30 seconds, for a total of 45 amplification cycles. Collect the FAM fluorescence signal at the end of each cycle. A Ct < 40 and a clear amplification curve are considered positive; a Ct ≥ 40 or no clear peak is considered negative.

[0055] Table 6 qPCR reaction system

[0056] Results of commercial qPCR kit testing: CT values ​​are shown in Table 7. Figure 3 shows that the S-shaped amplification curves for Cholesteroloma and Mycoplasma pyri are atypical, indicating negative results. The qPCR method in Example 1 of the present invention offers significant advantages over commercial qPCR kits. Compared to the commercial method's 20 μL template (10 ng / μL), the present method requires only 1 μL of sample DNA for detection. Compared to the commercial method's 45 cycles, the present method requires only 40 cycles. Furthermore, the present method amplifies typical S-shaped amplification curves for all 15 mycoplasmas, with stronger fluorescence intensity and smoother curves than the commercial qPCR kit, making it less prone to misidentification.

[0057] Table 7 CT values ​​of commercial qPCR kits for detecting 15 mycoplasmas

[0058] 2. Sensitivity

[0059] The E. coli carrying the positive plasmid in Example 1 was multiplied, the positive plasmid was extracted, and the concentration of the positive plasmid was measured using a spectrophotometer. The plasmid was diluted 10 times in sequence so that the plasmid concentration was 10 9 , 10 8 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 1 , 10 0 , 10 -1 1 μL of each positive plasmid was used as a template and tested using the qPCR method described in Example 1 to investigate the sensitivity of the method. Ten replicates were performed for each concentration.

[0060] The qPCR test results in Example 1 (Table 8) showed that the positive plasmid concentration was 10 -1 When the concentration of positive plasmid was 10 copies / μL, 3 out of 10 reactions could not stably detect the Ct value. 0 When the concentration of positive plasmid was 10 copies / μL, the Ct value could be stably detected and the Ct value was less than 38. -1 When the number of copies / μL was less than 10, the Ct value could not be stably expressed, so the sensitivity of the qPCR method was judged to be 10 0 copies / μL, and the Ct value threshold was 38.

[0061] Table 8 qPCR detection Ct values ​​of positive plasmids at various concentrations

[0062] When using other primers and probes for mycoplasma detection, such as primer MP03-F: 5'-GGTCGTCTACGTCAAAACTTGC-3', primer MP03-R: 5'-GCCATTTGGTCCCCGTCAAAG-3', and probe MP03-P: FAM-TACCTTGTTACGACTT-BHQ1, the broad spectrum is poor, and two of the 15 mycoplasmas tested cannot show a typical S-shaped curve, with a sensitivity of 10 2 copies / μL.

[0063] Example 3 Detection of Mycoplasma Contamination in Cell Culture Using qPCR Method

[0064] 106 cell samples from multiple laboratories were subjected to mycoplasma amplification detection using the qPCR method in Example 1, the culture method in the 2020 edition of the "Pharmacopoeia of the People's Republic of China", and a commercial qPCR method to examine the compliance rate of the qPCR method in Example 1 with the culture method in the 2020 edition of the "Pharmacopoeia of the People's Republic of China".

[0065] 1. qPCR method in Example 1

[0066] The supernatant from each cell sample was boiled to prepare the template. The specific steps are as follows: remove the supernatant from the cell culture to be tested, add it to a centrifuge tube, heat it to 100°C and boil it for 10 minutes, cool it, and centrifuge it for 5-6 seconds. The supernatant was then collected again (or DNA was extracted using a kit) as the sample DNA for the qPCR test.

[0067] 106 cell samples were tested using the qPCR method in Example 1. The results showed that 49 cell samples had CT values ​​less than 38 (Table 9) and had typical amplification curves, indicating that they were mycoplasma positive, as shown in Figure 4. The positive detection rate was 46.23%.

[0068] Table 9 CT values ​​of 106 cell samples detected by qPCR method in Example 1

[0069] 2. Detection of Mycoplasma in Cell Samples by Isolation and Culture

[0070] Each cell sample was cultured in liquid and solid form according to the culture method in the 2020 edition of the Veterinary Pharmacopoeia of the People's Republic of China. At the end of the culture period, if no mycoplasma grew in the culture medium inoculated with the cell sample, the cell sample was qualified; otherwise, it failed.

[0071] If mycoplasma grows, the color of the liquid culture medium will also change (pink or yellow); in solid culture medium, if mycoplasma grows after aerobic culture at 37°C for 30 days, pinpoint-shaped colonies will appear in the culture medium, and fried egg-like colonies can be observed under a microscope; in the experiment, known negative and positive samples are used as negative controls and positive controls, respectively.

[0072] After approximately 21 days of culture, the results showed that the liquid culture medium of 24 cell samples turned yellow (pH decreased), and the culture medium of 7 samples turned pink (pH increased). Therefore, these 31 cell samples were judged to be contaminated with mycoplasma. The culture medium with the color change was inoculated into solid culture medium. After approximately 30 days of culture, fried egg-like colonies formed on the solid culture medium. The positive detection rate was 29.25%.

[0073] 3. Detection of Mycoplasma in Cell Samples Using Commercial qPCR Kits

[0074] The commercial qPCR kit MycAway TM Mycoplasma Real-time qPCR Detection Kit (Yisheng Bio) was used to detect each cell sample, and the specific method was performed according to the instructions.

[0075] 106 cell samples were tested using a commercial qPCR kit. The results showed that 41 cell samples had a CT value less than 40 (Table 10) and a typical amplification curve, indicating that they were mycoplasma positive, as shown in Figure 5. The positive detection rate was 38.68%.

[0076] Table 10 CT values ​​of 106 cell samples tested using commercial qPCR kits

[0077] 4. Comparison of three methods

[0078] The qPCR method in Example 1 completed the detection within 1 hour, the commercial qPCR kit completed the detection in about 3 hours, and the isolation and culture method took 21 to 29 days to complete the detection of all cell samples.

[0079] Table 11 Comparison of detection performance of different methods

[0080] Mycoplasma testing was performed on 106 randomly collected cell samples. The results are shown in Table 11. The qPCR method in Example 1 detected 49 mycoplasma-positive samples, and the concordance rate with the classical culture method test results (number of cell samples tested positive by both methods / number of cell samples tested positive by the classical culture method * 100%) was 100%. The commercial qPCR kit detected 41 positive samples, which missed 4 samples compared with the classical culture method test results. The concordance rate between the commercial qPCR kit and the culture method was only 87.10%.

[0081] Therefore, the qPCR method in Example 1 is significantly superior to existing technologies in terms of broad spectrum, sensitivity and accuracy when detecting mycoplasma.

Claims

1. A composition for detecting mycoplasma, characterized in that An aqueous solution containing primers MF, primers MR and probe MP; the sequence of MF is shown in SEQ ID NO: 1; the sequence of MR is shown in SEQ ID NO: 2; the nucleotide sequence of probe MP is shown in SEQ ID NO: 3, the 5' end is connected to a fluorescent group FAM, and the 3' end is connected to a quenching group BHQ1.

2. The composition according to claim 2, characterized in that The molar concentration ratio of the primer MF, the primer MR and the probe MP is 1:0.8-1.2:0.8-1.

2.

3. A kit for detecting mycoplasma, characterized in that The invention comprises the composition according to claim 1.

4. The kit according to claim 3, characterized in that The molar concentration ratio of the primer MF, the primer MR and the probe MP in the kit is 1:0.8-1.2:0.8-1.

2.

5. The kit according to claim 4, characterized in that The kit also includes a positive plasmid, which is obtained by inserting a fragment with a sequence as shown in SEQ ID NO: 4 into a pUC57 plasmid vector.

6. A method for detecting mycoplasma using the composition of claim 1 for non-diagnostic purposes, characterized in that The steps include: (1) Extracting sample DNA; (2) Using sample DNA as template, qPCR detection was performed using primer MF, primer MR, and probe MP; (3) When the Ct value of the sample DNA detected by qPCR is less than 38 and there is a typical S-shaped amplification curve, it is judged as positive, indicating that mycoplasma is present in the sample; when the Ct value of the sample DNA detected by qPCR is ≥38 or there is no Ct value or no typical S-shaped amplification curve, it is judged as negative, indicating that there is no mycoplasma in the sample.

7. The method according to claim 6, characterized in that The reaction system in the qPCR detection includes: 12.5 μL of fluorescent PCR reaction solution, 1 μL of sample DNA, 3 μL of the composition according to claim 1, and 8.5 μL of ddH2O.

8. The method according to claim 7, characterized in that The qPCR reaction program was: 95°C for 3 min; 95°C for 15 sec, 60°C for 30 sec, for a total of 40 amplification cycles.

9. Use of the method of claim 1 in detecting mycoplasma contamination in biological products.

10. The use according to claim 9, characterized in that The biological product is a cell, a serum or a vaccine.

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