Compositions and kits for detecting mycoplasma

Abstract

The present application provides a composition and a kit for detecting mycoplasma, and belongs to the technical field of biotechnology. The composition for detecting mycoplasma is characterized by containing an aqueous solution of a primer M-F, a primer M-R and a probe M-P; the sequence of the primer M-F is shown in SEQ ID NO:1; the sequence of the primer M-R is shown in SEQ ID NO:2; and the nucleotide sequence of the probe M-P is shown in SEQ ID NO:3, wherein a fluorescent group FAM is connected to the 5' end, and a quenching group BHQ1 is connected to the 3' end. The composition of the present application has high sensitivity, strong specificity and wide detection range in the detection of mycoplasma.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a composition and kit for detecting mycoplasma. BACKGROUND

[0002] A big problem in cell culture is mycoplasma contamination. In 1956, researchers at Johns Hopkins reported mycoplasma contamination of HeLa cells used in their laboratory, which was the first time that mycoplasma was detected in cell culture. After mycoplasma contaminates cells, it can cause the cells to weaken in metabolism and slow down in proliferation, but because mycoplasma infection of cells is not lethal, it can often coexist with the cells for a long time and generally does not cause the cells to produce obvious morphological changes. When the contamination is early, the culture medium also does not become turbid, which makes it difficult to determine by the naked eye whether the cell culture is contaminated with mycoplasma, but the contaminated cells can undergo a series of biological changes, such as changes in cell membrane composition, chromosomal abnormalities, enzyme changes, and viral load changes, which can greatly mislead research work and seriously interfere with experimental results.

[0003] The main sources of mycoplasma as a cell culture contaminant are animal serum, trypsin, and aerosols. For mycoplasma gallisepticum (one of the most common contaminants), soil and other non-living sources must also be considered. Because the trypsin commonly available on the market is obtained from commercially available pig pancreas, porcine nasal mycoplasma 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 mycoplasma, and the study found that for the in vitro growth of mammalian cells, mycoplasma-contaminated cell cultures grew more slowly and had a shortened log phase.

[0004] The "Veterinary Pharmacopoeia of the People's Republic of China" specifies two methods for detecting mycoplasma: culture method and DNA fluorescent staining method, but the conventional culture method for detecting mycoplasma has the disadvantages of heavy workload and long cycle time, and for individual mycoplasma with more stringent nutritional requirements, there may be missed detection, and during the culture process, the long time span may also cause contamination leading to false positives. The DNA fluorescent staining method is highly sensitive, but the results are not easy to determine and are easily affected by the subjective judgment of the tester, and the time required is about 1 week, which is slightly shorter than the culture method, but there are still many limiting factors for application in research work. There is a lack of mycoplasma detection methods with high sensitivity, strong specificity, and wide detection range in the prior art. SUMMARY

[0005] The purpose of the present application is to provide a composition for detecting mycoplasma with high sensitivity, strong specificity, and wide detection range.

[0006] The purpose of the present application is achieved by the following technical solutions:

[0007] The composition for detecting mycoplasma is characterized by containing an aqueous solution of primer M-F, primer M-R and probe M-P; the sequence of the primer M-F is shown in SEQ ID NO: 1; the sequence of the primer M-R is shown in SEQ ID NO: 2; the nucleotide sequence of the probe M-P is shown in SEQ ID NO: 3, and a fluorescent group FAM is connected to the 5' end and a quenching group BHQ1 is connected to the 3' end.

[0008] In the present application, the molar concentration ratio of the primer M-F, the primer M-R and the probe M-P is 1:0.8-1.2:0.8-1.2.

[0009] The present application also provides a kit for detecting mycoplasma, comprising the composition.

[0010] In the present application, the molar concentration ratio of the primer M-F, the primer M-R and the probe M-P in the kit is 1:0.8-1.2:0.8-1.2.

[0011] In the present application, the kit further comprises a positive plasmid, which is obtained by inserting a fragment with the sequence shown in SEQ ID NO: 4 into a pUC57 plasmid vector.

[0012] The present application also provides a method for detecting mycoplasma using the composition for non-diagnostic purposes, characterized by comprising the following steps:

[0013] (1) extracting sample DNA;

[0014] (2) using the primer M-F, the primer M-R and the probe M-P to perform qPCR detection with the sample DNA as a template;

[0015] (3) when the Ct value of the qPCR detection of the sample DNA is <38 and there is a typical S-shaped amplification curve, it is judged as positive, indicating that there is mycoplasma in the sample; when the Ct value of the qPCR detection 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.

[0016] In the present application, the reaction system in the qPCR detection comprises: 12.5 μL of fluorescent PCR reaction solution, 1 μL of sample DNA, 3 μL of the composition of claim 1, and 8.5 μL of ddH2O.

[0017] In the present application, the qPCR reaction program is: 95℃ for 3 min; 95℃ for 15 sec, 60℃ for 30 sec, for a total of 40 amplification cycles.

[0018] The composition of the application has high sensitivity, strong specificity and wide detection range. 106 random cell samples from different regions and different laboratories are collected for detection. The coincidence rate of positive samples detected by the composition of the application and the culture method is 100%, and the detection time is significantly shortened. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 Detection results of the qPCR method in example 1, wherein (1) is the qPCR detection results of 15 mycoplasma; (2) is the qPCR detection results of cells, bacteria and viruses.

[0020] Figure 2 Electrophoresis map of detection results of mycoplasma by commercial nested mycoplasma detection PCR kit, wherein 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.

[0021] Figure 3 Detection results of mycoplasma by commercial qPCR kit, wherein (1) is the amplification curve of 15 mycoplasma samples, (2) is the amplification curve of M. pirum, and (3) is the amplification curve of A. laidlawii.

[0022] Figure 4 Amplification curve of 106 cell samples detected by the qPCR method in example 1.

[0023] Figure 5 Amplification curve of 106 cell samples detected by the commercial qPCR kit. DETAILED DESCRIPTION

[0024] Composition and kit for detecting mycoplasma and detection method

[0025] 1. A composition for detecting mycoplasma

[0026] In order to find a qPCR method for detecting mycoplasma with high sensitivity and universality, the applicant analyzes the whole genome alignment of 143 mycoplasma sequences published in the NCBI database, designs dozens of primers and probes, and finds that only one pair of primers (M-F and M-R) and probe M-P can detect 15 mycoplasma with high sensitivity.

[0027] The sequence of M-F (SEQ ID NO: 1) is as follows: 5'- ATCCATCCCCACGTTCTCGT-3'; the sequence of M-R (SEQ ID NO: 2) is as follows: 5'- TGCGGTGAATACGTTCTCGGG-3'; the nucleotide sequence of the probe M-P (SEQ ID NO: 3) is as follows: 5'- ACGGGCGGTGTGTACA-3', and the 5' end is connected with a fluorescent group FAM (carboxyfluorescein), and the 3' end is connected with a quenching group BHQ1 (succinimidyl ester).

[0028] The composition for detecting mycoplasma is an aqueous solution containing 10 μM of M-F, 10 μM of M-R and 10 μM of the probe M-P.

[0029] 2. The qPCR method for detecting mycoplasma

[0030] The qPCR method for detecting mycoplasma comprises the following steps:

[0031] (1) Extracting sample DNA;

[0032] (2) qPCR detection;

[0033] The qPCR reaction is carried out with the sample DNA as a template. The total reaction system of the qPCR is 25 μL: fluorescent PCR reaction solution (Novozyme, product number Q112-AA) 12.5 μL, sample DNA 1 μL, composition for detecting mycoplasma 3 μL, ddH2O 8.5 μL, and the reaction system is specifically as shown in Table 1. The PCR reaction tube added with the total reaction system of the qPCR is placed into the detection hole of the ABI fluorescent PCR instrument, the FAM channel detection (quenching group: BHQ-1) is selected, the reaction system is set to 25 μL, the cycle parameters are set to 95°C for 3 min; 95°C for 15 sec, 60°C for 30 sec, a total of 40 amplification cycles, and the FAM fluorescent signal is collected at the end of each cycle.

[0034] In addition, negative controls and positive controls are provided, wherein the negative controls replace the sample DNA with ddH2O, the positive controls replace the sample DNA with positive plasmid DNA, and the other qPCR detection methods are the same as those of the sample. The positive plasmid DNA used in the positive controls is obtained by connecting the gene fragment Spiroplasma (the sequence is shown in SEQ ID NO: 4) from the spiroplasma to the pUC57 plasmid vector through two enzyme cutting sites of BamHI and XhoI, and the positive plasmid is transformed into the E. coli competent XL10 strain through chemical transformation to proliferate.

[0035] Table 1 qPCR reaction system

[0036]

[0037]

[0038] (3) Result determination.

[0039] When the Ct value of qPCR detection of sample DNA is < 38 and there is a typical S-shaped amplification curve, it is determined to be positive, indicating that there is mycoplasma in the sample; when the Ct value of qPCR detection of sample DNA is ≥ 38 or there is no Ct value or no typical S-shaped amplification curve, it is determined to be negative, indicating that there is no mycoplasma in the sample.

[0040] Specificity and sensitivity of qPCR of Example 2

[0041] 1. Specificity

[0042] (1) The qPCR method in Example 1 was used to detect 15 kinds of mycoplasma, various bacteria, viral nucleic acids and different cells. The 15 kinds of mycoplasma are: A. laidlawii, M. fermentans, M. hyorhinis, M. oralis, M. arginini, M. pneumoniae, M. gallisepticum, M. synoviae, S. citri, M. hyosynoviae, M. hominis, M. pirum, M. flocculare, M. bovis, M. omiyatoense, a total of 15 kinds of mycoplasma. Various bacteria, viral nucleic acids and different cells include: S. pullorum, E. coli, S. aureus, P. fragi, yeast, PCV-2, PRV, PRRSV, Vero, PK-15, MDCK, Hep-2, RAW264.7, etc.

[0043] The qPCR method in Example 1 was used to detect the above common cells, viruses and bacteria, and no peak was generated. The qPCR method in Example 1 was used to detect the DNA of the above 15 kinds of mycoplasma, and the Ct value was < 38 (Table 2) and there was a typical S-shaped amplification curve. Figure 1 The above results prove that the qPCR method in Example 1 for detecting mycoplasma has good broad-spectrum and specificity.

[0044] Table 2 CT value of qPCR detection of 15 kinds of mycoplasma

[0045] No. Sample Name CT No. Sample Name CT 1 Neg Undet 10 MO 19.585 2 M. orale 21.753 11 Mhs 14.090 3 MS 11.681 12 M. arginini 17.749 4 Mf 27.571 13 S. citri 19.131 5 Mp 14.406 14 MG 22.151 6 Mb 27.371 15 M. pirum 18.373 7 M. fermentans 21.468 16 M. hyorhinis 21.559 8 Mh 19.990 17 Pos 21.922 9 A. laidlawii 17.394

[0046] Note: Undet in Table 2 means undetected CT value, Pos means positive control, Neg means negative control, and the same below.

[0047] (2) Commercial nested PCR method

[0048] The 15 Mycoplasma samples in (1) of the title of this example were detected by using the commercial nested Mycoplasma detection PCR kit GMyc-PCR Mycoplasma Test Kit (Yixing Biotech).

[0049] Operation steps: the first round of PCR reaction system is shown in Table 3, and the reaction program is shown in Table 4. After the first round of PCR amplification reaction, the amplification product was diluted 1000 times and used as a template for the second round of PCR reaction, and the second round of PCR reaction system is shown in Table 5, and the reaction program is the same as the first round.

[0050] Table 3 First round of PCR reaction system

[0051] Reagent Test Group Positive Control Negative Control GMyc-1st PCR Mix 25 μL 25 μL 25 μL Template DNA 4 μL 4 μL ddH2O 21 μL 20 μL 25 μL Positive Control Template 1 μL Total Volume 50 μL 50 μL 50 μL

[0052] Table 4 First round of PCR reaction conditions

[0053]

[0054] Table 5 Second round of PCR reaction system

[0055] Reagent Test Group Positive Control Negative Control GMyc-2nd PCR Mix 25 μL 25 μL 25 μL ddH2O 24 μL 24 μL 24 μL 1st Amplification Product diluted 1000 times 1 μL 1 μL 1 μL Total Volume 50 μL 50 μL 50 μL

[0056] Using the above-mentioned commercial nested Mycoplasma detection PCR kit to detect 15 kinds of Mycoplasma, the results are as follows Figure 2 Only 12 Mycoplasma strains can be detected, and this method needs two rounds of PCR reaction and agarose gel electrophoresis, and the operation is complicated, and the detection rate is 20% lower than that of the qPCR method of the present application.

[0057] (3) Commercial qPCR method

[0058] The 15 Mycoplasma samples in (1) of the title of this example were detected by using the commercial qPCR kit MycAway TM Mycoplasma Real-time qPCR Detection Kit (Yixing Biotech).

[0059] The components of the commercial qPCR reaction include 4x qPCR Reaction Buffer, Primer & Probe MIX, positive and negative controls, sterile nuclease-free water. The qPCR reaction system is shown in Table 6.

[0060] The reporter fluorescent group is FAM, the quencher fluorescent group is MGB, the reaction system is set to 40 μL, and the cycle parameters are set as follows: 95℃ for 5 min; 95℃ for 15 sec, 62℃ for 30 sec, a total of 45 amplification cycles, and the FAM fluorescence signal is collected at the end of each cycle. When Ct < 40 and there is a clear amplification curve, it is positive; when Ct ≥ 40 or there is no obvious peak, it is negative.

[0061] Table 6 qPCR reaction system

[0062] Component System (μL) 4x qPCR Reaction Buffer 10 Primer & Probe MIX 1 Template (10 ng / μL) 20 Sterile Nuclease-free Water Make up to 40 μL Total 40

[0063] The detection results of the commercial qPCR kit are shown in Table 7. The CT values are shown in Table 7, and the results are shown in Table 7. Figure 3 It can be seen that the S-type amplification curve of Mycoplasma sp. and Mycoplasma sp. is not typical, and is negative. The qPCR method in Example 1 of the present application has a significant advantage compared to the commercial qPCR kit. Compared with the commercial method of 20 μL template (10 ng / μL), the sample DNA only needs 1 μL template in the detection process of the present application. Compared with the commercial method of 45 cycles, the present application only needs 40 cycles. Moreover, the present application can amplify typical S-type amplification curves for all 15 kinds of mycoplasma, the fluorescence intensity is stronger than that of the commercial qPCR kit, the curve is smoother, and misjudgment is not easy.

[0064] Table 7 CT values of 15 kinds of mycoplasma detected by commercial qPCR kit

[0065] No. Sample CT No. Sample CT 1 Neg Undet 10 MO 11.621 2 M. orale 22.229 11 Mhs 13.263 3 MS 13.693 12 M. arginini 17.43 4 Mf 22.27 13 S. citri 11.27 5 Mp 29.855 14 MG 33.484 6 Mb 28.997 15 M. pirum 33.904 7 M. fermentans 19.764 16 M. hyorhinis 20.428 8 Mh 21.833 17 Pos 11.483 9 A. laidlawii 17.9

[0066] 2. Sensitivity

[0067] The E. coli carrying the positive plasmid in the proliferation example 1 was extracted, and the concentration of the positive plasmid was determined by a spectrophotometer. The plasmid was diluted by 10 times successively, so that the plasmid concentrations were 10 9 , 10 8 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 1 , 10 0 , 10 -1 copies / μL. 1 μL of the positive plasmid of each concentration was taken as a template, and the qPCR method in Example 1 was used for detection to investigate the sensitivity of the method. Ten parallel tests were performed for each concentration.

[0068] The qPCR detection results in Example 1 (Table 8) show that the positive plasmid concentration is 10 -1copies / μL, 3 out of 10 reactions could not stably detect Ct value. When the positive plasmid concentration was 10 0 copies / μL, Ct value could be stably detected, Ct value was less than 38, while when the positive plasmid concentration was 10 -1 copies / μL, Ct value could not stably appear, therefore the sensitivity of qPCR method was judged to be 10 0 copies / μL, Ct value threshold was 38.

[0069] Table 8 Ct value of qPCR method for detecting positive plasmid at each concentration

[0070]

[0071] When other primers and probes for detecting mycoplasma were used, such as primer MP03-F: 5'-GGTCGTCTACGTCAAAACTTGC-3', primer MP03-R: 5'-GCCATTTGGTCCCCGTCAAAG-3', and probe MP03-P: FAM-TACCTTGTTACGACTT-BHQ1, the broad-spectrum was poor, 2 out of 15 tested mycoplasma could not appear typical S-shaped curve, and the sensitivity was 10 2 copies / μL.

[0072] Example 3 Detection of mycoplasma contamination in cell culture by qPCR method

[0073] 106 cell samples from multiple laboratories were detected by qPCR method in Example 1, culture method in Chinese Pharmacopoeia 2020 edition, and commercial qPCR method for mycoplasma amplification detection, in order to investigate the coincidence rate of qPCR method in Example 1 and culture method in Chinese Pharmacopoeia 2020 edition.

[0074] 1. qPCR method in Example 1

[0075] The supernatant in each cell sample was prepared as a template by boiling method. The specific steps are as follows: the supernatant was taken from the cell culture to be detected and added to a centrifuge tube, heated to 100℃ and boiled for 10 min, cooled, centrifuged the supernatant for 5-6 s, and the supernatant was taken again (or DNA was extracted by using a kit) as sample DNA for qPCR detection method.

[0076] 106 cell samples were detected by qPCR method in Example 1, and the results showed that 49 cell samples had CT value less than 38 (Table 9), and had typical amplification curve, which were mycoplasma positive, as shown in Figure 4 The positive detection rate was 46.23%.

[0077] Table 9 CT values of 106 cell samples detected by qPCR method in Example 1

[0078]

[0079]

[0080] 2. Detection of mycoplasma in cell samples by isolation culture method

[0081] According to the culture method in the Veterinary Pharmacopoeia of the People's Republic of China (2020 edition), each cell sample was subjected to liquid and solid culture. At the end of the culture, if the culture medium inoculated with the cell sample had no mycoplasma growth, the cell sample was qualified, otherwise it was unqualified.

[0082] If there is mycoplasma growth, the color of the liquid culture medium will also change (pink or yellow); in the solid culture medium, 37°C aerobic culture for 30 days, if there is mycoplasma growth, pinhead-shaped colonies will appear in the culture medium, and fried egg-like colonies can be observed under a microscope; known negative and positive samples were used as negative and positive controls, respectively, in the experiment.

[0083] After about 21 days of culture, the results showed that the color of the liquid culture medium of 24 cell samples changed to yellow (pH value decreased), and the color of the culture medium of 7 samples changed to pink (pH value increased), indicating that these 31 cell samples were contaminated with mycoplasma, and the culture medium with color change was inoculated into solid culture medium, respectively, and after about 30 days of culture, fried egg-like colonies were formed on the solid culture medium. The positive detection rate was 29.25%.

[0084] 3. Detection of mycoplasma in cell samples by commercial qPCR kit

[0085] A commercial qPCR kit MycAway Mycoplasma Real-time qPCR Detection Kit (Yingsheng Biological) was used to detect each cell sample, and the specific method was operated according to the instruction manual. TM Mycoplasma Real-time qPCR Detection Kit (Yingsheng Biological) was used to detect each cell sample, and the specific method was operated according to the instruction manual.

[0086] Using a commercial qPCR kit to detect 106 cell samples, the results showed that the CT values of 41 cell samples were less than 40 (Table 10), and there were typical amplification curves, which were mycoplasma positive, as shown in Figure 5 The positive detection rate was 38.68%.

[0087] Table 10 CT values of 106 cell samples detected by commercial qPCR kit

[0088] No. CT No. CT No. CT No. CT 1 Undet 28 30.938 55 22.229 82 Undet 2 37.423 29 33.146 56 Undet 83 20.428 3 Undet 30 Undet 57 13.693 84 Undet 4 Undet 31 Undet 58 Undet 85 Undet 5 30.938 32 32.281 59 35.904 86 35.029 6 Undet 33 Undet 60 Undet 87 Undet 7 11.151 34 28.043 61 Undet 88 Undet 8 Undet 35 Undet 62 Undet 89 Undet 9 32.281 36 23.739 63 Undet 90 Undet 10 Undet 37 31.751 64 35.029 91 31.751 11 28.043 38 Undet 65 Undet 92 Undet 12 Undet 39 Undet 66 33.146 93 Undet 13 23.739 40 Undet 67 Undet 94 Undet 14 Undet 41 25.991 68 Undet 95 Undet 15 Undet 42 23.770 69 Undet 96 30.731 16 14.656 43 Undet 70 30.731 97 Undet 17 Undet 44 11.607 71 Undet 98 31.068 18 Undet 45 Undet 72 Undet 99 Undet 19 27.768 46 Undet 73 Undet 100 Undet 20 Undet 47 27.221 74 Undet 101 Undet 21 Undet 48 Undet 75 Undet 102 16.515 22 38.322 49 11.800 76 29.715 103 29.855 23 36.643 50 Undet 77 22.427 104 Undet 24 Undet 51 Undet 78 Undet 105 13.274 25 37.423 52 Undet 79 22.270 106 27.545 26 Undet 53 Undet 80 17.900 Pos 11.27 27 Undet 54 Undet 81 Undet Neg Undet

[0089] 4. Comparison of three methods

[0090] The qPCR method in Example 1 completed the detection within 1 h, the commercial qPCR kit completed the detection in about 3 h, and the separation culture method took 21-29 days to complete the detection of all cell samples.

[0091] Table 11 Comparison of detection performance of different methods

[0092] Method Time Consumed Detection Rate Agreement with Gold Standard (Isolation Culture Method) qPCR in Example 1 1h 46.23％ 100％ Isolation Culture Method 21～29d 29.25％ 100％ Commercial qPCR 3h 38.68％ 87.10％

[0093] Mycoplasma detection was performed on 106 collected random cell samples, and the results are shown in Table 11. The qPCR method in Example 1 detected 49 positive samples contaminated with mycoplasma, and the coincidence rate with the classic culture method (the number of cell samples detected as positive by both methods / the number of cell samples detected as positive by the classic culture method * 100%) was 100%. The commercial qPCR kit detected 41 positives, missed 4 compared with the classic culture method, and the coincidence rate of the commercial qPCR kit with the culture method was only 87.10%.

[0094] Therefore, the qPCR method in Example 1 is significantly superior to the prior art in terms of broad spectrum, sensitivity and accuracy when detecting mycoplasma.

Claims

1. A composition for detecting Mycoplasma, characterized in that it comprises An aqueous solution containing primer M-F, primer M-R and probe M-P; the sequence of M-F is shown as SEQ ID NO: 1; the sequence of M-R is shown as SEQ ID NO: 2; the nucleotide sequence of probe M-P is shown as SEQ ID NO: 3, with a fluorescent group FAM connected to the 5' end and a quenching group BHQ1 connected to the 3' end.

2. The composition of claim 1, wherein The molar concentration ratio of the primer M-F, primer M-R and probe M-P is 1:0.8-1.2:0.8-1.

2.

3. A kit for detecting Mycoplasma, characterized by The composition of claim 1.

4. The kit of claim 3, wherein The molar concentration ratio of the primer M-F, primer M-R and probe M-P in the kit is 1:0.8-1.2:0.8-1.

2.

5. The kit of claim 4, wherein The kit further comprises a positive plasmid, which is obtained by inserting a fragment with the sequence shown as SEQ ID NO: 4 into a pUC57 plasmid vector.

6. A method for detecting mycoplasma using the composition of claim 1 for a purpose other than diagnosis, characterized in that The kit further comprises a positive plasmid, which is obtained by inserting a fragment with the sequence shown as SEQ ID NO: 4 into a pUC57 plasmid vector. The kit further comprises a positive plasmid, which is obtained by inserting a fragment with the sequence shown as SEQ ID NO: 4 into a pUC57 plasmid vector. The kit further comprises a positive plasmid, which is obtained by inserting a fragment with the sequence shown as SEQ ID NO: 4 into a pUC57 plasmid vector. The kit further comprises a positive plasmid, which is obtained by inserting a fragment with the sequence shown as SEQ ID NO: 4 into a pUC57 plasmid vector.

7. The method of claim 6, wherein The kit further comprises a positive plasmid, which is obtained by inserting a fragment with the sequence shown as SEQ ID NO: 4 into a pUC57 plasmid vector.

8. The method of claim 7, wherein The kit further comprises a positive plasmid, which is obtained by inserting a fragment with the sequence shown as SEQ ID NO: 4 into a pUC57 plasmid vector. The kit further comprises a positive plasmid, which is obtained by inserting a fragment with the sequence shown as SEQ ID NO: 4 into a pUC57 plasmid vector.

10. Use according to claim 9, characterized in that The kit further comprises a positive plasmid, which is obtained by inserting a fragment with the sequence shown as SEQ ID NO: 4 into a pUC57 plasmid vector. The kit further comprises a positive plasmid, which is obtained by inserting a fragment with the sequence shown as SEQ ID NO: 4 into a pUC57 plasmid vector. The kit further comprises a positive plasmid, which is obtained by inserting a fragment with the sequence shown as SEQ ID NO: 4 into a pUC57 plasmid vector. The kit further comprises a positive plasmid, which is obtained by inserting a fragment with the sequence shown as SEQ ID NO: 4 into a pUC57 plasmid vector. The kit further comprises a positive plasmid, which is obtained by inserting a fragment with the sequence shown as SEQ ID NO: 4 into a pUC57 plasmid vector. The kit further comprises a positive plasmid, which is obtained by inserting a fragment with the sequence shown as SEQ ID NO: 4 into a pUC57 plasmid vector. The kit further comprises a positive plasmid, which is obtained by inserting a fragment with the sequence shown as SEQ ID NO: 4 into a pUC57 plasmid vector. The kit further comprises a positive plasmid, which is obtained by inserting a fragment with the sequence shown as SEQ ID NO: 4 into a pUC57 plasmid vector. The kit further comprises a positive plasmid, which is obtained by inserting a fragment with the sequence shown as SEQ ID NO: 4 into a pUC57 plasmid vector. The kit further comprises a positive plasmid, which is obtained by inserting a fragment with the sequence shown as SEQ ID NO: 4 into a pUC57 plasmid vector. The kit further comprises a positive plasmid, which is obtained by inserting a fragment with the sequence shown as SEQ ID NO: 4 into a pUC57 plasmid vector. The kit further comprises a positive plasmid, which is obtained by inserting a fragment with the sequence shown as SEQ ID NO: 4 into a pUC57 plasmid vector. The kit further comprises a positive plasmid, which is obtained by inserting a fragment with the sequence shown as SEQ ID NO: 4 into a pUC57 plasmid vector. The kit further comprises a positive plasmid, which is obtained by inserting a fragment with the sequence shown as SEQ ID NO: 4 into

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