阅读说明：本技术 一种hela细胞污染检测方法 (HELA cell pollution detection method ) 是由 胡策 涂继刚 于 2019-10-10 设计创作，主要内容包括：本发明提供了检测Hela细胞污染的引物组,所述引物组包括2对HPV引物序列和2对STK11引物序列。本发明还提供一种HELA细胞污染检测方法。本发明同时利用HELA细胞的两种特性,共同验证细胞系是否有HELA细胞污染。本发明只要用最简单和廉价的琼脂糖凝胶电泳,无需使用昂贵的荧光染料,无需使用荧光PCR、毛细管电泳仪、激光扫描等昂贵大型设备,耗时短、成本低。(The invention provides a primer group for detecting Hela cell pollution, which comprises 2 pairs of HPV primer sequences and 2 pairs of STK11 primer sequences. The invention also provides a HELA cell contamination detection method. The invention simultaneously utilizes two characteristics of the HELA cells to jointly verify whether the cell line is polluted by the HELA cells. The invention only uses the simplest and cheapest agarose gel electrophoresis, does not need to use expensive fluorescent dye, does not need to use expensive large-scale equipment such as fluorescent PCR, capillary electrophoresis apparatus, laser scanning and the like, and has short time consumption and low cost.)

The application of STK11 complete gene deletion in detecting Hela cell contamination.

2. A primer group for detecting Hela cell contamination is characterized by comprising 2 pairs of HPV primer sequences and 2 pairs of STK11 primer sequences, wherein the HPV primer sequences are as follows:

the sequence of the upstream primer is shown as SEQ ID NO. 1;

the sequence of the downstream primer is shown as SEQ ID NO. 2;

the sequence of the upstream primer is shown as SEQ ID NO. 3;

the sequence of the downstream primer is shown as SEQ ID NO. 4;

the sequence of the STK11 primer is as follows:

the sequence of the upstream primer is shown as SEQ ID NO. 5;

the sequence of the downstream primer is shown as SEQ ID NO. 6;

the sequence of the upstream primer is shown as SEQ ID NO. 7;

the sequence of the downstream primer is shown as SEQ ID NO. 8.

3. A HELA cell contamination detection method is characterized by comprising the following steps:

(1) extracting the genome DNA of a sample to be detected;

(2) taking the genomic DNA of a sample to be detected as a first round PCR template, taking the genomic DNA of MS751 and HELA cells as a negative control and a positive control of PCR respectively, and carrying out first round PCR by using primer combinations of SEQ ID NO. 1-2 and SEQ ID NO. 5-6;

(3) taking the first round PCR product as a second round PCR template, and carrying out second round PCR by using primer combinations of SEQ ID NO. 3-4 and SEQ ID NO. 7-8;

(4) and detecting the second round product by agarose gel electrophoresis, and judging whether the cell to be detected is polluted by the Hela cell.

4. The method for detecting HELA cell contamination according to claim 3, wherein the first round PCR reaction system is: adding 10 XPCR buffer, primers SEQ ID NO 1-2 and SEQ ID NO 5-6, DNTP (10um), DNA template, PfuDNA polymerase and dd H2O to 20 mul; the reaction conditions are as follows: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 50s, annealing at 57 ℃ for 40s, extension at 72 ℃ for 45s, 28 cycles, final extension at 72 ℃ for 5min, and heat preservation at 4 ℃.

5. The method of claim 3, wherein the second round of PCR reaction system is: 10 XPCR buffer, primers SEQ ID NO 3-4 and SEQ ID NO 7-8, DNTP (10um), first PCR product, Pfu DNA polymerase, dd H₂Supplementing O to 15.2 μ l; the reaction conditions are as follows: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 57 ℃ for 30s, extension at 72 ℃ for 25s, 28 cycles, final extension at 72 ℃ for 5min, and heat preservation at 4 ℃.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a HELA cell contamination detection method.

Background

The HeLa cell line was derived from cervical cancer cells from Heliytta cells (Henrietta cells), a U.S. woman in 1952. The hela cells are widely used in the medical field for tumor research, biological experiments or cell culture, and become very important tools in medical research. As the first human cervical cancer cell line determined to be capable of being cultured in vitro, HeLa cells are widely used in the study of cervical cancer, and play an important role in the biological study of cervical cancer cells and the diagnosis and treatment of cervical cancer. Furthermore, HeLa cells are widely used as research models in cell biology for basic biological research.

The HeLa cell strain is a cell line with unlimited proliferation capacity, can be continuously passed, can be abnormally and rapidly proliferated, can not be aged and killed, and can be infinitely divided; this cell line is very infectious compared to other cancer cells. Therefore, it is one of the important sources of cell cross-contamination. Many other cell lines have been demonstrated to be contaminated and completely replaced. The use of wrong and contaminated cell lines for research experiments will lead to wrong research results, which in turn leads to huge economic and social losses.

The existing detection method utilizes the characteristic that HELA cells are infected by HPV virus, and amplifies a section of sequence of the HPV virus by a PCR method, thereby judging whether HELA is polluted or not. However, this method cannot effectively distinguish other cells infected with HPV virus, such as cervical cancer cells infected with HPV virus. Therefore, the method of determining whether or not HELA is contaminated by merely detecting HPV infection cannot obtain an accurate determination result.

Therefore, finding a detection method capable of accurately distinguishing HELA cells infected by HPV virus from other cervical cancer cells is an urgent technical problem to be solved in the technical field.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a method for detecting HELA cell contamination. The inventor finds that HeLa cells have a homozygous whole-gene mutation through a cell database (http:// cancer. sanger. ac. uk/cosmetic/sample/overview. (as shown in figures 1 and 2). Therefore, the invention simultaneously utilizes the characteristics of HELA cell HPV virus infection and whole gene deletion to jointly detect whether the sample is polluted by HELA. According to the technical scheme, a section of sequence of HPV and a section of sequence on two sides of the deletion of STK11 whole gene are amplified simultaneously by a PCR method, so that whether HELA is polluted or not is judged, and whether HELA is polluted by other cells infected by HPV virus or not is distinguished.

The first purpose of the invention is to provide the application of STK11 whole gene deletion in detecting Hela cell contamination.

The second purpose of the invention is to provide a primer group for detecting Hela cell contamination, which comprises 2 pairs of HPV primer sequences and 2 pairs of STK11 primer sequences, wherein the HPV primer sequences are as follows:

upstream primer 5 'acgagccgaaccacaacg 3' (SEQ ID NO:1)

Downstream primer 5 'agctctgcctgttcgcaa 3' (SEQ ID NO:2)

Upstream primer 5 'tgtgtgtccgtggtgtgc 3' (SEQ ID NO:3)

The downstream primer 5 'agctctgcctgttcgcaa 3'; (SEQ ID NO:4)

The sequence of the STK11 primer is as follows:

upstream primer 5 'GCGTCTCCGAGGACCAATG 3' (SEQ ID NO:5)

Downstream primer 5 'GGCTCAACACCGTGACTGC 3' (SEQ ID NO:6)

Upstream primer 5 'GGATGGCAGGTTCAACCAA 3' (SEQ ID NO:7)

The downstream primer 5 'CACCGTGACTGCCGACCT 3' (SEQ ID NO: 8).

The third purpose of the invention is to provide a method for detecting HELA cell contamination, which comprises the following steps:

(1) extracting the genome DNA of a sample to be detected;

(2) taking the genomic DNA of a sample to be detected as a first round PCR template, taking the genomic DNA of MS751 and HELA cells as a negative control and a positive control of PCR respectively, and carrying out first round PCR by using primer combinations of SEQ ID NO. 1-2 and SEQ ID NO. 5-6;

(3) taking the first round PCR product as a second round PCR template, and carrying out second round PCR by using primer combinations of SEQ ID NO. 3-4 and SEQ ID NO. 7-8;

(4) and detecting the second round product by agarose gel electrophoresis, and judging whether the cell to be detected is polluted by the Hela cell.

Preferably, the first round PCR reaction system is: 10 XPCR buffer, primers SEQ ID NO 1-2 and SEQ ID NO 5-6, DNTP (10um), DNA template, Pfu DNA polymerase, dd H₂Supplementing O to 20 μ l; the reaction conditions are as follows: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 50s, annealing at 57 ℃ for 40s, extension at 72 ℃ for 45s, 28 cycles, final extension at 72 ℃ for 5min, and heat preservation at 4 ℃.

Preferably, the second round PCR reaction system is: 10 XPCR buffer, primers SEQ ID NO 3-4 and SEQ ID NO 7-8, DNTP (10um), first PCR product, Pfu DNApolymerase, dd H₂Supplementing O to 15.2 μ l; the reaction conditions are as follows: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 57 ℃ for 30s, extension at 72 ℃ for 25s, 28 cycles, final extension at 72 ℃ for 5min, and heat preservation at 4 ℃.

The invention has the beneficial effects that: the invention utilizes the uniqueness of the complete gene deletion of the HELA cell STK11 to combine the characteristic that the HELA cell is infected by HPV virus, and simultaneously amplifies a sequence before and after the complete gene deletion site of STK11 and a sequence of HPV to verify whether a cell line is polluted by the HELA cell. The invention can accurately distinguish HELA cells infected by HPV virus from other cells, such as cervical cancer cells. Can effectively detect whether the sample is polluted by the HELA cells.

Drawings

Fig. 1 is a web screenshot of the integrated information of HELA cells in the cell database.

Fig. 2 is a web screen shot of mutation information of HELA cells in the cell database.

FIG. 3 shows the results of measurement of the samples of examples.

Detailed Description

In order to more concisely and clearly demonstrate technical solutions, objects and advantages of the present invention, the following detailed description of the present invention is provided with reference to specific embodiments and accompanying drawings.

1. Design of PCR primers

2 pairs of HPV primer sequences and 2 pairs of STK11 primer sequences are obtained by designing and artificially synthesizing the gene sequence after the STK11 complete gene deletion and the HPV gene sequence, and the primer sequences are shown in the table 1:

table 1:

2. detection method

(1) Genomic DNAs of a sample to be tested, MS751 cell and Hela cell were extracted and quantified, respectively.

(2) Genomic DNA was used as a template for the first round of PCR and divided into 5 groups, which were grouped as follows:

sample DNA, sample DNA + 1% HeLa DNA, MS751 DNA and HeLaDNA.

Performing a first round of PCR with the primer set A; the primers in the group A in the step are as follows:

upstream primer 5 'acgagccgaaccacaacg 3' (SEQ ID NO:1)

Downstream primer 5 'agctctgcctgttcgcaa 3' (SEQ ID NO:2)

Upstream primer 5 'GCGTCTCCGAGGACCAATG 3' (SEQ ID NO:5)

The downstream primer 5 'GGCTCAACACCGTGACTGC 3' (SEQ ID NO: 6).

The first PCR amplification reaction system is shown in Table 2:

table 2:

the conditions for the first PCR amplification were set as follows:

(3) taking the first round PCR product as a second round PCR template, and carrying out second round PCR by using a B group primer combination, wherein the B group primer in the step is as follows:

upstream primer 5 'tgtgtgtccgtggtgtgc 3' (SEQ ID NO:3)

Downstream primer 5 'agctctgcctgttcgcaa 3' (SEQ ID NO:4)

Upstream primer 5 'GCGTCTCCGAGGACCAATG 3' (SEQ ID NO:7)

Downstream primer 5 'GGCTCAACACCGTGACTGC 3' (SEQ ID NO:8)

Second PCR reaction system, as shown in Table 3:

table 3:

the conditions for the second PCR amplification were set as follows:

(4) and detecting the second round product by agarose gel electrophoresis, and judging whether the cell to be detected is polluted by the Hela cell.

3. The result of the detection

The results obtained by the detection of the method are shown in fig. 3, the HELA cells serving as positive control have target bands at the positions of HPV bands and STK11 bands, so that the primers designed by the invention can simultaneously amplify two obvious target bands, and the detection method of the invention can effectively embody two characteristics of the HELA cells, namely the characteristics of HPV infection and STK11 whole gene deletion. In comparison with MS751 cells as a negative control, the MS751 cells showed only the target band at the HPV band position, indicating that the MS751 cells were infected only with HPV virus and not with HELA cells. The presence of the band of interest at both the HPV band position and the STK11 band position in MS751+ 1% HeLa indicates that MS751+ 1% HeLa was infected with both HPV virus and HELA cells. The presence of the band of interest in both the HPV band position and the STK11 band position in sample + 1% HeLa indicates that sample + 1% HeLa was infected with both HPV virus and HELA cells. No target band appeared in both the HPV band position and the STK11 band position in the sample, indicating that the sample was neither infected with HPV nor contaminated with HELA cells.

As is clear from the results shown in FIG. 3, the detection method of the present invention can accurately detect a sample contaminated with HELA cells and other cells infected with HPV only and not contaminated with HELA cells.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

SEQUENCE LISTING

<110> Guangzhou Jinie Europe Biotech Co., Ltd

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