CN114891862B - LAMP and Multi-LAMP-based reagent set for rapidly detecting cell species and cross contamination - Google Patents

LAMP and Multi-LAMP-based reagent set for rapidly detecting cell species and cross contamination Download PDF

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CN114891862B
CN114891862B CN202210705223.2A CN202210705223A CN114891862B CN 114891862 B CN114891862 B CN 114891862B CN 202210705223 A CN202210705223 A CN 202210705223A CN 114891862 B CN114891862 B CN 114891862B
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artificial sequence
lamp
species
dna
cell
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CN114891862A (en
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沈超
赵慧仪
赵佳曦
皮睿琦
杨媚媚
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Wuhan University WHU
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention discloses a reagent set for rapidly detecting cell species and cross contamination based on LAMP and Multi-LAMP, belonging to the fields of life science and medicine. The reagent set of the present invention comprises primers for identifying 14 species shown in nucleotide sequences SEQ ID NO.1-56, 14 species of DNA, LAMP reaction reagent, etc., wherein 14 species are respectively cow, syrian hamster, cat, dog, human, mouse, chicken, pig, chinese hamster, rat, rabbit, guinea pig, green monkey, rhesus monkey. According to the invention, sample DNA does not need to be extracted, the sample can be directly subjected to LAMP reaction in a water bath after being subjected to rapid pyrolysis, and the result is readable by naked eyes. The invention can detect 13 kinds of cells in a tube at the same time.

Description

LAMP and Multi-LAMP-based reagent set for rapidly detecting cell species and cross contamination
Technical Field
The invention belongs to the fields of life science and medicine, and mainly relates to a rapid detection reagent set for cross contamination of experimental cell species and other species.
Background
Cells are an important model for in vitro studies in the fields of life sciences and medicine, and in order to ensure the correctness and reproducibility of the relevant studies, the cell species must be correct and free of contamination by other species. When primary cells are separated, cell lines are established, materials are obtained by mistake, and cross contamination can be caused by the sharing of consumable materials. Immortalized cells have unlimited passage capability, so that operation errors can be easily introduced into other cells in the passage process, and pollution is caused. Early in the research related to cell culture, the importance of cell species origin identification and cross-contamination detection was recognized, and once the misidentified cells were used, the related research was ineffective, resulting in waste of various resources, time and money. Erroneous experimental conclusions the consequences can be very serious if used clinically.
The existing technology for identifying cell species and detecting cross contamination mainly comprises an isozyme method and a PCR method. Isozymes detection has been the standard method of species identification over the past fifty years, based on differences in the number and size of electrophoretic bands of different species isozymes (glucose-6-phosphate dehydrogenase G6PD, lactate dehydrogenase LD, nucleoside phosphorylase NP, malate dehydrogenase MD). Isozymes analysis requires optimization of experimental conditions, including electrophoresis, color development time to determine the appropriate band migration distance, and selection of the appropriate combination of enzymes to determine the desired species. Some species are similar in band and are difficult to distinguish, so analysis of the electrophoresis results requires comparison with a set of known species to determine species origin. In addition, commercial kits have stopped selling in 2015, and the drugs for formulating running buffers are difficult to purchase in the regulatory domain. These factors limit the use of isozymes electrophoresis for cell species identification.
The PCR method for identifying the cell species has been developed in the last 20 years, and the method obtains different size strips by amplifying the species-specific genes through a PCR instrument, judges the source of the cell species through agarose gel electrophoresis, has higher detection speed compared with an isozyme method, and related reagents are easy to purchase, but a PCR instrument and a DNA imager are needed.
Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification technology, 4 primers are designed aiming at a target gene, and the experimental result can be judged visually after a constant temperature reaction is carried out for tens of minutes. Compared with the PCR method, the method is simpler, has higher sensitivity, does not need a PCR instrument with more than ten thousand yuan, and can meet the experimental requirements by a common water bath. LAMP has not been used for cell species identification and cross-contamination detection.
Disclosure of Invention
The invention aims to provide a reagent set for rapidly detecting cell species and cross contamination based on LAMP and Multi-LAMP, and also aims to provide a using method of the reagent set.
The aim of the invention is achieved by the following technical scheme:
a rapid detection cell species and cross-contamination reagent set based on LAMP and Multi-LAMP comprises LAMP primers for 14 species shown in Table 1 below.
TABLE 1.14 LAMP primers for species
Further, the reagent set also comprises DNA of different species.
Further, the reagent set also comprises LAMP reaction reagents. The LAMP reaction reagent comprises Bst DNA polymerase, dNTPs and buffer solution. The buffer preferably contains 2.5mM Mg 2+ Is a Tris-HCl buffer.
Further, the reagent set also comprises a dye. Dyes commonly used in LAMP reactions include malachite Green, SYBR Green I, picogreen, hydroxybromophenol blue, and the like. Further, the dye is malachite green.
Further, the reagent set further comprises a cell lysate. The formula of the cell lysate is preferably as follows: 2% Triton X-100,1% SDS,5% sucrose, 25mM Tris-HCl, pH=7.4.
A method for identifying known cell species and cross-contamination using the above reagent set, comprising the steps of:
(1) The cells to be examined were collected, and a cell lysate was added thereto, and the lysed liquid was directly used for the following reaction.
(2) Taking 6 EP pipes;
wherein 3 EP tubes are used for LAMP reaction, LAMP primer, bst DNA polymerase, dNTP, buffer solution and malachite green of the species of the cell to be detected are respectively added, then water is respectively added as negative control, DNA of the species of the cell to be detected is added as positive control, and the cracked liquid is added as a sample;
the other 3 EP tubes were used for MultiLAMP reaction, respectively adding 13 species LAMP primer mixtures of the non-examined cell belonging species, bst DNA polymerase, dNTPs, buffer solution, malachite green, respectively adding water as negative control, adding 13 species DNA mixtures of the non-examined cell belonging species as positive control, and adding the lysed liquid as sample.
(3) The prepared 6-tube reagent is placed at 60-65 ℃ for incubation for 35-40 minutes.
(4) After the reaction is finished, observing the color of each tube, and if the color of the sample for LAMP reaction is consistent with that of the positive control, the cells to be detected are the cells of the belonging species; the color of the sample used for the Multi-LAMP reaction is consistent with that of the negative control, so that the cells to be detected are free from other species of cell pollution.
The method for identifying unknown cell species and cross contamination by using the reagent group comprises the following steps:
(1) The cells to be examined were collected, and a cell lysate was added thereto, and the lysed liquid was directly used for the following reaction.
(2) Taking 42 EP tubes, and respectively adding Bst DNA polymerase, dNTP, buffer solution, malachite green and LAMP primers of corresponding species according to a group of every 3 EP tubes, wherein the group corresponds to one species, namely a negative control, a positive control and a sample tube; and respectively adding water, DNA of the corresponding species and the liquid after the cleavage into the negative control, the positive control and the sample tube.
(3) The prepared 42-tube reagent is placed at 60-65 ℃ for incubation for 35-40 minutes.
(4) After the reaction is finished, determining the species of the cells to be detected and whether cross contamination exists according to the colors of the sample, positive controls and negative controls of different species.
The invention has the advantages and beneficial effects that:
1. the sample DNA does not need to be extracted, and the sample can be directly used for LAMP reaction after rapid lysis.
2. And a large-scale equipment PCR instrument and a DNA imager are not needed, the reaction is carried out in a water bath kettle, and the result is readable by naked eyes.
3. Cells of 13 species can be detected simultaneously in one tube.
Drawings
FIG. 1 shows the results of LAMP and Multi-LAMP experiments on human cells to be examined in example 1.
FIG. 2 shows the LAMP results of 14 species positive controls (template is species DNA) in example 2.
FIG. 3 shows the LAMP results of 14 species negative controls (template: ultrapure water) in example 2.
FIG. 4 shows the LAMP results of unknown cells to be examined in example 2.
Detailed Description
The invention designs a species-specific LAMP primer for the species of 14 common cells (bovine, syrian hamster, cat, dog, human, mouse, chicken, pig, chinese hamster, rat, rabbit, guinea pig, green monkey, rhesus monkey) by selecting cytochrome oxidase and ATP synthase as target genes, as shown in Table 1 above.
Generally, the LAMP reaction requires the design of 4-6 primers to amplify the gene of interest. In order to avoid false positives in the LAMP reaction, the primers mentioned in Table 1 were all screened in large numbers and were determined to have sufficient species specificity.
For rapid identification of 14 species of cells (bovine, syrian hamster, cat, dog, human, mouse, chicken, pig, chinese hamster, rat, rabbit, guinea pig, green monkey, rhesus monkey), 14 LAMP reagent sets and 14 Multi-LAMP reagent sets were prepared, the compositions of which are shown in table 2. The MultiLAMP reaction is to mix primers of a plurality of species in proportion and then add the mixed primers to the same tube, and when DNA templates of the corresponding species appear, the amplification reaction starts. In order to prevent the primer from crosslinking and affecting the pairing of the primer and the target template, the sensitivity of the primer is improved, the primer sequences of all species are subjected to a large number of comparison and adjustment, the primer concentrations are subjected to a large number of screening, 2 groups of primers and 3 groups of primers are mixed firstly, 5 groups of primers and 6 groups of primers are mixed, and finally 13 groups of primers are mixed together.
For the identification of cells of unknown species origin, the composition of the LAMP reaction reagent set is shown in Table 3.
TABLE 2 LAMP reaction reagent set for species identification
TABLE 3 LAMP reaction reagent set for unknown species identification
The LAMP reaction premix in tables 2 and 3 has the following concentration: 1U Bst enzyme/50. Mu.L, 0.25mM dNTP,0.1% malachite green, 2.5mM Mg 2+ 200nM of the corresponding species primer.
The concentrations of the components of the Multi-LAMP reaction premix in Table 2 above were: 1U Bst enzyme/50. Mu.L, 0.25mM dNTP,0.1% malachite green, 2.5mM Mg 2+ 100-200nM of the various primers. The ratio of each primer in each cross-contamination detection reagent set was varied, as shown in Table 4. In order to prevent cross-linking between primers, non-specific binding of primers to templates, mixed primers capable of simultaneously detecting 13 species must be mixed according to the ratio in Table 4, which is found by trial and error; if the primers are not mixed in this ratio, DNA of a certain species or of several species cannot be detected.
TABLE 4 mixing ratio of primers for cross contamination detection (in the table, the ratio relationship is molar ratio)
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In each LAMP reaction reagent set, cell lysates were also prepared in addition to the components mentioned in tables 2 and 3. The formula of the cell lysate is as follows: 2% Triton X-100,1% SDS,5% sucrose, 25mM Tris-HCl, pH=7.4.
The following examples serve to further illustrate the invention but are not to be construed as limiting the invention. The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated.
EXAMPLE 1 identification of human cell species and Cross contamination
Experimental reagent: human cells to be detected, a human cell LAMP reagent group and ultrapure water.
Experimental equipment: water bath, sample adding gun and gun head.
The experimental steps are as follows:
(1) Collecting cells to be detected, wherein the number of the cells is 1 multiplied by 10 6 ～3×10 6 。
(2) 100 μl of the cell lysate was added, and the mixture was lysed at room temperature for 5 minutes, and the lysed solution was used directly for LAMP reaction.
(3) 6 clean 1.5mL EP tubes were taken and labeled "human-", "human+", "human sample", "mix-", "mix+", and "mix sample", respectively.
(4) 49. Mu.L of reagent 2 (premix for human cell LAMP reaction: enzyme, dNTP, buffer, malachite green, human LAMP primer) was added to each of the "human-", "human+", and "human sample" tubes, and 49. Mu.L of reagent 4 (premix for non-human cell Multi-LAMP reaction: enzyme, dNTP, buffer, malachite green, mixture of non-human 13 species LAMP primers) was added to each of the "mix-", "mix+", and "mix sample" tubes.
(5) 1. Mu.L of ultrapure water was added to each of the "human-" and "mix-" tubes as a negative control; 1. Mu.L of reagent 1 (positive control: human DNA) was added to the "human+" tube, and 1. Mu.L of reagent 3 (positive control: non-human 13 species DNA mixture) was added to the "mixed+" tube as a positive control; 1 μl of the lysed sample was added to each of the "human sample" and "mixed sample" tubes.
(6) The prepared 6-tube reagent is put into a water bath kettle and incubated for 40 minutes at 65 ℃.
(7) After the reaction was completed, the change in color per tube was observed. As shown in fig. 1, the color of the cell to be detected "human sample" is consistent with that of the "human+" tube, which indicates that the cell to be detected is a human cell; the color of the cell to be detected 'mixed sample' is consistent with that of the 'mixed-' tube, which indicates that the cell to be detected has no pollution of other species cells.
EXAMPLE 2 identification of unknown cell species and Cross contamination
Experimental reagent: unknown cells to be detected, LAMP reagent groups of unknown species cells, and ultrapure water.
Experimental equipment: water bath, sample adding gun and gun head.
The experimental steps are as follows:
(1) Collecting cells to be detected, wherein the number of the cells is 1 multiplied by 10 6 ～3×10 6 。
(2) 100 μl of the cell lysate was added, and the mixture was lysed at room temperature for 5 minutes, and the lysed solution was used directly for LAMP reaction.
(3) 42 clean 1.5mL EP tubes, 14 negative controls, 14 positive controls, and 14 sample tubes were labeled separately.
(4) All EP tubes were added 49. Mu.L of LAMP reaction premix for each of 14 species, for each of 3 groups (negative, positive, sample tube).
(5) 1. Mu.L of ultrapure water was added to each of the 14 negative control tubes as a negative control; adding DNA of the corresponding species into 14 positive control tubes to serve as positive control; in 14 sample tubes, 1. Mu.L of the lysed sample was added, respectively.
(6) The prepared 42-tube reagent is put into a water bath kettle and incubated for 40 minutes at 65 ℃.
(7) After the completion of the reaction, the change in color per tube was observed (fig. 2, 3, 4). As shown in FIG. 4, the cells to be examined were only consistent with the color of the "mouse+" tube, indicating that the cells to be examined were mouse cells and were free of contamination by other species of cells.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Sequence listing
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<120> LAMP and Multi-LAMP based reagent set for rapid detection of cell species and cross contamination
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tgggcataga tgtagacaca cgaaaacttt aactccagtg ggaa 44
<210> 53
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 53
cttagcaaac tcatccctag a 21
<210> 54
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 54
ggttaaattt acgcccatga a 21
<210> 55
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 55
gcgaatacag ctcctattga tagaacgtac tacacgatac atactacg 48
<210> 56
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 56
tatggggggc tttatacact gagatgaagt gggctttggc 40

Claims (7)

1. A kit for detecting cell species and cross-contamination, comprising: LAMP primers comprising the species shown in the following table:
2. the kit of claim 1, wherein: LAMP reagents are also included.
3. The kit of claim 2, wherein: the LAMP reaction reagent comprises Bst DNA polymerase, dNTPs and buffer solution.
4. A kit according to claim 3, wherein: the buffer solution contains 2.5mM Mg 2+ Is a Tris-HCl buffer.
5. The kit of claim 1, wherein: also comprises dye; the dye is selected from malachite Green, SYBR Green I, picogreen and hydroxy bromophenol blue.
6. The kit of claim 1, wherein: cell lysates are also included.
7. The kit of claim 6, wherein: the formula of the cell lysate is as follows: 2% Triton X-100,1% SDS,5% sucrose, 25mM Tris-HCl, pH=7.4.
CN202210705223.2A 2022-06-21 2022-06-21 LAMP and Multi-LAMP-based reagent set for rapidly detecting cell species and cross contamination Active CN114891862B (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110358816A (en) * 2019-08-13 2019-10-22 湖北国际旅行卫生保健中心 A kind of primer sets, kit and application for chicken derived cell PCR detection
CN112941201A (en) * 2021-03-01 2021-06-11 武汉珈创生物技术股份有限公司 Mixed primer for multi-cell species identification and cross contamination detection and use method thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110358816A (en) * 2019-08-13 2019-10-22 湖北国际旅行卫生保健中心 A kind of primer sets, kit and application for chicken derived cell PCR detection
CN112941201A (en) * 2021-03-01 2021-06-11 武汉珈创生物技术股份有限公司 Mixed primer for multi-cell species identification and cross contamination detection and use method thereof

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