LAMP (loop-mediated isothermal amplification) primer composition for rapidly screening pollutant target molecules

Abstract

The invention provides an LAMP (Loop-Mediated Isothermal Amplification) primer composition for rapidly screening pollutant target molecules and a detection method of the LAMP primer composition. The LAMP primer composition provided by the invention can be used for detecting lung toxicity effect target molecules caused by exposure of gas mixed pollutants. A plurality of specific primers are designed for a specific region of a target gene, and isothermal amplification is performed under the action of DNA polymerase, so that the operation steps are simplified. A target sequence can be efficiently amplified at high specificity under an isothermal condition. The kit has the characteristics of strong specificity, high sensitivity, convenience and rapidness, and is suitable for application and popularization. At present, the technology has not been reported in the aspect of target molecule screening of the pulmonary toxicity effect caused by mixed pollution of gaseous pollutants.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology applications, and in particular relates to a LAMP primer composition for rapid screening of pollutant target molecules. Background Art

[0002] Gaseous pollutants are one of the typical pollutants commonly found in the environment. Gaseous pollutants are one of the typical pollutants commonly found in the environment, posing a serious threat to human health and ecosystems. These pollutants mainly come from a variety of human activities such as industrial activities, transportation, energy production and household combustion. There are many types of gaseous pollutants, including benzene, halides, alkanes, etc. Long-term exposure to high concentrations of benzene can cause blood diseases such as leukemia and anemia. Benzene can also damage the immune system and increase the risk of infection and disease. Inhaling high concentrations of benzene in a short period of time can cause dizziness, nausea, vomiting and coma, and in severe cases can lead to death. Chlorides may irritate the respiratory tract and skin, causing respiratory diseases and skin inflammation. Bromides and iodides have little effect on health at low concentrations, but exposure to high concentrations can also cause health problems. The harm of alkanes to the human body is mainly reflected in their flammability and irritation to the respiratory system. High concentrations of methane can cause hypoxia, dizziness, nausea and even suffocation; ethane and propane may cause explosions, posing a threat to life and property safety. In addition, alkanes can also form photochemical reactions in the atmosphere to produce harmful substances such as ozone, further aggravating air pollution.

[0003] In summary, benzene, halides and alkanes pose multiple threats to human health and the environment. They can not only directly damage human health, but also aggravate the impact of other pollutants through complex chemical reactions, forming a complex pollution effect. Therefore, controlling the emission and exposure of these harmful substances is of great significance for protecting human health and environmental safety. People are also inevitably exposed to complex mixtures of harmful gases. Therefore, the study of combined toxicity caused by mixed exposure to different gases and the rapid detection of multiple target molecules have become research hotspots in this field. In response to the problem of the combined toxic effects of mixed pollutants and the unclear target molecules, polymerase chain reaction (PCR) is currently a widely used in vitro nucleic acid amplification method. Since this technology relies on thermal cycling steps, it still has the following problems: First, the instruments and equipment are expensive and the operation is cumbersome, and the technical requirements for the experimenters are high, and they need to master certain experimental operations and data processing capabilities; second, the experimental conditions are demanding, and complex temperature control procedures are required, which is time-consuming.

[0004] In view of this, the purpose of the present invention is to provide a LAMP primer combination for rapid screening of pollutant target molecules, which can amplify the target sequence efficiently and specifically under isothermal conditions by using a specific primer combination designed for a specific region of the target gene. There are no reports on the use of LAMP (isothermal nucleic acid amplification detection technology) in the screening of target molecules for the pulmonary toxicity effects caused by pollution. Summary of the invention

[0005] The present invention aims to solve the problem of unclear target molecules of pulmonary toxicity caused by exposure to mixed gas pollution. By establishing a BEAS-2B cell exposure model for toxic effect evaluation, transcriptomics sequencing combined with bioinformatics analysis technology is used to clarify the multiple target molecules related to pulmonary toxicity. The specific primers are designed in combination with isothermal amplification to achieve high-throughput screening of target molecules of pulmonary toxicity caused by it.

[0006] In order to achieve the above technical objectives, the present invention proposes the following technical solutions: The first aspect of the present invention provides a LAMP primer composition, which can be used for rapid screening of mixed pollutant target molecules. The LAMP primer composition includes a LAMP primer mixture, magnesium sulfate, dNTP, Bst DNA polymerase, sterile enzyme-free water, RNA sample, and buffer.

[0007] Furthermore, the LAMP primer mixture includes LAMP outer primers, LAMP inner primers and LAMP loop primers.

[0008] Furthermore, the LAMP external primers include primers with sequences as shown in SEQ ID NOs: 1, 2, 7, and 8.

[0009] Furthermore, the LAMP inner primers include primers with sequences as shown in SEQ ID NOs: 3, 4, 9, and 10.

[0010] Furthermore, the LAMP loop primers include primers with sequences as shown in SEQ ID NOs: 5, 6, and 11.

[0011] Furthermore, the concentration of the LAMP primer mixture is 25×.

[0012] Furthermore, the target molecules include target molecules for lung toxicity effects caused by exposure to mixed gaseous pollutants.

[0013] Furthermore, the target molecules include one or more of GPX4 and FTH1.

[0014] Glutathione peroxidase 4 (GPX4) is a member of the glutathione peroxidase family that uses glutathione as an electron donor to reduce highly dangerous lipid hydroperoxides in the membrane to less toxic lipid alcohols. Its function is to reduce hydroperoxides in membrane lipids and lipoproteins, thereby protecting mitochondrial function and inhibiting cellular ferroptosis. GPX4 is a major inhibitor of iron poisoning because it has a unique function that can reduce complex hydroperoxides including phospholipid hydroperoxides and cholesterol hydroperoxides to their corresponding counterparts, thereby blocking the lipid peroxidation chain reaction. Studies have shown that GPX4 is an enzyme required for the health and survival of motor neurons.

[0015] FTH1 is ferritin heavy chain 1, also known as FHC (ferritin heavy chain), FTH, FTHL6, HFE5, PIG15, PLIF, etc. It has iron oxidase activity and plays an important role in the storage and transportation of iron in organisms. 2+ Oxidized to Fe 3+ , scavenging free radical reactions mediated by ferrous ions, protecting cells from oxidative damage, and controlling cell homeostasis. In organisms, iron is essential for normal cell growth, proliferation, and energy metabolism, while excess iron is harmful. For example, excessive Fe 2+ Excessive ROS can be produced through the Fenton reaction, disrupting the oxidative balance in cells, leading to oxidative stress and causing damage to DNA, proteins, lipids, etc.

[0016] Furthermore, the LAMP primer composition includes 1 μL of primer mixture, 1.5 μL of magnesium sulfate, 3.5 μL of dNTP, 1 μL of Bst DNA polymerase, 16.5 μL of sterile enzyme-free water, 50 ng of RNA sample, and 2.5 μL of 10-fold buffer.

[0017] The second aspect of the present invention provides a method for rapid screening of mixed pollutant target molecules, the method comprising the steps of detecting with the LAMP primer composition described in the first aspect of the present invention.

[0018] Furthermore, the steps of the method include: 1) preparing a control group: 2.5 μL 10-fold buffer, 1.5 μL magnesium sulfate, 3.5 μL dNTP, 1 μL primer mixture, 1 μL Bst DNA polymerase and 16.5 μL sterile enzyme-free water to form a mixture; 2) reverse transcribing the control group and the LAMP primer composition described in the first aspect of the present invention in a PCR instrument at 65° C. for 30 min; after the reverse transcription is completed, preparing a nucleic acid gel with a concentration of 1.2%, and loading 10 μL of each sample for gel electrophoresis; 3) comparing the bands of the control group and the LAMP primer composition.

[0019] Advantages and beneficial effects of the present invention: multiple specific primers are designed for specific regions of the target gene, and constant temperature amplification is performed under the action of DNA polymerase, thereby simplifying the operation steps. The target sequence can be amplified efficiently and specifically under isothermal conditions. The method has the characteristics of strong specificity, high sensitivity, convenience and speed, and is suitable for application and promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a curve diagram of cell viability detection after exposure to dichloromethane, toluene and ethylbenzene.

[0021] Figure 2 This is a bar graph of cell viability detection after exposure to dichloromethane, toluene, and ethylbenzene.

[0022] Figure 3 This is a diagram showing the effects of pollutant exposure on the BEAS-2B cell gene chip results.

[0023] Figure 4 This is the result of LAMP detection of GPX4 gene.

[0024] Figure 5 This is the result of LAMP detection of FTH1 gene. DETAILED DESCRIPTION

[0025] In the present invention, LAMP (loop-mediated isothermal amplification) is a new nucleic acid amplification technology that does not require expensive instruments and can complete amplification in a short time (usually within one hour) at a constant temperature (60-65°C). The reaction can produce a large amount of amplification products, namely white precipitates of magnesium pyrophosphate, and the presence or absence of the target molecule can be determined by visually observing the presence or absence of the white precipitate. In some embodiments, the amplification products can be detected by electrophoresis, and the presence and content of the target molecule can be determined based on the brightness of the band.

[0026] The core technology of LAMP lies in the design of primers. Highly specific primers are the key to the success of LAMP experiments. When designing primers, it is necessary and primary to determine the six specific regions F3, F2, F1, B1, B2, and B3 on the target gene. Next, four primers are designed based on these six specific regions, which are divided into one pair of external primers and one pair of internal primers. The external primers limit the range of amplification and provide templates for the internal primers.

[0027] The FIP primer (Forward Inner Primer) consists of the F2 region and the F1c region. The F2 region is complementary to the F2c region at the 3' end of the target gene, and the F1C region is identical to the Flc region at the 5' end of the target gene. The F3 primer (Forward Outer Primer) consists of the F3 region and is complementary to the F3c region of the target gene. The BIP primer (Backward Inner Primer) consists of the B1c and B2 regions. The B2 region is complementary to the B2c region at the 3' end of the target gene, and the B1c region is identical to the Blc region at the 5' end of the target gene. The B3 primer (Backward Outer Primer) consists of the B3 region and is complementary to the B3c region of the target gene. The upstream loop primer Loop F can be designed between the 3' end of F2c and the 5' end of F1c, and the downstream loop primer Loop B can be designed between the 3' end of B1 and the 5' end of B2 to increase the reaction speed and detection rate.

[0028] Primer design needs to consider issues such as Tm value, spacing, GC content, terminal stability and secondary structure. Regarding Tm value, F1c / B1c is 64~66℃, F2 / B2, F3 / B3 are 59~61℃, and the Tm value of LoopF / LoopB is 64~66℃. Regarding spacing, the distance between the 5 end of F2 and the 5 end of B2 is 120-160 bp, the distance between the 5 end of F2 and the 5 end of F1c is 40-60 bp, and the distance between the 5 end of F2 and the 3 end of F3 is 0-60 bp. Generally, the GC content is 40-65%, and the effect is better when the GC content is 50-66%. Regarding terminal stability, the ΔG of F2 / B2, F3 / B3, the 3 end of LoopF / LoopB and the 5 end of F1c / B1c is ≤-4 kcal / mol. Avoid repeating single or double bases more than three times in primers (such as ACCC, ATATAT) to avoid the formation of primer dimers or hairpin structures, which may cause nonspecific amplification.

[0029] In LAMP technology, correct primer design is a prerequisite for successful amplification, and the selection of high-quality key enzyme raw materials is a necessary condition for successful LAMP amplification. This technology requires the use of strand displacement Bst DNA polymerase to perform amplification reactions at a constant temperature of 60-65°C, achieving 10% of the target sequence within 15-60 minutes. 9 -10 10 times amplification.

[0030] The present invention is further described below in conjunction with the embodiments. The following description is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change it into an equivalent embodiment with equivalent changes. Any simple modification or equivalent change made to the following embodiments based on the technical essence of the present invention without departing from the content of the present invention falls within the protection scope of the present invention.

[0031] Example

[0032] 1. Experimental materials: PCR primers and LAMP primers of target genes were synthesized by Shanghai Sangon Biotechnology Co., Ltd., reagent standards were purchased from DRE Company, and Bst 3.0 DNA Polymerase (NEB, USA), Magnesium Sulfate (MgSO4) Solution (NEB, USA), and Deoxynucleotide (dNTP) Solution Mix (NEB, USA) used in the experiment. If the specific conditions are not specified in the examples, the conventional conditions or the conditions recommended by the manufacturer are followed. The reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased commercially.

[0033] 2. Experimental methods: 1. First, determine the pollutant exposure IC 50 The specific process is as follows: The cell titer method was used to determine the effects of dichloromethane, toluene, and ethylbenzene alone and in combination on cell viability. The density of logarithmic phase BEAS-2B cells was adjusted to 1.6×10 6 / mL, 100μL was inoculated in each well of a 96-well plate and incubated for 24h. When the cell density was about 70% to 80%, the culture medium was discarded and culture medium containing different concentrations was added to continue culturing; after 24h of culturing, 50μL of cell titer reagent was added to each well, and its luminescence value was measured with an enzyme marker within 15 min, and the cell viability change curve was drawn, such as Figure 1 , 2 shown.

[0034] 2. Take cells in the logarithmic growth phase and use 1×10 6 / mL density was inoculated in a large dish, and control and mixed exposure treatments were carried out according to the experimental groups. The culture dish was gently shaken and placed in an incubator for exposure for 24 hours; after the exposure time, the culture plate was removed, the poison solution was discarded, and the cells were washed 3 times with pre-cooled PBS, placed on ice, and 3mL TRIZOL lysis solution was added to each dish. After sufficient lysis, it was transferred to a 1.5 mL RNAse-free EP tube and stored at -80℃; the extraction and quality inspection of total RNA, the construction and sequencing of the library, the filtering and quality control of the data, and the comparison with the reference genome and other bioinformatics analysis were all performed by Beijing Novogene Technology Co., Ltd. in accordance with standard experimental operations and data analysis methods. By comparing the data of the experimental group and the control group samples, the differentially expressed genes were screened out, and the differentially expressed genes were compared with databases such as GO and KEGG. The results are shown in the figure. Figure 3 As shown, the functional annotation of differentially expressed genes revealed for the first time that mixed exposure to human BEAS-2B cells could significantly activate the ferroptosis signaling pathway.

[0035] 3. Take cells in the logarithmic growth phase and use 1×10 6 / mL was planted in a 6-well plate, and 2mL of cell suspension was added to each well. After the cell density was appropriate, drug exposure was performed according to the experimental group, and the culture plate was gently shaken and placed in an incubator for exposure for 24 hours; after the exposure time, the culture plate was removed, the poison solution was discarded, and the cells were washed twice with pre-cooled PBS, RNA was extracted from the cells using the reagent Trizol, and the RNA sample was reverse transcribed into cDNA according to the instructions of the Prime ScriptTM RT kit. The conditions for reverse transcription were: 37℃, 30min; 85℃, 10sec; 4℃ storage; after cooling, -20℃ storage.

[0036] 4. The core technology of LAMP lies in the design of primers. Primers with strong specificity are the key to the success of LAMP experiments. When designing primers, it is necessary and primary to determine the six specific regions F3, F2, F1, B1, B2, and B3 on the target gene. Next, four primers are designed based on these six specific regions, which are divided into one pair of external primers and one pair of internal primers. The external primers limit the range of amplification and provide templates for the internal primers. The external primers include an upstream external primer F3 composed of the F3 region on the target gene and a downstream external primer (B3) composed of the B3 region on the target gene; the internal primers include a forward internal primer (FIP) composed of F1c and F2 and a reverse internal primer (BIP) composed of B1c and B2, as shown in Table 1.

[0037] Table 1 Primer sequence list

[0038] After the primer design is completed, dissolve each tube of primer in DEPC water according to the instructions, and then prepare Primer MIX (25×). The configuration system is shown in Table 2.

[0039] Table 2 Primer MIX configuration system (25×)

[0040] The configuration of the LAMP amplification reaction system is shown in Table 3.

[0041] Table 3 LAMP amplification reaction system

[0042] After the reaction system is configured, reverse transcription is performed in a PCR instrument at 65°C for 30 minutes. After reverse transcription is completed, a 1.2% nucleic acid gel is prepared and 10 μL of each sample is loaded for gel electrophoresis. The experimental results are shown in Figure 2. Figure 5 It is shown that this method has good feasibility.

[0043] According to the present invention, the optimal loading amount of the sample is 50 ng.

[0044] According to the present invention, the temperature of the isothermal amplification reaction is 65° C.; the optimal amplification reaction time is 30 min.

[0045] According to the present invention, the optimal concentration of the nucleic acid gel is 1.2%; the optimal loading volume is 10 μL.

[0046] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. A LAMP primer composition, which can be used for rapid screening of mixed pollutant target molecules, characterized in that: The LAMP primer composition comprises a LAMP primer mixture, magnesium sulfate, dNTP, Bst DNA polymerase, sterile enzyme-free water, an RNA sample, and a buffer.

2. The LAMP primer composition according to claim 1, characterized in that The LAMP primer mixture includes LAMP outer primers, LAMP inner primers and LAMP loop primers.

3. The LAMP primer composition according to claim 2, characterized in that The LAMP external primers include primers with sequences as shown in SEQ ID NOs: 1, 2, 7, and 8.

4. The LAMP primer composition according to claim 2, characterized in that The LAMP inner primers include primers with sequences as shown in SEQ ID NOs: 3, 4, 9, and 10.

5. The LAMP primer composition according to claim 2, characterized in that The LAMP loop primers include primers with sequences as shown in SEQ ID NOs: 5, 6, and 11.

6. The LAMP primer composition according to claim 2, characterized in that The concentration of the LAMP primer mixture was 25×.

7. The LAMP primer composition according to claim 1, characterized in that The target molecules include target molecules for lung toxicity effects caused by exposure to mixed gaseous pollutants; Preferably, the target molecules include GPX4 and FTH1.

8. The LAMP primer composition according to claim 1, characterized in that The LAMP primer composition includes 1 μL of primer mixture, 1.5 μL of magnesium sulfate, 3.5 μL of dNTP, 1 μL of Bst DNA polymerase, 16.5 μL of sterile enzyme-free water, 50 ng of RNA sample, and 2.5 μL of 10-fold buffer.

9. A method for rapid screening of mixed pollutant target molecules, characterized in that: The method comprises the steps of performing detection using the LAMP primer composition according to any one of claims 1 to 3.

10. The method according to claim 9, characterized in that The steps of the method include: 1) Prepare the control group: 2.5 μL 10x buffer, 1.5 μL magnesium sulfate, 3.5 μL dNTP, 1 μL primer mixture, 1 μL Bst DNA polymerase and 16.5 μL sterile enzyme-free water to form a mixture; 2) The control group and the LAMP primer combination according to any one of claims 1 to 3 were reverse transcribed in a PCR instrument at 65°C for 30 min; after the reverse transcription was completed, a nucleic acid gel with a concentration of 1.2% was prepared, and the sample volume of each group was 10 μL for gel electrophoresis; 3) Compare the bands of the control group and the LAMP primer combination.

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