CN103276084A - Method for detecting bastard halibut LITAF gene expression by applying reverse transcription-polymerase chain reaction (RT-PCR) - Google Patents

Abstract

The invention discloses a method for detecting LITAF gene expression of flounder by using RT-PCR. This method lays the foundation for studying the regulation mechanism and immunological function of LITAF gene expression in flounder. TNF-α plays a very important role in the process of immune response and killing foreign pathogenic bacteria. LITAF is a transcription factor of TNF-α gene, which directly regulates the expression of TNF-α gene. Therefore, the expression abundance of LITAF gene reflects the activity of the immune system of flounder to a certain extent, and can be used as an immune monitoring index and an evaluation index of immune preparation quality in the prevention and treatment of flounder diseases. By detecting the change of the LITAF gene expression of the flounder: it is possible to judge early whether the flounder is infected with a disease, and to take preventive and treatment measures in time to avoid irreversible losses caused by serious development of the situation; at the same time, it is possible to evaluate the quality of fish immune preparations and determine the disease of flounder Which immune preparation is more effective in prevention and treatment. The invention provides a technical platform for the relative quantitative analysis of the LITAF gene at the mRNA level.

Description

RT-PCR detection method of LITAF gene expression in flounder

This application is supported by the National High-tech Development Program (863) project (2012AA10A401), the National Science and Technology Support Program project (2011BAD13B07, 2012BAD26B04) and the Tianjin Normal University Talent Research Startup Fund (Project No.: 5RL120).

technical field

The invention relates to the field of biological technology, and relates to a specific primer for detecting the expression of the flounder LITAF gene, and more specifically to a method for detecting the expression of the flounder LITAF gene by using a fluorescent RT-PCR technique. It is mainly used for the study of the expression regulation mechanism of the LITAF gene of the flounder and the study of the immunological function, as well as the detection of the early pathogenic infection of the flounder and the evaluation of the effect of the fish immune preparation.

Background technique

In recent years, with the improvement of people's living standards, fish has become more and more popular as a high-protein and low-fat food. Due to the gradual expansion of the scale of aquaculture around the world, the trade of aquatic products at home and abroad and the cross-regional exchange of aquatic seed have become increasingly frequent, and the chances of the spread of aquaculture animal pathogens have greatly increased. The modern aquaculture industry is characterized by highly intensive and high-density production, which leads to the deterioration of the fishery water environment and the proliferation of bacteria in the water body, which will trigger the stress response of the farmed animals and lead to the suppression of the body's immune system. Due to the interaction of the above factors, the outbreaks of aquaculture animal diseases in the world tend to be more frequent and the prevalence tends to be more widespread, thus bringing great economic losses to the aquaculture industry. By using modern biological technology as the technical support of aquaculture, it is an urgent problem to be solved in aquatic science and technology to master the disease control technology of economic animals. Therefore, fish immunology has received more and more attention internationally in recent years.

The flounder ( Paralichthys olivaceus ) belongs to the order Scutellaria, the family Flounder, and the genus Flounder, commonly known as "flounder" and "left mouth". Japan, Russia's Far East coastal areas. In the past 10 years, due to overfishing, the resources of flounder have gradually been exhausted, and the aquaculture industry has been developed rapidly. Because of its delicious meat and rich nutrition, flounder has gradually become one of the important marine economical fishes in northern my country. High-density intensive farming has led to the outbreak and prevalence of various fish diseases, such as ascites disease, viral neuronecrosis, parasitic diseases, etc., which has caused huge economic losses to the breeding enterprises and seriously restricted the flounder breeding industry. healthy development. Among the various diseases of flounder, the harm of ascites disease is the most serious. The disease is caused by pathogenic bacteria, which occurs from fry to adult fish, and the mortality rate is as high as 50%-80%. Due to the lack of understanding of the immune regulation mechanism of flounder, there is no effective immune control technology and treatment.

In the history of immunology research, cellular immunity and humoral immunity have always been the main content of research. In contrast, the research on innate immunity is relatively lagging behind. In the 1990s, Janeway et al. proposed the related concepts of "pathogen-associated molecular patterns" and "pattern recognition receptors", which marked that the research on innate immunity has entered a new stage. Fishes belong to lower vertebrates and have the basic characteristics of immunity. There are two types of immune responses in their bodies: innate immune response and acquired immune response. But compared with mammals, research on innate immunity in fish has only just begun.

TNF-α is a factor that exists in serum and can kill tumor cells or cause hemorrhage and necrosis of tumor tissues in vivo, and plays a very important role in innate immunity and inflammatory responses. As a transcription factor of TNF-α gene, LITAF participates in the immune regulation of fish by directly regulating the transcription process of TNF-α and controlling the change of its expression level, which reflects the immune ability of fish to a certain extent. LITAF, as the regulatory gene of TNF-α, is expressed in the early stage of bacterial infection of flounder, and the expression time is early and the response intensity is strong. Therefore, it can be used as an important index for early detection of pathogenic infection in flounder and an index for evaluating the effect of fish immune preparations.

Fluorescent RT-PCR technology is an extremely effective method for quantifying nucleic acids in the field of molecular biology. This technology realizes the leap of PCR from qualitative to quantitative. Its principle is mainly based on the 3′-5′ exonuclease activity of DNA Taq enzyme and fluorescence energy transfer technology. Compared with conventional PCR, fluorescent RT-PCR has stronger specificity and sensitivity, can effectively solve the problem of PCR contamination, and has a high degree of automation, which ensures the reliability and repeatability of the results. Therefore, fluorescent RT-PCR has been widely used in the fields of molecular biology and medical research. With the further development of fluorescent RT-PCR kits, fluorescent RT-PCR technology has also been widely used in the diagnosis of animal diseases and the evaluation of immune preparations in recent years.

Contents of the invention

The purpose of the invention is to disclose a method for detecting the expression of the LITAF gene of the flounder by using the fluorescent RT-PCR technique.

In order to achieve the above object, the technical scheme adopted in the present invention is as follows:

Design a specific primer for detection of LITAF gene expression in flounder flounder by fluorescent RT-PCR technology, which is characterized in that it includes specific upstream and downstream primers that meet the characteristics of fluorescent RT-PCR reaction: LITAF-q-F: 5'-CGCTGCCAAGTCACTGTTCTC-3'; LITAF-q-R: 5′-CCACATCCTTACAGGCATCCA-3′; and the upstream and downstream primers specific to the flounder β-actin gene as an internal reference gene: β-actin-F: 5′-AGGTTCCGTTGTCCCG-3′; β-actin-R: 5'-TGGTTCCTCCAGATAGCAC-3'.

The method for rapid detection of LITAF gene expression in flounder flounder with specific primers of the present invention is characterized in that the detection is performed using a kit composed of the following reagents.

The components of the kit are as follows:

*Purchased from Dalian Bao Biological Company (product number: DRR420A), containing SYBR Green I, Ex Taq enzyme, dNTP and reaction buffer. the

The specific detection steps are as follows:

(1) Use the following primers to detect the gene:

The sequence-specific upstream and downstream primers that meet the characteristics of fluorescent RT-PCR reactions:

LITAF-q-F: 5′-CGCTGCCAAGTCACTGTTCTC-3′

LITAF-q-R: 5′-CCACATCCTTACAGGCATCCA-3′

And the specific upstream and downstream primers of the flounder β-actin gene as the internal reference gene:

β-actin-F: 5′-AGGTTCCGTTGTCCCG-3′

β-actin-R: 5′-TGGTTCCTCCAGATAGCAC-3′

Each primer was prepared into a 10 μmol/L storage solution, and the working concentration was 0.4 μmol/L.

(2) Extraction and purification of total RNA: Purified total RNA of head kidney of flounder was extracted from head kidney tissue or cells of flounder by using various common RNA extraction methods and purification methods.

(3) Synthesis of the first strand of cDNA: the first strand of cDNA was synthesized using the total RNA of the head kidney of the flounder fish as a template, and the reaction system was 20 μL;

(4) Fluorescence RT-PCR: using the first strand of cDNA synthesized above as a template, and the LITAF-q-F and LITAF-q-R, β-actin-F and β-actin-R in the above (1) as specific primers, carry out For fluorescence RT-PCR amplification reaction, 3 parallel tubes were set up for each sample, and the Ct values of the 3 parallel tubes obtained after amplification were averaged;

(5) Calculation of the relative expression of LITAF gene in head kidney tissue of flounder; after completion of fluorescent RT-PCR, calculate △Ct, 2 ^-(△Ct) , 2 ^{-( △△Ct)} value, the calculation method is as follows:

△Ct = Ct - Ct internal reference (β-acin)

△△Ct = △Ct - △Ct (healthy flounder at 0 time)

The expression level of LITAF gene relative to β-actin gene in head kidney of flounder was represented by 2 ^-(△Ct) ;

The expression level of LITAF gene in the head kidney of flounder relative to the LITAF gene in healthy flounder was represented by 2 ^-(△△Ct) .

The more detailed experimental method of the present invention is as follows:

(1) Primer design: According to the full-length sequence of the LITAF gene, use Primer Premier 5 software to design specific primers suitable for fluorescent RT-PCR detection. The primer sequences are as follows:

LITAF-q-F: 5'-CGCTGCCAAGTCACTGTTCTC-3'

LITAF-q-R: 5'-CCACATCCTTACAGGCATCCA-3'

The expected length of the PCR product of LITAF is 139bp. The results of agarose gel electrophoresis show that the length of the amplified product is consistent with the expected length of the product, and it is a single band, indicating that the designed primers have strong specificity and are suitable for fluorescent RT. -PCR detection, the results of agarose gel electrophoresis are shown in Figure 1.

At the same time, according to the cds sequence of β-actin (EU090804) provided by NCBI, design primers for internal reference of fluorescent RT-PCR. The primer sequences are as follows:

β-actin-F: 5'-AGGTTCCGTTGTCCCG-3'

β-actin-R: 5’-TGGTTCCTCCAGATAGCAC-3’

The expected length of the PCR product of β-actin is 150bp. The results of agarose gel electrophoresis showed that the length of the amplified product was consistent with the expected product length, and it was a single band, indicating that the designed primers had strong specificity and were suitable for use as internal reference amplification in fluorescent RT-PCR detection . The results of agarose gel electrophoresis are shown in Figure 2.

(2) Flounder infection experiment: 50 healthy flounder (body length 9-11cm) that had not been vaccinated recently were kept in the aquarium for a week and then injected with bacteria. Each tail was injected with 100 μL of live bacteria (10 ⁶ slow Edwardsiella), the head kidney tissue was dissected from flounder at different time periods.

(3) Stimulation experiment of head and kidney cells of flounder: 3 healthy flounder (body length 45cm-50cm) that have not been vaccinated recently were put to death after temporary breeding in the aquarium. The cells were then cultured in the primary culture, stimulated with LPS after overnight culture, and the cells were lysed at different time periods.

(4) Extraction and purification of total RNA: using various common RNA extraction and purification methods, the purified total RNA of the head kidney of the flounder was extracted from the head kidney of the flounder.

(5) Synthesis of the first strand of cDNA: The first strand of cDNA was synthesized using the GoScript ^TM Reverse Transcription System kit from Promega, and the reverse transcription primer used the Random Primer or Oligo (dT) ₁₅ Primer provided in the kit. The total RNA of head kidney tissue or cells was used as a template to synthesize the first strand of cDNA, and the reaction volume was 20 μL.

(6) Fluorescent RT-PCR: Fluorescent RT-PCR was carried out using the SYBR Premix Ex Taq ^TM II kit from Baobio Company. Using the first strand of cDNA synthesized above as a template, LITAF-qF and LITAF-qR, β-actin-F and β-actin-R in the above (1) as specific primers, carry out fluorescent RT-PCR amplification reaction, Three parallel tubes were set up for each sample, and the Ct values of the three parallel tubes obtained after amplification (that is, the number of reaction cycles experienced when the fluorescent signal in each reaction tube reached the set threshold) were averaged.

The fluorescent RT-PCR amplification reaction system is set as follows:

The fluorescent RT-PCR amplification parameters were set as follows:

95°C 30sec;

95°C 5 sec, 60°C 30 sec (40 cycles);

55°C—95°C 30 sec (melting curve).

(7) Calculation of the relative expression of LITAF gene in head kidney tissue or cells infected with flounder: after the completion of fluorescent RT-PCR, calculate the △Ct, 2 ^{-(△Ct) of} flounder pathogen infection for a period of time according to the Ct value , 2 ^-(△△Ct) value, the calculation method is as follows:

△Ct = Ct - Ct internal reference (β-acin)

△△Ct = △Ct - △Ct (healthy flounder at 0 time)

The expression level of LITAF gene relative to β-actin gene in head kidney of flounder was represented by 2 ^-(△Ct) ;

The expression level of LITAF gene in the head kidney of flounder relative to the LITAF gene in healthy flounder was represented by 2 ^-(△△Ct) .

2 ^-(△Ct) value represents the expression fold of LITAF gene relative to β-actin gene. 2 ^-(△△Ct) value represents the expression fold of LITAF gene relative to healthy flounder LITAF gene. The β-actin gene is a housekeeping gene, which is expressed in all types of cells. The expression of the housekeeping gene is only affected by the interaction between the promoter sequence or the promoter and RNA polymerase, not regulated by other mechanisms, and affected by environmental factors Small and relatively constant expression levels. Therefore, housekeeping genes are often used as reference genes when determining the expression level of target genes. In this experiment, when the value of 2 ^-(△△Ct) or 2 ^-(△△Ct) is larger, it indicates that the expression level of LITAF gene is higher.

Figure 3 shows the changes of LITAF gene expression in the head kidney tissue of flounder after intraperitoneal injection of Edwardsiella tarda, and Figure 4 shows the changes of LITAF gene expression in head kidney cells stimulated by LPS. It can be seen from the figure that the expression level of the LITAF gene is always lower than that of the housekeeping gene β-actin. The results in Fig. 3 show that the expression level of LITAF gene in the head kidney tissue of healthy flounder was 2×10 ^-3 times that of the housekeeping gene when uninfected, and the expression level of LITAF gene in the head kidney tissue of flounder after intraperitoneal injection of Edwardsiella tarda was between It increases within a short period of time, and is 4×10 ^-3 -6×10 ^-3 times of the housekeeping gene. Four hours after infection, the expression level of LITAF gene was 3.3 times that of healthy flounder. The above results suggest that when the relative expression of the LITAF gene increases, it means that the flounder is already in the state of pathogenic infection, and the innate immune system in the flounder has been activated. At this time, the infection is in the early stage, and the flounder cannot be observed with the naked eye. Have any symptoms of infection.

The results in Figure 4 show that the expression of LITAF gene in the head kidney cells of the healthy flounder without LPS stimulation is 1×10 ^-3 times that of the housekeeping gene. The rapid up-regulation was 3×10 ^-3 to 7×10 ^-3 times that of the housekeeping gene. After 2 hours of infection, the expression level of LITAF gene was 5.3 times that before stimulation. The above results suggest that the time required for the expression of LITAF gene in the head kidney cells of flounder flounder to change is relatively short and the difference is significant, which is suitable for detecting the immune effect of immune preparations.

Therefore, the expression level of LITAF gene reflects the immune activity of flounder to a certain extent. By detecting the relative expression of LITAF gene in the head kidney tissue or cells of flounder, it can be detected early whether the flounder is infected with pathogens, and preventive treatment measures can be taken in time. , to avoid irreparable losses caused by the serious development of the situation. The culture method of primary cells of flounder head kidney is simple, and the time required for LITAF gene response is short and the difference is significant, which can be used as a method to identify the quality of fish immune preparations.

Compared with modern technology, the method for rapid detection of flounder LITAF gene expression by specific primers provided by the invention has the following beneficial effects:

(1) Compared with conventional PCR, the fluorescent RT-PCR technology adopted in the present invention has stronger specificity and sensitivity, can effectively solve the problem of PCR pollution, and has a high degree of automation, which ensures the reliability and repeatability of the results . At the same time, fluorescent RT-PCR technology also eliminates the follow-up cumbersome steps such as electrophoresis, scanning, and software quantitative analysis of conventional PCR, which greatly shortens the experimental time. The entire reaction process of fluorescent RT-PCR is carried out in one tube, which eliminates the electrophoresis process of conventional PCR and greatly reduces the possibility of contamination in the experiment.

(2) The change of LITAF gene expression reflects the activity of the immune system of the flounder to a certain extent. At this time, no symptoms of infection can be observed in the flounder. Therefore, by detecting the changes of LITAF gene expression in the head kidney tissue or cells of the flounder, we can know whether the flounder is infected with pathogens early, and take preventive and treatment measures in time to avoid irreparable losses caused by the serious development of the situation. The large amount of expression of LITAF gene takes a short time at the cellular level, which can quickly determine the activity of fish immune enhancers, and is conducive to the rapid selection of immune enhancers with better effects.

(3) TNF-α plays an important role in the innate immune response and inflammatory response in the body. As a transcription factor of TNF-α, LITAF is expressed in the early stage of pathogen invasion. Therefore, the LITAF gene can be used as a tool for detecting bacterial infection of flounder, and once its high expression is found, broad-spectrum preventive measures should be taken immediately to gain valuable time for further diagnosis and treatment; at the same time, the high expression of the LITAF gene is required The time is short, so the effect of the fish immune booster can be quickly judged.

Description of drawings:

Figure 1 shows the electrophoresis results of the fluorescent RT-PCR product of the flounder LITAF gene: the length of the LITAF product is 139bp, and the Marker bands are 100, 250, 500, 750, 1000, 2000bp from bottom to top;

Figure 2 shows the results of electrophoresis of the fluorescent RT-PCR product of the internal reference gene β-actin in flounder: the length of the β-actin product is 150 bp, and the Marker bands are 100, 250, 500, 750, 1000, 2000 bp from bottom to top;

Figure 3 shows the differential expression of LITAF gene in head and kidney tissues of healthy flounder and intraperitoneal injection of Edwardsiella tarda;

Fig. 4 shows the differential expression of LITAF gene in healthy flounder head kidney cells and LPS-stimulated head kidney cells of flounder.

Detailed ways:

The present invention is illustrated below in conjunction with the examples, the scheme of the examples described here does not limit the present invention, and those skilled in the art can improve and change it according to the spirit of the present invention, and these described improvements and changes should be regarded as Within the scope of the present invention, the scope and spirit of the invention are defined by the claims; reagents used therein are commercially available.

Example 1

A kit for detecting the expression of the flounder LITAF gene:

Component I SYBR Premix Ex Taq II^*(Ex Taq fluorescent PCR mix) Component II LITAF gene upstream specific primer LITAF-q-F Component III LITAF gene downstream specific primer LITAF-q-R Component IV β-actin gene-specific upstream primer β-actin-F Component V β-actin gene-specific downstream primer β-actin-R

*Purchased from Dalian Bao Biological Company (product number: DRR420A), containing SYBR Green I, Ex Taq enzyme, dNTP and reaction buffer.

Example 2

Flounder infection experiment:

Edwardsiella tarda (provided by the Tianjin Aquatic Animal Disease Prevention and Control Center, available externally) was inoculated in Luriai-Bertani liquid medium with a pH value of 7.5 and 2% NaCl, and cultured at 28°C with constant temperature shaking (150rpm/min) 24h. The OD value was detected at 550nm, and the concentration of the bacterial solution and the total number of bacterial cells were calculated. Centrifuge (3000rpm/min) for 5 minutes to collect the bacteria, wash the bacteria three times with PBS, and prepare a bacterial solution with a concentration of 10 ⁷ cells/mL.

Fifty healthy flounder (body length 9-11cm) that had not been vaccinated recently were kept in the aquarium for a week and then injected with bacteria. Each fish was intraperitoneally injected with 100 μL of live bacteria (10 ⁶ Edwardsiella tarda), at different times The flounder was sacrificed and the head and kidney tissues were dissected.

Stimulation experiment of flounder head kidney cells:

Three healthy flounder (body length 45cm-50cm) that have not been vaccinated recently were put to death after temporary breeding in the aquarium. The head and kidney tissues were dissected and separated into single cells by mechanical dispersion for primary culture. After overnight culture, they were used Cells were lysed at different time periods stimulated by LPS.

Example 3

Extraction and purification of total RNA

(1) Take 100 mg head kidney tissue of flounder, add it to a homogenizer, and add 1000 μL Trizol reagent (Invitrogen Company) to it, and grind it thoroughly. If it is a cell, add 1000 μL Trizol to the culture well and pipette repeatedly to lyse the cells. Then transfer it to a 1.5 mL RNase-free centrifuge tube;

(2) Centrifuge at 12000rcf for 10min at 4°C;

(3) Take the supernatant, add it to a new centrifuge tube, and let it rest for a few minutes to completely lyse the nucleoprotein;

(4) Add 200 μL of chloroform to the supernatant, shake vigorously for 15 sec, and let stand at room temperature for 2-3 min;

(5) Centrifuge at 12000 rcf for 15 min at 4°C. After centrifugation, the product is divided into three layers: the lower phase is mainly protein, the middle phase is mainly DNA, and the upper phase is RNA;

(6) Extract the upper phase (rather less than more, do not touch the middle phase and the lower phase) into a new centrifuge tube, add 500 μL of isopropanol solution (RNA: isopropanol volume ratio ≈ 1:1) ), mix gently, and let stand at room temperature for 15 minutes to precipitate RNA;

(7) Centrifuge at 4°C and 12000 rcf for 10 min, discard the supernatant, then place the centrifuge tube upside down on filter paper, and dry the centrifuge tube;

(8) Add 1mL of 75% ethanol to the centrifuge tube, blow gently to wash the precipitate;

(9) Centrifuge at 4°C and 7500 rcf for 6 minutes, discard the supernatant, and place the centrifuge tube upside down on the filter paper to dry it;

(10) Add an appropriate amount of RNase free H ₂ O to the centrifuge tube to dissolve the RNA;

(11) Put the RNA solution in a constant temperature water bath, and bathe in a water bath at 55°C for 5 minutes to accelerate dissolution. The resulting solution was total RNA and stored at -80°C.

(12) The extracted total RNA can be further purified with DNaseI enzyme (Invitrogen Company) to remove the residual DNA in the RNA. The specific method is as follows: In a 200μL RNase-free centrifuge tube, add 1μL 10×DNase Reaction Buffer, 1μL DNase I (1U /μL), 2μg RNA, DEPC water to make up to 10μL volume. Place at room temperature for 15 minutes, add 1 μL EDTA, mix well, and keep at 65°C for 10 minutes. Purified total RNA can be obtained.

Example 4

Fluorescence RT-PCR detection of LITAF gene:

(1) Primer design:

According to the full-length sequence of the LITAF gene, use Primer Premier 5 software to design specific primers suitable for fluorescent RT-PCR detection. The primer sequences are as follows:

LITAF-q-F: 5'-CGCTGCCAAGTCACTGTTCTC-3'

LITAF-q-R: 5'-CCACATCCTTACAGGCATCCA-3'

The expected length of the PCR product of LITAF is 139bp. The results of agarose gel electrophoresis show that the length of the amplified product is consistent with the expected length of the product, and it is a single band, indicating that the designed primers have strong specificity and are suitable for fluorescent RT. -PCR detection. The results of agarose gel electrophoresis are shown in Figure 1.

At the same time, according to the cds sequence of flounder β-actin (EU090804) provided by NCBI, primers for internal reference of fluorescent RT-PCR were designed. The primer sequence is as follows

β-actin-F: 5'-AGGTTCCGTTGTCCCG-3'

β-actin-R: 5'-TGGTTCCTCCAGATAGCAC-3'

The expected length of the PCR product of β-actin is 150bp. The results of agarose gel electrophoresis showed that the length of the amplified product was consistent with the expected product length, and it was a single band, indicating that the designed primers had strong specificity and were suitable for use as internal reference amplification in fluorescent RT-PCR detection . The results of agarose gel electrophoresis are shown in Figure 2.

(2) cDNA first-strand synthesis:

Using the purified total RNA of flounder head kidney as described in Example 2 as a template, the GoScript ^™ Reverse Transcription System kit of Promega was used, and the reverse transcription primer used the Random Primer or Oligo (dT) ₁₅ Primer provided by the kit to The total RNA of flounder head kidney tissue or cells was used as the template to synthesize the first strand of cDNA, and the reaction volume was 20 μL.

(3) Fluorescent RT-PCR:

Fluorescent RT-PCR was carried out using the SYBR Premix Ex Taq ^TM II kit from Bao Bio Company. Using the first strand of cDNA synthesized in the above (2) as a template, and the LITAF-qF and LITAF-qR, β-actin-F and β-actin-R in the above (1) as specific primers, perform fluorescent RT-PCR amplification. For the amplification reaction, 3 parallel tubes were set up for each sample, and the Ct value of the 3 parallel tubes obtained after amplification (that is, the number of cycles experienced when the fluorescent signal in each reaction tube reached the set threshold value) was averaged.

The fluorescent RT-PCR amplification reaction system is set as follows:

Ingredients per tube Volume (μL) SYBR Premix Ex Taq II (Ex Taq fluorescent PCR mix) 10 PCR Forward Primer 0.8 PCR reverse primer 0.8 cDNA first strand 2 dH ₂ O 6.4 Total volume (μL) 20

The fluorescent RT-PCR amplification parameters were set as follows:

95°C 30sec;

95°C 5 sec, 60°C 30 sec (40 cycles);

55°C—95°C 30 sec (melting curve).

(4) Calculation of the relative expression of LITAF gene in head kidney tissue or cells infected with flounder:

After the completion of fluorescent RT-PCR, the △Ct, 2 ^-(△Ct) and 2 ^-(△△Ct) values of flounder pathogenic infection were calculated according to the Ct value, and the calculation method was as follows:

△Ct = Ct - Ct internal reference (β-acin)

△△Ct = △Ct - △Ct (healthy flounder at 0 time)

The expression level of LITAF gene relative to β-actin gene in head kidney of flounder was represented by 2 ^-(△Ct) ;

The expression level of LITAF gene in the head kidney of flounder relative to the LITAF gene in healthy flounder was represented by 2 ^-(△△Ct) .

2 ^-(△Ct) value represents the expression fold of LITAF gene relative to β-actin gene. 2 ^-(△△Ct) value represents the expression fold of LITAF gene relative to healthy flounder LITAF gene. The β-actin gene is a housekeeping gene, which is expressed in all types of cells. The expression of the housekeeping gene is only affected by the interaction between the promoter sequence or the promoter and RNA polymerase, not regulated by other mechanisms, and affected by environmental factors Small and relatively constant expression levels. Therefore, housekeeping genes are often used as reference genes when determining the expression level of target genes. In this experiment, when the value of 2 ^-(△△Ct) or 2 ^-(△△Ct) is larger, it indicates that the expression level of LITAF gene is higher.

Figure 3 shows the changes of LITAF gene expression in the head kidney tissue of flounder after intraperitoneal injection of Edwardsiella tarda, and Figure 4 shows the changes of LITAF gene expression in head kidney cells stimulated by LPS. It can be seen from the figure that the expression level of the LITAF gene is always lower than that of the housekeeping gene β-actin. The results in Fig. 3 show that the expression level of LITAF gene in the head kidney tissue of healthy flounder was 2×10 ^-3 times that of the housekeeping gene when uninfected, and the expression level of LITAF gene in the head kidney tissue of flounder after intraperitoneal injection of Edwardsiella tarda was between It increases within a short period of time, and is 4×10 ^-3 -6×10 ^-3 times of the housekeeping gene. Four hours after infection, the expression level of LITAF gene was 3.3 times that of healthy flounder. The above results suggest that when the relative expression of the LITAF gene increases, it means that the flounder is already in the state of pathogenic infection, and the innate immune system in the flounder has been activated. At this time, the infection is in the early stage, and the flounder cannot be observed with the naked eye. Have any symptoms of infection.

The results in Figure 4 show that the expression of LITAF gene in the head kidney cells of the healthy flounder without LPS stimulation is 1×10 ^-3 times that of the housekeeping gene. The rapid up-regulation was 3×10 ^-3 to 7×10 ^-3 times that of the housekeeping gene. After 2 hours of infection, the expression level of LITAF gene was 5.3 times that of the unstimulated one. The above results suggest that the time required for the expression of LITAF gene in head kidney cells to change is relatively short and the difference is significant, which is suitable for detecting the immune strength of fish immune preparations.

Therefore, the expression level of LITAF gene reflects the immune activity of flounder to a certain extent. By detecting the relative expression of LITAF gene in the head kidney tissue or cells of flounder, it can be detected early whether the flounder is infected with pathogens, and preventive treatment measures can be taken in time. , to avoid irreparable losses caused by the serious development of the situation. The primary cells of flounder head kidney are convenient to culture, and the time required for LITAF gene response is short and the difference is significant, which can be used as a method to identify the quality of fish immune preparations.

Example 5

The actual use of the kit (see Example 4 for specific steps)

(1) Kit composition

Component I SYBR Premix Ex Taq II^*(Ex Taq fluorescent PCR mix) Component II LITAF gene upstream specific primer LITAF-q-F Component III LITAF gene downstream specific primer LITAF-q-R Component IV β-actin gene-specific upstream primer β-actin-F Component V β-actin gene-specific downstream primer β-actin-R

*Purchased from Dalian Bao Biological Company (product number: DRR420A), containing SYBR Green I, Ex Taq enzyme, dNTP and reaction buffer.

(2) The detection status of the kit:

Figure 3 shows the changes of LITAF gene expression in the head kidney tissue of flounder after intraperitoneal injection of Edwardsiella tarda, and Figure 4 shows the changes of LITAF gene expression in head kidney cells stimulated by LPS. It can be seen from the figure that the expression level of the LITAF gene is always lower than that of the housekeeping gene β-actin. The results in Fig. 3 show that the expression level of LITAF gene in the head kidney tissue of healthy flounder was 2×10 ^-3 times that of the housekeeping gene when uninfected, and the expression level of LITAF gene in the head kidney tissue of flounder after intraperitoneal injection of Edwardsiella tarda was between It increases within a short period of time, and is 4×10 ^-3 -6×10 ^-3 times of the housekeeping gene. Four hours after infection, the expression level of LITAF gene was 3.3 times that of healthy flounder. The above results suggest that when the relative expression of the LITAF gene increases, it means that the flounder is already in the state of pathogenic infection, and the innate immune system in the flounder has been activated. At this time, the infection is in the early stage, and the flounder cannot be observed with the naked eye. Have any symptoms of infection.

The results in Figure 4 show that the expression of LITAF gene in the head kidney cells of the healthy flounder without LPS stimulation is 1×10 ^-3 times that of the housekeeping gene. The rapid up-regulation was 3×10 ^-3 to 7×10 ^-3 times that of the housekeeping gene. After 2 hours of infection, the expression level of LITAF gene was 5.3 times that of the unstimulated one. The above results suggest that the time required for the expression of LITAF gene in head kidney cells to change is relatively short and the difference is significant, which is suitable for detecting the immune strength of fish immune preparations.

(3) Conclusion

The expression of LITAF gene reflects the immune activity of flounder to a certain extent. By detecting the relative expression of LITAF gene in the head kidney tissue or cells of flounder, it can be detected early whether the flounder is infected with pathogens, and preventive and treatment measures can be taken in time to avoid The serious development of the situation caused irreparable losses. The primary cells of flounder head kidney are convenient to culture, and the time required for LITAF gene response is short and the difference is significant, which can be used as a method to identify the quality of fish immune preparations.

<110> Tianjin Normal University

<120> A method for detection of LITAF gene expression in flounder by RT-PCR

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<170> PatentIn version 3.5

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Claims (3)

1. an Auele Specific Primer that adopts fluorescent RT-PCR technology to detect lefteye flounder LITAF genetic expression is characterized in that comprising the specificity upstream and downstream primer that meets the fluorescence RT-PCR reaction characteristics: LITAF-q-F:5 '-CGCTGCCAAGTCACTGTTCTC-3 '; LITAF-q-R:5 '-CCACATCCTTACAGGCATCCA-3 '; And as interior lefteye flounder β with reference to gene-actin gene specific upstream and downstream primer: β-actin-F:5 '-AGGTTCCGTTGTCCCG-3 '; β-actin-R:5 '-TGGTTCCTCCAGATAGCAC-3 '.

2. test kit for detection of lefteye flounder LITAF genetic expression is characterized in that it is participated in forming by the described Auele Specific Primer of claim 1:

。

3. method that adopts the described test kit of claim 2 to detect is characterized in that the test kit moiety is as follows:

Concrete detection step is as follows:

Use following primer to carry out the detection of this gene:

This sequence-specific upstream and downstream primer that meets the quantitative fluorescent PCR reaction characteristics:

LITAF-q-F：5′-CGCTGCCAAGTCACTGTTCTC-3′

LITAF-q-R：5′-CCACATCCTTACAGGCATCCA-3′

And as interior lefteye flounder β with reference to gene-actin gene specific upstream and downstream primer:

β-actin-F：5′-AGGTTCCGTTGTCCCG-3′

β-actin-R：5′-TGGTTCCTCCAGATAGCAC-3′；

Wherein every primer is mixed with the storage liquid of 10 μ mol/L respectively, and working concentration is 0.4 μ mol/L;

Total RNA extraction and purification: adopt various general RNA extracting method and purification process, from lefteye flounder head-kidney tissue or cell, extract the total RNA of lefteye flounder head-kidney that obtains purifying;

CDNA first chain is synthetic: be synthetic cDNA first chain of template with the total RNA of lefteye flounder head-kidney, reaction system is 20 μ L;

Fluorescence RT-PCR: be template with above-mentioned synthetic cDNA first chain, LITAF-q-F in above-mentioned (1) and LITAF-q-R, β-actin-F and β-actin-R are Auele Specific Primer, carry out the fluorescence RT-PCR amplified reaction, each sample is established 3 parallel pipes, and the Ct value of 3 parallel pipes of amplification back gained is taken the mean;

The fluorescence RT-PCR amplification reaction system arranges as follows:

Every pipe composition Volume (μ L) SYBR ^@Premix Ex Taq II(Ex Taq fluorescent PCR mixture) 10 The PCR forward primer 0.8 The PCR reverse primer 0.8 CDNA first chain 2 dH ₂O 6.4 Cumulative volume (μ L) 20

Fluorescence RT-PCR amplification parameter arranges as follows:

95℃ 30 sec；

95 ℃ of 5sec, 60 ℃ of 30sec, 40 circulations;

55 ℃ of-95 ℃ of 30sec, solubility curve;

The relative expression quantity of LITAF gene calculates in the head-kidney tissue of infection lefteye flounder or the cell: after fluorescence RT-PCR is finished, calculate the △ Ct, 2 of lefteye flounder pathogen infection under each time period according to the Ct value ^{-(△ Ct)}, 2 ^{-(△ △ Ct)}Value, method of calculation are as follows:

Reference in △ Ct=Ct-Ct (β-actin)

Healthy Paralichthys olivaceus during △ △ Ct=△ Ct-△ Ct(0)

With 2 ^{-(△ Ct)}The LITAF gene is with respect to β-actin expression of gene amount in the numeric representation lefteye flounder head-kidney;

With 2 ^{-(△ △ Ct)}The LITAF gene is with respect to healthy Paralichthys olivaceus LITAF expression of gene amount in the numeric representation lefteye flounder head-kidney.

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