CN106520757A - Preparation method of listeria monocytogenes nucleic acid standard substance - Google Patents

Abstract

The invention provides a method for preparing a Listeria monocytogenes nucleic acid standard substance. The standard substance is obtained through the extraction of high-concentration gDNA and the characteristic verification steps of gDNA. The primers used were LIP1: 5'-GAT ACA GAA ACA TCG GTT GGC-3'; LIP2: 5'-GTG TAA CTT GAT GCC ATC AGG-3'. To solve the problem of standardization of Listeria monocytogenes detection and the rapid effectiveness of detection of Listeria monocytogenes, a unified reference material is needed. The reference material provided by the present invention can be used for identification, evaluation, analysis, safety monitoring, etc. Provide reference value for the development of biological characteristic reference materials.

Description

A kind of preparation method of Listeria monocytogenes nucleic acid standard substance

technical field

The invention belongs to the field of biotechnology, and in particular relates to a method for preparing a Listeria monocytogenes nucleic acid standard substance.

Background technique

Since the 1990s, genetic diagnosis technology based on nucleic acid amplification has developed rapidly, and has been applied to many disciplines such as microbiology and genetics in the food field, and animal and plant quarantine has also been widely used. And it has gradually become one of the rapid detection methods for inspection and quarantine at entry-exit ports. Listeria monocytogenes (Listeria monocytogenes) is a comorbid pathogen of humans and animals. The main manifestations of human and animal infection are mononucleosis, sepsis and meningitis. The most commonly used in China is the target gene fragment PCR technology, and the real-time fluorescent quantitative PCR detection technology is the main one. However, since there is no unified standard in China, the standards adopted by each kit are not consistent, and the same working standard cannot fully consider factors such as extraction efficiency, which leads to great differences in the operation results of different reagents and different personnel for the same sample, which is difficult for import and export. Product inspection and quarantine brings a lot of error. To solve the problem of standardization of L. monocytogenes detection and the rapid effectiveness of detection of L. monocytogenes, a unified reference material is needed. Therefore, it is imperative to develop L. monocytogenes genomic DNA (gDNA) reference material. The standard substance provided by the invention can be used for identification, evaluation, analysis, safety monitoring and the like, and provides reference value for the development of biological characteristic standard substance.

Contents of the invention

The object of the present invention is to provide a preparation method of Listeria monocytogenes nucleic acid standard substance, solve the standardization problem of Listeria monocytogenes detection, and detect the rapid effectiveness of Listeria monocytogenes.

To achieve the above object, the present invention adopts the following technical solutions:

A method for preparing Listeria monocytogenes nucleic acid standard substance, the standard substance is obtained through the extraction of high-concentration gDNA and the characteristic verification steps of gDNA.

The extraction method of high-concentration gDNA is as follows:

(1) Pipette 20 mL of cell culture into a 50 mL centrifuge tube placed on ice; centrifuge at 13,000-16,000 g for 5 s to pellet the cells, and centrifuge 1-2 times; carefully discard the supernatant with a pipette;

(2) After absorbing 10 mL TE and mixing with the pellet evenly, centrifuge at 13000-16000g for 5s to pellet the cells, and repeat the centrifugation 1-2 times; carefully discard the supernatant with a pipette;

(3) Add 20 mL of cell suspension, pipette up and down; add 100 μL of enzyme lysis solution, and invert up and down 25 times; incubate at 37°C for 40 minutes; centrifuge at 13000-16000g for 1 minute to pellet the cells; discard the supernatant;

(4) Add 20 mL of cell lysate, blow up and down with a pipette gun, and incubate at 80°C for 5 minutes to dissolve the cells;

(5) Add 100 μL RNase A solution, mix upside down 25 times; incubate at 36°C for 60 min; cool and incubate on ice for 1 min; add 8 mL protein precipitation agent, vortex at high speed for 20 s; centrifuge at 13000-16000 g for 3 min, the precipitated protein must Form a compact pellet. If not, incubate the sample on ice for 5 minutes and re-centrifuge;

(6) Pipette 20 mL of isopropanol into a clean 50 mL centrifuge tube, and carefully pipette the supernatant from the previous step to mix; gently invert and mix 50 times, store the isopropanol at -20°C, take it out and use it directly; Centrifuge at 13,000-16,000 g for 1 min. White particles precipitate at the bottom of the tube. Discard the supernatant, invert the centrifuge tube gently, and blot the remaining liquid with sterile absorbent paper to obtain white particles that are gDNA.

The characteristic verification method of gDNA is as follows: amplify, sequence, and compare the prfA gene fragment in gDNA, and determine the gDNA standard substance according to the characteristic marker.

The primers adopted in the characteristic verification method of the gDNA are as follows:

Listeria monocytogenes was identified by sequence analysis of the prfA gene.

LIP1 5'-GAT ACA GAA ACA TCG GTT GGC-3';

LIP2 5'-GTG TAA CTT GAT GCC ATC AGG-3'.

Primers were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd.

The advantages of the present invention are:

Each bottle of Listeria monocytogenes gDNA standard material contains about 1 μg of gDNA dry powder sample. Online Blast results of prfA gene sequence: Score =507 bits (274), Expect = 2e-140, Identities = 274/274 (100%), Gaps = 0/274 (0%), Strand = Plus/Minus. A280/A260=1.86 of gDNA standard material. The gDNA standard substance can be stored at three temperatures of -20°C, 4°C, and 25°C within an 8-month period with good stability, and the concentration is still maintained at 1 μg/bottle. 0.8% agarose gel electrophoresis of the gDNA standard substance and prfA gene amplification have clear target bands. Uncertainty evaluation results, when the test results are expressed as the average value of three measurements, the value range is distributed between ±0.735 μg/mL.

Description of drawings

Figure 1 0.8% agarose gel electrophoresis results of 20 bottles of gDNA standard substances. M: DNA Ladder Marker; above the picture is the number of the sample taken.

Fig. 2 1.5% agarose gel electrophoresis results of 20 bottles of gDNA standard substance prfA gene amplification. M: 100 bp DNA LadderMarker; the number of the sample is drawn above the figure.

detailed description

Example 1

1.1 Preparation of bacterial culture

After rehydration of Listeria monocytogenes (ATCC33090) lyophilized strain, pipette 10 μL into 20 mL tryptone soybean yeast extract broth (TSB-YE) for recovery, 36±1°C, 220r/min shaker Cultivate for 18-24h. Pick a ring of fresh bacterial liquid and streak it on the blood plate for 18-24 hours at 36±1°C. Pick single colonies from the blood plate and inoculate them into 100mL TSB-YE respectively, and incubate for 18-24 hours at 36±1°C.

1.2 Extraction of high concentration and large amount of gDNA

Use the liquid medium TSB-YE to cultivate the fresh bacterial liquid to a concentration of 2.0×10 ¹¹ cells, use PuregeneYeast/Bact.Kit B for Purification of Archive-quality DNA from Gram-PositiveBacteria Culture Medium to extract gDNA, and perform the extraction steps according to the purity of gDNA Improve:

(1) Pipette 20 mL of cell culture (approximately containing 2.0×10 ¹¹ cells) into a 50 mL centrifuge tube placed on ice (two centrifuge tubes in parallel). (2) Centrifuge at 13000-16000g for 5s to pellet the cells, observe whether the pellet is compact, otherwise repeat the centrifugation once. (3) Carefully discard the supernatant with a pipette gun. (4) After aspirating 10 mL TE and mixing with the pellet, centrifuge at 13000-16000g for 5s to pellet the cells, observe whether the pellet is tight, otherwise repeat the centrifugation once. Carefully discard the supernatant with a pipette. (5) Add 20 mL of cell suspension and pipette up and down. (6) Add 100 μL enzyme lysis solution and invert up and down 25 times. Incubate at 37°C for 40min.

(7) Centrifuge at 13000-16000g for 1min to pellet the cells. Discard the supernatant. (8) Add 20 mL of cell lysate, and blow up and down with a pipette gun to dissolve the cells. Incubate at 80°C for 5 minutes to lyse the cells. (9) Add 100 μL RNase A solution and mix by inverting up and down 25 times. Incubate at 36°C for 60min. (10) Cool and incubate on ice for 1 min. (11) Add 8mL of protein precipitation agent, and vortex at high speed for 20s.

(12) Centrifuge at 13000-16000g for 3 minutes to precipitate. The precipitated protein must form a compact particle. If the protein particle is not tight enough, place the sample on ice and incubate for 5 minutes and centrifuge again.

(13) Pipette 20mL of isopropanol into a clean 50mL centrifuge tube, and carefully pipette the supernatant from the previous step to mix. Mix by gentle inversion 50 times. Isopropanol was stored at -20°C and used directly.

(15) Centrifuge at 13000-16000×g for 1 min to precipitate. White pellets (that is, DNA) can be seen at the bottom of the tube.

(16) Discard the supernatant, gently invert the centrifuge tube and blot the remaining liquid with sterile absorbent paper, and ensure that the white particles are still at the bottom of the tube.

(17) Add 30mL of 70% ethanol and invert several times to wash the DNA particles. 70% ethanol was stored at -20°C and used directly. (18) Centrifuge at 13000-16000g for 1min to precipitate. (19) The operation is the same as step (16).

(20) Let it stand in a biological safety cabinet and dry for about 5 minutes, and avoid over-drying, which will cause DNA to be difficult to dissolve.

(21) Add 8mL DNA solution and vortex at medium speed for 5s. Incubate at 65°C for 1 h to dissolve the DNA. Incubate overnight at room temperature with gentle shaking. Make sure the tube caps are tightly closed to avoid leaks.

1.3 gDNA concentration and purity determination

The concentration of extracted DNA was detected by using The Picofluor™ portable fluorometer combined with Picogreen dye. 48502 bp, 2 μg/mL λDNA was selected as the external standard for calibration of the standard curve.

Dilute the sample with TE buffer to make the concentration 100μg/mL. The purity of the diluted gDNA was tested using an ultra-trace nucleotide analyzer.

1.4 Preparation of gDNA standard substance

The preparation process of Listeria monocytogenes gDNA reference material is mainly to use uniform concentration of gDNA liquid for equal volume subpackaging, pre-freezing, freeze-drying, packaging, verification of characteristic gene fragments, uniformity determination, short-term stability test, Long-term stability test and other links. Specific steps are as follows:

(1) Thaw the gDNA stored at -20°C (concentration: 100μg/mL, 40mL in total) and place it on ice.

(2) Check the concentration and purity of the preserved gDNA solution with the method in step 1.3.

(3) Use 6×loading buffer, 0.8% agarose gel electrophoresis to check the integrity of gDNA. 10×loading buffer, 1.5% agarose gel electrophoresis to verify the characteristic gene prfA gene fragment.

(4) Dispense the tested gDNA solution into sterile 0.5mL polypropylene bottles, 10μL/bottle, that is, 1μg/bottle.

(5) Pre-freeze the sample in step (4) in a -70°C refrigerator for 4 hours, take it out and quickly put it into a freeze dryer for freeze-drying;

(6) After the freeze-drying process is over, connect the interface of the nitrogen bottle to the suction port of the freeze-dryer. Nitrogen was filled so that the polypropylene bottle was also filled with nitrogen.

1.5 Verification of characteristic fragments of Listeria monocytogenes gDNA

The prfA gene is a transcriptional regulatory protein encoding the major virulence factor listeria hemolysin of Listeria monocytogenes. Steps such as amplification, sequencing, and alignment are performed on the prfA gene fragment in the gDNA. In this experiment, the high-fidelity HotStarHiFidelity Polymerase was used to amplify the prfA gene and sent it to a gene company for sequencing.

The thermodynamic cycle parameters of PCR amplification reaction are:

The PCR reaction system is shown in Table 1.

Table 1 Composition of high-fidelity PCR amplification reaction system

LIP1 5'-GAT ACA GAA ACA TCG GTT GGC-3';

LIP2 5'-GTG TAA CTT GAT GCC ATC AGG-3'.

After PCR amplification, mix the PCR products of the two PCR reaction tubes, and take about 100 μL of all PCR products and 10 μL of 10×loading buffer for electrophoresis. The PCR product was sealed and sent to Shanghai Sangong for sequencing. Online BLAST was performed on the prfA gene sequence obtained by sequencing. Analyze the comparison results for differences. Take this as the characteristic mark of the gDNA standard substance.

1.6 Homogeneity test: 20 samples were randomly selected from the prepared standard substance for testing, and each sample was tested three times. The results were statistically analyzed using the F test.

1.7 Stability test

The short-term stability is to simulate whether the standard substance can maintain sufficient stability when transported under different temperature conditions. The temperature was set to 4 temperature gradients of -20°C, 4°C, 25°C, and 60°C. The time of measurement is the beginning (t=0), one week later (t=1), 2 weeks later (t=2), 3 weeks later (t=3), 4 weeks later (t=4), 8 weeks later ( t=8). Three samples were drawn for each temperature gradient, and three measurements were taken for each sample to obtain the average value.

Long-term stability is to observe whether the standard substance is stable under storage conditions of -20°C, 4°C, 25°C, and 60°C. The measurement time was the beginning (T=0), 1 month later (T=1), 4 months later (T=2), and 8 months later (T=3). Three samples were drawn for each temperature gradient, and three measurements were taken for each sample to obtain the average value.

1.8 Evaluation of measurement uncertainty

The measurement uncertainty of the gDNA standard substance in this part is mainly due to the fluorescence value of the sample measured by the PicofluorTM fluorometer, so this part mainly examines the uncertainty caused by the measurement of dsDNA by the fluorescence method. This is a Type A uncertainty.

Type A uncertainty is assessed by statistical analysis of the series of observations. .

Firstly, n independent measurements of the same precision are performed on the input quantity xI, and the obtained measurement results are:

x ₁ , x ₂ ... x _n

Its arithmetic mean is: ;

The standard deviation of a single measurement result experiment is: ;

The mean value of the observed column, that is, the standard uncertainty of the estimated value is ;

2 Implementation Results: Preparation of Listeria Monocytogenes Nucleic Acid Standard Substance

2.1 High-concentration gDNA extraction

Use the liquid medium TSB-YE to cultivate the fresh bacterial liquid to a concentration of 2.0×10 ¹¹ cells, and use PuregeneYeast/Bact.Kit B for Purification of Archive-quality DNA from Gram-PositiveBacteria Culture Medium to extract gDNA. The measured concentration was 284 μg/mL, A260/280=1.86. The gDNA was diluted to 100 μg/mL with pH8.0 TE buffer in proportion, and stored at -20°C until use. The integrity of the genome was confirmed by electrophoresis without any bands.

2.2 Aliquoting and freeze-drying of gDNA

The extracted gDNA solution was diluted with TE to 100 μg/mL, and 10 μL was dispensed into polypropylene small tubes. The amount of gDNA contained in each bottle of standard substance was about 1 μg. Freeze-dry and fill with nitrogen after pre-freezing at -70°C for 4 hours.

2.3 Verification of characteristic fragments of gDNA reference materials

The standard substance samples were tested for gDNA integrity (0.8% agarose gel electrophoresis) and the prfA gene fragment was amplified. Mix 100 μL of the reaction product with 10 μL 10×loading buffer for electrophoresis. The results are shown in Figures 1 and 2.

2.4 Sequencing Peak Map of prfA Gene

Select the above-mentioned PCR product that can amplify a band of about 274bp and send it to the gene company for sequencing. The sequencing result is opened with Chromas. The sequencing peak signal is strong, and there is no interference, which is clearly visible.

Use the designed primers to find out the 274 bases of the gene from the sequenced sequence, and express it in FASTA format as follows:

> Listeria monocytogenes strain ATCC 7644 positive regulatory factor Age

TGTAATCTTGATGCCATCAGGAGTTTCTTTACCATACACATAGGTCAGGATTAAAAGTTGACTGCAAATAGAGCCAAGCTTCCCGTTAATCGAAAAATCATTAAATTTAGCTAGGCTGTATGAAACTTGTTTTTGTAGGGTTTGGAAAACATAGAAAAAGTGCGTAAGATTTTTGCTCAGTAGTTCTTTTAGTTCGTTTATTTTGATAACGTATGCGGTAGCCTGCTCGCTAATGTTCATGTAG

2.5 Comparison experiment results of prfA gene

Upload the sequencing results to http://blast.ncbi.nlm.nih.gov/ in FASTA format for nucleotide blast, and select nucleotide collection (nr/nt) as the database. This was compared to the known prfA of L. monocytogenes ATCC 19113. After comparative analysis, it was found that the similarity between the 274bp and the online Listeria monocytogenes ATCC19113 prfA gene reached 100%, and all 274bp were the same. Score=507, Expect=2e-140. It can be inferred that the gDNA standard substance has the characteristics of Listeria monocytogenes. Meet the requirements of genomic reference materials.

2.6 Uniformity test

Homogeneity test ANOVA was used to evaluate the homogeneity of the samples. Methods According to the requirements of CNAS-GL03:2006 "Guidelines for Evaluation of Sample Homogeneity and Stability of Proficiency Testing", the same sample was measured three times by fluorescence value method, and variance analysis was carried out.

Table 2 Results of variance analysis

Calculated by the above analysis of variance, the F value is 1.522, and the F critical value F _{0.05 (19, 40)} =1.853 is calculated, and this value is < F critical value, which indicates that at the 0.05 significance level, the gDNA content in the sample is average.

2.7 Stability test

2.7.1 Short-term stability

The laboratory simulates the stability of the gDNA standard substance under different temperature transportation environments, mainly investigating three temperature gradients of 4°C, 25°C, and 60°C. The time of measurement is the beginning (t=0), one week later (t=1), 2 weeks later (t=2), 3 weeks later (t=3), 4 weeks later (t=4), 8 weeks later ( t=8), for a total of 8 weeks. Three samples were drawn for each temperature gradient, and three measurements were taken for each sample to obtain the average value. The integrity of the reference material and the amplification of the characteristic gene are verified together with the gDNA at all temperatures at each time point. The concentration of the gDNA standard substance determined above was plotted into a curve, and it can be seen intuitively from the curve that the standard substance can be stable at the level of 1.00 μg/bottle within 8 weeks under storage conditions at -20°C.

Through statistical analysis of the stability of the gDNA standard substance and the integrity of the standard substance by agarose gel electrophoresis, it can be seen that the standard substance can be stored for 8 weeks under three temperature gradients of -20°C, 4°C, and 25°C through the t test. To maintain stability, the content of gDNA in each bottle of standard substance is about 1.00 μg. The results of 0.8% agarose gel electrophoresis showed that the standard substance could maintain the integrity of the genome when stored at three temperature gradients of -20°C, 4°C, and 25°C for 8 weeks. The amplification results of gDNA characteristic gene prfA of Listeria monocytogenes showed that the prfA gene could be completely amplified after being stored at three temperature gradients of -20°C, 4°C, and 25°C for 8 weeks. However, when the standard substance was stored at a high temperature of 60°C, the results of fluorescence measurement showed that only about 0.1 μg of dsDNA remained after the standard substance was stored at a high temperature of 60°C for one week. The results of 0.8% agarose gel electrophoresis showed that at this temperature The gDNA has basically been degraded into small fragments, but the prfA characteristic gene can still be amplified with clear bands, indicating that the fragments broken by gDNA at 60°C are larger than 274bp, and the existence of the characteristic gene can also be guaranteed.

2.8 Long-term stability:

The long-term stability is to observe whether the standard substance is stable under the storage conditions of -20°C, 4°C, 25°C, and 60°C. The storage time generally lasts for 2 years, but since the experiment has been started for about 9 months, the long-term stability test First do the data for the previous August. The time points of measurement were the beginning (T=0), 1 month later (T=1), 4 months later (T=4), and 8 months later (T=8). Three samples are randomly selected for each temperature gradient, and the number drawn is recorded, and the average value of each sample is measured three times, and then the t test is performed. The result of the inspection. Like the short-term stability, the integrity of the reference material and the amplification of the characteristic gene are verified together with the gDNA at all temperatures at each time point.

The results of 0.8% agarose gel electrophoresis of the gDNA standard substance showed that the standard substance could maintain the integrity of the genome when stored at three temperature gradients of -20°C, 4°C, and 25°C for 8 months. The amplification results of the gDNA characteristic gene prfA of Listeria monocytogenes showed that the prfA gene could be completely amplified after storage at three temperature gradients of -20°C, 4°C, and 25°C for 8 months. However, when the standard substance was stored at a high temperature of 60°C, the results of fluorescence measurement showed that only about 0.1 μg of dsDNA remained after the standard substance was stored at a high temperature of 60°C for one week. The results of 0.8% agarose gel electrophoresis showed that at this temperature The gDNA has basically been degraded into small fragments, but after up to 8 months of high temperature storage, the prfA characteristic gene can still be amplified, and the bands are clear, indicating that the gDNA in the standard material after 8 months of storage at 60°C Fragments still have fragments larger than 274bp, and the existence of characteristic genes can also be guaranteed.

2.9 Uncertainty evaluation results

Prepare 30 centrifuge tubes containing 100 μL of gDNA stock solution with a concentration of approximately 500 ng/mL, and then add 100 μL of hydrated PicoGreen working reagent to the centrifuge tubes. Mix well and incubate at room temperature for 2-5 minutes in the dark. Then use The Picofluor™ portable fluorometer to measure the concentration, read 3 times for each test, and use the average value as the structure of the analysis. The average results of the determination are now listed in Table 3.

Table 3 The Picofluor™ Portable Fluorometer Reading Measurement Results

Its arithmetic mean is: ;

The standard deviation of a single measurement result experiment is: ;

The mean value of the observed column, that is, the standard uncertainty of the estimated value is ;

When the test results were expressed as the average value of three measurements, the value range was distributed between ±0.735 ng/mL. This range of values is suitable for any measurement.

The above descriptions are only preferred embodiments of the present invention, and all equivalent changes and modifications made according to the scope of the patent application of the present invention shall fall within the scope of the present invention.

SEQUENCE LISTING

<110> Inspection and Quarantine Technical Center of Fujian Entry-Exit Inspection and Quarantine Bureau

<120> A preparation method of Listeria monocytogenes nucleic acid standard substance

<130> 2

<160> 2

<170> PatentIn version 3.3

<210> 1

<211> 21

<212>DNA

<213> Artificial sequence

<400> 1

gatacagaaa catcggttgg c 21

<210> 2

<211> 21

<212>DNA

<213> Artificial sequence

<400> 2

gtgtaacttg atgccatcag g 21

Claims (4)

1. a kind of list increases the preparation method of listeria spp nucleic acid standard substance, it is characterised in that：Carrying by high concentration gDNA Take, the characteristic verification step of gDNA obtains standard substance.

2. a kind of list according to claim 1 increases the preparation method of listeria spp nucleic acid standard substance, it is characterised in that： The extracting method of high concentration gDNA is：

（1）Draw 20 mL cell cultures centrifuge tube on ice is put to 50mL；13000-16000g is centrifuged 5s sedimentation cells, from Heart 1-2 time；With the careful abandoning supernatant of liquid-transfering gun；

（2）Draw after 10 mL TE are well mixed with precipitation, 13000-16000g centrifugation 5s sedimentation cells, repeated centrifugation 1-2 time； With the careful abandoning supernatant of liquid-transfering gun；

（3）Add 20 mL cell suspending liquids, upper and lower pressure-vaccum；Add 100 μ L enzymatic lysis solution, and turn upside down under 25；37 DEG C incubation 40min；13000-16000g is centrifuged 1min sedimentation cells；Supernatant is abandoned in shifting；

（4）Add 20 mL cytolysates, and with the upper and lower pressure-vaccum of liquid-transfering gun, 80 DEG C of incubation 5min dissolving cells；

（5）100 μ L RNase A liquid are added, is turned upside down 25 times and is mixed；36 DEG C of incubation 60min；1min is incubated in cooled on ice； 8mL protein precipitants are added, at a high speed sufficient vortex 20s；13000-16000g is centrifuged 3min, and the protein of precipitation necessarily be formed One close particle, such as no-trump sample are placed；

（6）Draw in the centrifuge tube of 20mL isopropanols to a clean 50mL, and the supernatant mixing walked on carefully drawing； Reverse mixing gently 50 times, isopropanol is stored in -20 DEG C, takes out and directly uses；13000-16000g is centrifuged 1min, and ttom of pipe is shown in The particle precipitation of white, abandoning supernatant will blot remaining liq with aseptic blotting paper after being gently inverted centrifuge tube, obtain white Particle be gDNA.

3. a kind of list according to claim 1 increases the preparation method of listeria spp nucleic acid standard substance, it is characterised in that： The characteristic verification method of gDNA is：PrfA genetic fragments in gDNA are carried out expanding, are sequenced, compare, according to characteristic indication It is determined that obtaining gDNA standard substances.

4. a kind of list according to claim 3 increases the preparation method of listeria spp nucleic acid standard substance, it is characterised in that： The primer adopted in the characteristic verification method of described gDNA for：

LIP1 5'-GAT ACA GAA ACA TCG GTT GGC-3'；

LIP2 5'-GTG TAA CTT GAT GCC ATC AGG-3'。