CN113249500A - Method for rapidly detecting vibrio vulnificus in clinical blood - Google Patents
Method for rapidly detecting vibrio vulnificus in clinical blood Download PDFInfo
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Abstract
The invention discloses a method for rapidly detecting vibrio vulnificus in clinical blood, which comprises the steps of adding an upstream primer, a downstream primer and a fluorescent probe to a vvhA gene conserved region in a strain DNA to be detected, and carrying out a fluorescent RPA reaction in real time. The invention aims to establish a vibrio vulnificus rapid detection method taking a vhhA gene as a target spot based on real-time RPA. On the basis, the real-time RPA method is optimized, and the reproducibility of the method in practical application is evaluated through the detection of artificially infected blood samples.
Description
Technical Field
The invention belongs to the technical field of medical treatment, and particularly relates to a method for rapidly detecting vibrio vulnificus in clinical blood.
Background
Vibrio vulnificus is a thermophilic halophilic gram-negative marine bacterium that can cause severe infections in people around the world, particularly in states and islands along the sea. Many Vibrio vulnificus-infected individuals require intensive care or amputation, with about one-fifth of the infected individuals dying, sometimes within one or two days after the disease. If a patient is unable to receive antibiotic treatment within three days, the mortality rate may be 100%. Vibrio vulnificus is one of the most common marine pathogens, can be found in seafood such as oyster, and can cause infection of people due to eating seafood. A report by the united states Food and Drug Administration (FDA) shows that from 1992 to 2007 459 total cases with a mortality rate of 51.7%. In the northern hemisphere, most cases occur in the warm water month of 5 to 10 months. Worse still, as a thermophilic halophilic bacterium, its outbreak area is expanding with global warming. In view of this, it is desirable to establish a fast and accurate detection method to better control the propagation.
The traditional vibrio vulnificus detection method is based on biochemical analysis detection, and needs long-time pre-enrichment, preliminary morphological identification and final biochemical confirmation. As a gold standard for clinical testing, this method, while relatively accurate, is time consuming and laborious, typically requiring 3-4 days to achieve the final result. Considering that Vibrio vulnificus infected patients can survive for only 3 days, timely diagnosis is critical for rescuing patients, and the conventional method is obviously not suitable for diagnosis of clinical samples. Therefore, many efforts have been made to reduce the detection time. The nucleic acid detection technology has the characteristics of strong specificity, short analysis time and the like, and seems to be more suitable for the rapid detection of vibrio vulnificus. Among them, PCR has been widely used for the rapid detection of Vibrio vulnificus, and has high specificity and sensitivity. However, this method relies on sophisticated thermal cyclers and trained personnel, limiting its application in field testing scenarios. Recently, LAMP method is reported to be sensitive to Vibrio vulnificus rapidly, but the reaction time and temperature are about 1h, 63 ℃. The need for a simple, rapid, and accurate method for the early diagnosis of Vibrio vulnificus infection remains.
Piepenburg and his colleagues invented a new isothermal gene amplification strategy, Recombinase Polymerase Amplification (RPA), in 2006 and has proven to be a simple, rapid, specific, sensitive and cost-effective method to identify different pathogens. RPA replaces DNA synthesis by recombinase (T4uvsX) -primer complex driven autonomous cycle, thereby completing nucleic acid amplification, rather than thermal cycle as in conventional PCR, which allows PRA to complete nucleic acid amplification at lower temperature (37-42 ℃) in shorter time (10-20 minutes). This greatly reduces the energy consumption and cost required for the nucleic acid detection system itself. Amplification products of the RPA can be identified by obtaining fluorescent signals from the amplification products, which are synchronized with the nucleic acid amplification process, which further shortens the analysis time and also avoids the problem of contamination caused by open tubes in nucleic acid amplification detection. Therefore, the advantages of real-time fluorescent RPA in clinical diagnosis are more prominent, and attract the attention of many researchers.
Disclosure of Invention
The invention provides a method for rapidly detecting vibrio vulnificus in clinical blood in order to overcome the defects of the prior art.
In order to achieve the purpose, the invention adopts the following technical scheme: a method for rapidly detecting Vibrio vulnificus in clinical blood comprises
Adding an upstream primer, a downstream primer and a fluorescent probe to a vvhA gene conserved region in the DNA of a strain to be detected, and carrying out a fluorescent RPA reaction in real time.
The invention aims to establish a vibrio vulnificus rapid detection method taking a vhhA gene as a target spot based on real-time RPA. On the basis, the real-time RPA method is optimized, and the reproducibility of the method in practical application is evaluated through the detection of artificially infected blood samples.
Preferably, the upstream primer is RPA-F1, wherein the sequence of RPA-F1 is: CGGCAAAGTAGGTGCGGAAGTGAACAAAGA are provided.
Preferably, the downstream primer is RPA-R4, and the sequence of the RPA-R4 is as follows: CTCAATGATGAACGGTTGTTGATGCGATAG are provided.
Preferably, the fluorescent Probe is an RPA-Probe, and the sequence of the RPA-Probe is as follows: TGGCGAAGTCAGTGGCTCATTTACCTACAAC [ FAM-dT ] a [ THF ] bC [ BHQ1-dT ] C CGAAGACCTTGGTGTT (C3 Spacer) d.
Preferably, the reaction system of the real-time fluorescent RPA reaction is a 20-L reaction system comprising 11.8-L buffer solution, 4.48-L deionized water, 0.84-L upstream and downstream primers, 1-L LMg2+, 0.24-L probe and 0.8-L standard plasmid.
Preferably, the temperature at which the fluorescent RPA reaction is carried out in real time is 35-40 ℃.
Preferably, the temperature at which the fluorescent RPA reaction is carried out in real time is 38 ℃.
Preferably, in the reaction system of the fluorescent RPA reaction, the concentration of the upstream primer and the downstream primer is 10 μ M, and the concentration of the fluorescent probe is 10 μ M.
Preferably, in the reaction system, the standard plasmid is prepared by amplifying a 498bp fragment of the vvhA gene in a DNA fragment of the strain to be tested by using an upstream primer vvhA-F and a downstream primer vvhA-R. The amplification conditions were 25. mu.L of 2 XGoldstar TaqMan mixture, 1. mu.L (10. mu.M) of each primer, 22. mu.L of ddH2O, 2.5 μ L genomic template, amplification cycle conditions: denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30s, denaturation at 52 ℃ for 30s, and elongation at 72 ℃ for 45s for 35 cycles, and extension at 72 ℃ for 7min, and introducing the product after the above amplification cycle reaction into PMD19-T vector and sequencing the vector to determine the insertion of target cells.
Compared with the prior art, the invention has extremely high specificity, sensitivity and rapidness which are as low as 1.2 multiplied by 102At cfu/mL infection concentration, the real-time fluorescence RPA method can detect Vibrio vulnificus within 16min, whereas the traditional real-time fluorescence PCR detection requires about 45 min. And the detection time decreased with increasing cell number. At higher concentrations (1.2X 10)6cfu/mL) real-time fluorescent RPA method can obtain positive results within 3.44min at the shortest. Meanwhile, the real-time RPA method invented by the inventor can be combined with a portable device, and is a very useful method for monitoring the infection of the vibrio vulnificus in the clinical diagnosis of the vibrio vulnificus outbreak field, especially under the condition of limited resources.
Drawings
FIG. 1 optimal primer and probe combination analysis for real-time fluorescent RPA detection of Vibrio. The best combination was selected using 7 candidate primer combinations and 1 probe. NTC represents a negative control.
FIG. 2 fluorescence curves of temperature optimization experiments. Real-time fluorescent RPA was performed at 37, 38, 39 ℃ respectively. NTC represents a negative control.
FIG. 3 real-time fluorescent RPA-specific detection. 1. Vibrio vulnificus ATCC 27562; 2. vibrio vulnificus HMO 2; 3. vibrio vulnificus HMYJ; 4. vibrio fluvialis LMG 7894; 5. vibrio parahaemolyticus CGMCC 1.1997; 6. vibrio harveyi CGMCC 1.1599; 7. vibrio harveyi NBV 269; 8. vibrio harveyi 9-S29; 9. listeria anguillarum ATCC 19264; 10. edwardsiella tarda ATCC 15947; 11. pseudomonas aeruginosa CGMCC 1.1785; 12. escherichia coli ATCC 25922; 13. e.coli HM 7039; 14. e.coli HM 11022; 15. escherichia coli HM 4769; 16. listeria monocytogenes ATCC 19115; 17. salmonella typhimurium ATCC 13311; 18. klebsiella pneumoniae ATCC 33495; 19. klebsiella pneumoniae HM 7837; 20 klebsiella pneumoniae HM 9872; 21. klebsiella pneumoniae HM 10474; 22. no template negative control.
FIG. 4 sensitivity analysis of real-time fluorescent RAP detection of Vibrio vulnificus. A. Average CQ value (n 3), R of real-time PCR and logDNA concentration20.9859 (linear regression). Mean CQ values (n 3) of real-time reaction of RPA with logDNA concentration, R20.9227 (linear regression) and 0.9962 (quadratic polynomial regression). C. Mean CQ values, R of real-time RPA reactions with standard plasmid log copy number (n-8)20.8386 (linear regression) and 0.9816 (quadratic polynomial regression)
Fig. 5. Probit regression was performed on the data from 8 replicates of different concentration gradients using the "glm" software package in r.
Detailed Description
In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention.
In one embodiment, a method for rapidly detecting Vibrio vulnificus in clinical blood comprises
Step one, extracting DNA of a strain to be detected from a sample to be detected, circularly amplifying the DNA of the strain to be detected by using primers vvhA-F and vvhA-R, further amplifying a 498bp fragment of vvhA gene of the strain to be detected, introducing a circularly amplified product into a PMD19-T vector, and sequencing to determine whether a target gene is inserted so as to obtain a standard plasmid.
Step two, performing real-time fluorescence RPA reaction on the standard plasmid obtained in the step two, specifically, the reaction system of the real-time fluorescence RPA reaction is that a 20-microliter reaction system comprises: 11.8. mu.L buffer, 4.48. mu.L deionized water, 0.84. mu.L (10. mu.M) of each of the upstream and downstream primers, 1. mu.L of Mg2+0.24. mu.L of probe (10. mu.M) and 0.8. mu.L of standard plasmid. In the step, the upstream primer is RPA-F1, the downstream primer is RPA-R4, and the Probe is RPA-Probe. In this example, the upstream primer was selected as RPA-F1 and the downstream primer was selected as RPA-R4, while in the remaining examples, the primers used to perform the real-time fluorescent RPA reaction were selected as shown in Table 2.
Further, in the second step, the reaction temperature of the real-time fluorescence RPA reaction is 35-40 ℃, and after optimization, 38 ℃ is the optimal reaction temperature. Furthermore, it can be found that the reaction temperature of the real-time fluorescent RPA reaction can be between 35 ℃ and 40 ℃, so that the method can be applied to portable detection equipment, and the detection of vibrio vulnificus is not required to be limited to a laboratory or a condition requiring a professional detection environment.
The method for rapidly detecting Vibrio vulnificus in clinical blood described above is examined below in combination with practical circumstances.
1. Materials and methods
1.1 strains and culture conditions
The 21 strains involved in the invention include 3 strains of Vibrio vulnificus, 5 other strains of Vibrio and 13 other strains. The reference strain (Ref) is purchased to China general microbiological culture Collection center or China Marine microbiological culture Collection center. Other strains were isolated from clinical specimens or aquaculture water and confirmed by 16S rRNA sequencing. Vibrio and Listonella anguillarum were cultured in 2216E medium, and the remaining strains were cultured in LB broth. Each strain was incubated on a shaker at 37 ℃ overnight for 18 h.
TABLE 1 related strains are involved in this study
1.2 extraction of genomic DNA and construction of plasmid standards
Genomic DNA of each strain was extracted with a special bacterial genomic DNA extraction reagent (Omega Bio-tek, Inc., Norcross, USA). The quantity and quality of DNA of each extracted strain (A260/A280) was measured using a nucleic acid concentration measuring apparatus Nanodrop ND-1000(Thermo Fisher Scientific, Waltham, MA USA). The ratio of A260/A280 of DNA samples of various strains is 1.8-2.0, and the samples are stored in a refrigerator at-20 ℃.
To obtain a standard plasmid, we amplified a 498bp fragment of the Vibrio vulnificus vvhA gene (Genebank accession No.: KC 821520.1; position: 375-872bp) using the primer pair vvhA-F and vvhA-R (Table 2). The amplification conditions were:
25 μ L of 2 XGoldstar TaqMan mix, 1 μ L (10 μ M) of each primer, 22 μ L ddH2O, 2.5 μ L genomic template, amplification cycle conditions: denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30s, denaturation at 52 ℃ for 30s, and denaturation at 72 ℃ for 45s for 35 cycles, and extension at 72 ℃ for 7 min. The amplification product was introduced into the PMD19-T vector and sequenced to confirm correct insertion of the target gene. Standard plasmids were quantitated using a NanoDrop ND-1000 spectrophotometer (Thermo Science, USA). Plasmid copy number calculation was as described previously. The standard plasmid prepared was then run from 100-106Copies/. mu.L were serially diluted and stored at-20 ℃.
TABLE 2 primers and probes designed in this study
FAM-dT,6 carboxyfluorescein labeled thymidine nucleotide; THF, tetrahydrofuran spacer; BHQ1-dT, quenching group, C3-spacer, C3 spacer modified blocking group
1.3 real-time fluorescent PCR detection of Vibrio vulnificus
For real-time PCR of Vibrio vulnificus, reference was made to the relevant literature and some minor modifications were made to the reaction conditions. Briefly, real-time PCR was performed on a Roche480 thermocycler (Roche, USA) using the primers P-vvhA-F and P-vvhA-R (Table 2). The reaction system contained 12.5. mu.L of 2 XSSYGB Green I mix, 0.6. mu.L of each primer (10. mu.M), 2. mu.L of serially diluted genomic DNA (or 2. mu.L of non-nucleic acid water as a blank). The reaction conditions are as follows: denaturation at 95 ℃ for 10min, then at 95 ℃ for 15s and 60 ℃ for 90s for 40 cycles.
1.4 Condition optimization for real-time fluorescent RPA detection of Vibrio vulnificus
4 and 1 fluorescent probes (Table 2) were designed for each of the upstream and downstream primers in the conserved region of vvhA gene and synthesized by Shanghai Biosynthesization Ltd. The optimal primer pair is screened out by a twist Amp Exo kit for the subsequent optimization of a reaction system. The real-time fluorescent RPA reaction conditions are as follows: the 20. mu.L reaction system included: 11.8. mu.L buffer, 4.48. mu.L deionized water, 0.84. mu.L (10. mu.M) of each of the upstream and downstream primers, 1. mu.L of buffer, 1. mu.L of deionized water, and 1. mu.L of the upstream and downstream primers2+0.24. mu.L of probe (10. mu.M) and 0.8. mu.L of standard plasmid. The DNA template was replaced with deionized water as a negative control. Fluorescent signals are collected by a Roche fluorescent quantitative PCR instrument, and the reaction temperature of the real-time fluorescent RPA is screened by respectively setting three reaction temperatures of 37 ℃,38 ℃ and 39 ℃.
1.5 comparative analysis of specificity and sensitivity of real-time fluorescent RPA and PCR detection of Vibrio vulnificus
To evaluate the specificity of real-time fluorescent RPA detection of vibrio vulnificus, real-time fluorescent quantitation of RPA was performed to amplify the strains listed in table 1 (including vibrio vulnificus and other non-vibrio vulnificus), respectively. To analyze the sensitivity of real-time fluorescent RPA, 10-fold dilutions of vibrio vulnificus genomic DNA and standard plasmid were prepared, respectively. The content of genome DNA is within the range of 10 ng-0.1 pg as a template, a regression curve is established in each reaction (20 mu L), and real-time fluorescence quantitative analysis is carried out. Fluorescent quantitative PCR was performed using genomic DNA of the same dilution series. All ofThe tests were repeated 3 times. Meanwhile, plasmid standard products with different concentration gradients are taken as templates (10)0-106copies/. mu.L), real-time RPA detection was performed, 8 sets of each concentration gradient in parallel, for analysis.
1.6 detection of plasma samples of artificially infected Vibrio vulnificus
In order to evaluate the detection effect of the real-time fluorescence RPA method on vibrio vulnificus in clinical samples, the diagnosis effect of the real-time fluorescence quantitative RPA method and the real-time fluorescence quantitative PCR method on artificially polluted plasma samples is compared. Plasma samples will be prepared at different concentrations (1.2X 10) after being negative by real-time fluorescent quantitative PCR detection01.2X 106 CFU/mL) of Vibrio vulnificus 100. mu.L into 900. mu.L of plasma samples which are indeed considered negative, plasma samples after infection with Vibrio vulnificus at different concentrations were formed. Then, DNA was extracted from each plasma sample using a bacterial DNA kit (Omega, USA), and amplification detection was performed by the subsequent real-time fluorescent quantitative RPA method or real-time fluorescent quantitative PCR method.
1.7 statistical analysis
In order to analyze the sensitivity, the amplification results of the real-time fluorescence RPA method and the real-time fluorescence PCR method are respectively subjected to linear regression. In order to further improve the regression accuracy, the real-time fluorescence RPA is further subjected to second-order polynomial regression on the basis of the original linear regression. All regressions were calculated in Microsoft Excel (Microsoft, USA). The data obtained with 6 concentration gradients in the standard plasmid were subjected to probit regression analysis using the "glm" software package of r. to obtain the copy number corresponding to the real-time fluorescent RPA method for detecting vibrio vulnificus with an overall accuracy of 95%.
2. Results of the experiment
2.1 determination of primer pairs and reaction conditions for real-time fluorescent RPA detection of Vibrio vulnificus
Combinations of primer pairs and probes from set 7 of Table 2 were screened. All combinations were amplified differently in 20 minutes from the DNA of Vibrio vulnificus in the reaction system. In comparison, the primer combination (RPA-F1/RPA-R4) produced the strongest fluorescent signal in the shortest time (FIG. 1). Therefore, we selected this primer set for subsequent Vibrio vulnificus detection with associated real-time fluorescent RPA. For the reaction temperature, we set three reaction temperatures of 37 ℃,38 ℃ and 39 ℃ and the results show that real-time fluorescent RPA has better amplification effect at 38 ℃ than at 37 ℃ and 39 ℃ (fig. 2).
2.2 specificity analysis of real-time fluorescent RPA detection of Vibrio vulnificus
Real-time fluorescence quantitative RPA is used for respectively amplifying the genome DNA of vibrio vulnificus and other bacteria. As a result, as shown in FIG. 3, no cross reaction with DNA of other bacterial strains was observed. In fact, a positive signal is only produced when one of the three Vibrio vulnificus species is detected. This indicates that the primer/probe combination we selected is the specific primer/probe combination for Vibrio vulnificus in this study.
2.3 sensitivity analysis of real-time fluorescent RPA detection of Vibrio vulnificus
The regression analysis is respectively carried out on the data of detecting vibrio vulnificus with different concentration gradients by real-time fluorescence RPA and real-time fluorescence PCR. The results show that the logarithmic DNA concentration of real-time fluorescence PCR and real-time fluorescence RAP detection is linearly related to CQ value, and the related coefficients are respectively R20.9859 and R20.9227 (fig. 4A and B). Detection with real-time fluorescent RPA 106～101Copies/. mu.L of the standard plasmid, the logarithmic copy number is linearly related to the CQ value, the correlation coefficient R20.8386. To further improve the regression accuracy, quadratic polynomial regression was performed on real-time RPA. As expected, the correlation coefficient R of logDNA concentration to CQ value2To 0.9962. Similarly, the correlation coefficient R of the logarithmic copy number of the standard plasmid to the CQ value2To 0.9816. In terms of sensitivity, FIG. 4 vividly shows that the lowest detection limit of real-time fluorescent RPA is consistent with the real-time fluorescent PCR reaction, both reaching 0.1 pg/. mu.L (FIGS. 4A and 4B). Meanwhile, according to the results shown in FIG. 4C, the sensitivity of the standard plasmid to real-time RPA was 10 copies/. mu.L. Probability regression prediction is carried out by using 8 times of repeated experiment results of different standard plasmid concentrations in the research, and the standard plasmid copy concentration of the real-time fluorescence RPA detection Vibrio vulnificus LOD is about 1.58 multiplied by 10 under the condition that 95 percent of LOD is present2Copy (fig. 5).
2.4 detection evaluation analysis of artificially infected blood samples
In order to evaluate the practical application of fluorescent real-time RPA detection vibrio in clinic, blood samples with different concentrations are artificially infected, and the positive detection results of plasma samples after the real-time RPA method and the real-time PCR method are compared. The results showed that the real-time fluorescent RPA detection results were consistent with the real-time fluorescent PCR detection results (table 3). However, real-time RPA has an overwhelming advantage in saving time. At a low level of 1.2X 102At the added concentration of cfu/mL, the real-time RPA can detect Vibrio vulnificus within 16min, while the real-time fluorescence PCR detection requires about 45 min. The detection time decreased with increasing cell number. The real-time fluorescent RPA method at higher concentrations can obtain positive results at a minimum of 3.44min (table 3). These results strongly suggest the significant advantage of real-time RPA technology in terms of rapidity and sensitivity.
TABLE 3 comparison of real-time fluorescent RPA and real-time PCR detection of artificially infected samples.
In general, the invention is a method for rapidly detecting vibrio vulnificus in clinical plasma samples based on a fluorescence real-time RPA technology. The method has high specificity, sensitivity and rapidness as low as 1.2 × 102Under the infection concentration of cfu/mL, the real-time fluorescence RPA method can detect the vibrio vulnificus within 16min, while the traditional real-time fluorescence PCR detection needs about 45 min. And the detection time decreased with increasing cell number. At higher concentrations (1.2X 10)6cfu/mL) real-time fluorescent RPA method can obtain positive results within 3.44min at the shortest. Meanwhile, the real-time RPA method of our invention can be combined with portable equipment, and in the clinical diagnosis of the vibrio vulnificus outbreak field, especially under the condition of limited resources, the real-time RPA method can be used for monitoring the infection of the vibrio vulnificusA very useful method.
It is to be understood that the described embodiments are merely a few embodiments of the invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Claims (9)
1. A method for rapidly detecting Vibrio vulnificus in clinical blood comprises
Adding an upstream primer, a downstream primer and a fluorescent probe to a vvhA gene conserved region in the DNA of a strain to be detected, and carrying out a fluorescent RPA reaction in real time.
2. The method for rapidly detecting Vibrio vulnificus in clinical blood according to claim 1, wherein the upstream primer is RPA-F1, wherein the sequence of RPA-F1 is:
CGGCAAAGTAGGTGCGGAAGTGAACAAAGA。
3. the method for rapidly detecting Vibrio vulnificus in clinical blood according to claim 1, wherein the downstream primer is RPA-R4, and the sequence of RPA-R4 is:
CTCAATGATGAACGGTTGTTGATGCGATAG。
4. the method for rapidly detecting Vibrio vulnificus in clinical blood according to claim 1, wherein the fluorescent Probe is RPA-Probe, and the sequence of the RPA-Probe is:
TGGCGAAGTCAGTGGCTCATTTACCTACAAC[FAM-dT]a[THF]bC[BHQ1-dT]cCGAAGACCTTGGTGTT(C3 Spacer)d。
5. the method as claimed in claim 1, wherein the real-time fluorescent RPA reaction system comprises 11.8 μ L buffer, 4.48 μ L deionized water, and upstream and downstream primers 0.84 μ L and 1 μ L of deionized water in 20 μ L of the reaction system2+0.24 μ L ofProbes and 0.8. mu.L of standard plasmid.
6. The method for rapid detection of Vibrio vulnificus in clinical blood as claimed in claim 1, wherein the temperature for performing the fluorescent RPA reaction in real time is 35-40 ℃.
7. The method for rapid detection of Vibrio vulnificus in clinical blood as claimed in claim 6, wherein the temperature for performing the fluorescent RPA reaction in real time is 38 ℃.
8. The method of claim 5, wherein the concentration of the upstream and downstream primers is 10 μ M and the concentration of the fluorescent probe is 10 μ M in the fluorescent RPA reaction system.
9. The method of claim 5, wherein the standard plasmid is prepared by amplifying 498bp of vvhA gene in DNA fragment of the test strain with upstream primer vvhA-F and downstream primer vvhA-R under the conditions of 25 μ L of 2 XGoldstar TaqMan mixture, 1 μ L (10 μ M) of each primer, 22 μ L of ddH2O, and 2.5 μ L of genome template, and the amplification cycle is as follows: denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30s, denaturation at 52 ℃ for 30s, and elongation at 72 ℃ for 45s for 35 cycles, and extension at 72 ℃ for 7min, wherein the product obtained after the amplification cycle is introduced into PMD19-T vector and sequenced to confirm the insertion of target cells.
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