CN117867174A - One-step PCR detection method for white spot syndrome virus - Google Patents
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Abstract
The invention discloses a PCR primer for detecting white spot syndrome virus (White spot syndrome virus, WSSV), the sequence of which is shown in SEQ ID NO. 2-3, the primer can amplify 196bp fragments of main envelope protein VP19 genes of the WSSV virus, the invention also discloses a one-step PCR method for detecting the WSSV, the method can specifically detect the WSSV, and the method is a rapid and extremely high-sensitivity detection method, compared with a nested PCR standard detection method, the amplification reaction time is shortened by 66.13%, and the minimum detection limits of theoretical and diagnostic sensitivity are respectively 1.83 copies/mu L and 0.82 fg/mu L, which are respectively improved by 100 and 1000 times.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a PCR detection primer for white spot syndrome virus (White spot syndrome virus, WSSV), and a one-step PCR detection method for WSSV.
Background
WSSV is a cyclic double-stranded DNA virus with a capsule, can infect various economic crustaceans such as procambarus clarkia, penaeus vannamei and the like, is a crustacean virus which is seriously damaged so far, and is listed in the "aquatic animal health act" issued by the world animal health organization (World Organization for Animal Health, WOAH), and the "first, second and third animal epidemic disease species directory" issued by the agricultural rural department determines the virus as the second epidemic disease of aquatic animals.
Besides the broad spectrum of infected hosts, WSSV is also very complex in its transmission pathways, including intraspecies horizontal transmission, intraspecies vertical transmission and interspecies transmission. In addition, studies have shown that WSSV exists in hosts during a latent infection period, and rapidly bursts once the infection threshold is reached, resulting in mortality rates as high as 100% within 7-10 days. These infection and morbidity characteristics make WSSV difficult to treat, and the current intensive studies on its pathogenic mechanism remain inadequate, impeding the development of effective therapies. Therefore, prevention is one of the key measures currently available, and establishing a rapid, convenient and sensitive detection method is also an important point of research.
The detection method of WSSV mainly comprises histopathological diagnosis, electron microscope observation, immunological detection, nucleic acid molecule detection and the like. Nucleic acid molecule detection is currently the most commonly used method for detecting WSSV, wherein the nested polymerase chain reaction (nested PCR) has high sensitivity and high specificity. The nested PCR detection method developed in 1996 is incorporated into the WSSV detection standard of WOAH in 2009, but the method needs to sequentially and respectively perform two PCR reactions by using two pairs of primers, is complex in operation, takes long time, and is easy to cause nonspecific amplification to influence the accuracy of results. In contrast, fluorescent quantitative PCR is more sensitive, takes less time, and a single virus copy is minimally detectable, but it requires a specific fluorescent kit and an expensive fluorescent quantitative PCR instrument, and its application in practical production is limited by high cost and operational expertise. The one-step PCR method can rapidly amplify WSSV in actual production, realizes mass sample detection in a short time and has low cost, but the method has the lowest sensitivity detection limit of 10 at present 2 The sensitivity is still low for each viral copy. Accordingly, the present invention provides a WSSV-The step-type PCR detection method has the advantages of simple experimental operation, short time consumption, strong specificity, high sensitivity and high detection rate, can be applied to the rapid large-scale detection of WSSV in economic crustaceans such as procambarus clarkia, shows wide practical production applicability, and can enable white spot syndrome to be found early and prevented early.
Disclosure of Invention
The invention aims to provide a PCR primer for detecting WSSV and application thereof.
To achieve the above objective, the applicant designed a pair of specific primers V1 based on the WSSV virus genome (Accession No. AF332093) major envelope protein VP19 gene, wherein the sequences of the primers V1 are shown as SEQ ID NO. 2-3, the primers can amplify a 196bp fragment of the VP19 gene, and the nucleotide sequence of the VP19 gene is shown as SEQ ID NO. 1.
The primer can only amplify target fragments from genomic DNA of WSSV virus, and has no amplification product for other common aquatic pathogens, so the primer can be used for specific detection of WSSV. Other aquatic pathogens in the present invention include four viruses (IHHNV, CMNV, RGV and SVCV) and five bacteria (Aeromonas veronii, aeromonas caviae, citrobacter freundii, citrobacter braakii, acinetobacter junii).
The primer has the advantage of high amplification efficiency, can amplify target fragments from a very small amount of samples, and can remarkably improve the sensitivity of PCR detection.
Therefore, the PCR primer provided by the invention can be applied to the preparation of a diagnostic kit for procambarus clarkia white spot syndrome.
The invention further provides a kit which is a PCR kit for diagnosing procambarus clarkia white spot syndrome, and the kit contains the PCR primer.
Further, the kit also contains a PCR reaction mixture and ddH 2 O and a positive control, wherein the positive control is genomic DNA extracted from gill tissue of procambarus clarkia artificially infected with WSSV.
The invention also provides a method for detecting WSSV, in particular to a one-step PCR method for detecting WSSV, which comprises the following steps:
1) Extracting genome DNA of a sample to be detected;
2) Amplifying by using the extracted genome DNA as a template and the PCR primer;
3) And (5) detecting the PCR amplification product by agarose gel electrophoresis.
Preferably, the amplification reaction system is 2x Taq MasterMix 10. Mu.L, 1. Mu.L of DNA template, 1. Mu.L of each of the upstream primer and the downstream primer, ddH 2 O 7μL。
Further preferably, the amplification reaction procedure is a 95℃pre-denaturation for 4min; denaturation at 95℃for 30s, annealing at 60℃for 30s, extension at 72℃for 30s, 30 cycles total; finally, the extension is carried out at 72 ℃ for 5min.
Compared with the existing method, the method has the advantages of rapidness, convenience, low cost and simple operation, and has high sensitivity and detection rate, so that the method has high application value in actual production, wherein the detection theoretical sensitivity of the method to standard plasmids can reach single copy (1.83 copies/. Mu.L), the diagnostic sensitivity to clinical samples is 0.82fg, and the detection rate to clinical samples is 90.48%.
The method provided by the invention can be applied to disease diagnosis of procambarus clarkia white spot syndrome, and can be applied to WSSV virus laboratory screening and identification aiming at non-disease diagnosis.
Compared with the prior art, the invention has the following advantages:
1) The invention designs a pair of primers V1 (V1-F and V1-R) based on WSSV main envelope protein VP19, and is a new target sequence selection.
2) The primer designed by the invention can be applied to specific detection of WSSV of economic crustaceans such as procambarus clarkia.
3) The one-step PCR detection method of procambarus clarkia provided by the invention has theoretical sensitivity up to single copy (1.83 copies/. Mu.L) in standard plasmid template detection, is consistent with the sensitivity of fluorescent quantitative PCR, and is 100 times higher than the sensitivity of WOAH nest PCR.
4) The one-step PCR detection method for procambarus clarkia provided by the invention has the diagnostic sensitivity of 0.82fg in clinical sample detection, and the sensitivity of WOAH nest PCR is 0.82pg, which are different by 1000 times.
5) The one-step PCR detection method of procambarus clarkia provided by the invention has the detection rate of 90.48% in clinical sample detection, and the detection rate is 10% higher than that of WOAH nested PCR.
6) The one-step PCR detection method for procambarus clarkia provided by the invention really has the advantages of low cost, easiness in operation, quick detection, high sensitivity and the like, and can be applied to early diagnosis of procambarus clarkia WSSV or quick detection of large-scale quarantine samples.
Drawings
FIG. 1 is a one-step PCR amplification map in example 1. M indicates DNA marker2000,1 indicates positive control, and 2 indicates blank control.
FIG. 2 is a schematic diagram of a one-step PCR assay for WSSV specific amplification in example 2. M refers to DNA marker2000,1 to positive control, 2 to infectious subcutaneous and hematopoietic necrosis virus (IHHNV), 3 to candidiasis virus (CMNV), 4 to frog iridovirus (RGV), 5 to Spring Viremia of Carp Virus (SVCV), 6 to Aeromonas veronii,7 to Aeromonas caviae,8 to Citrobacter freundii,9 to Citrobacter braakii,10 to Acinetobacter junii,11 to blank control.
FIG. 3 shows the result of theoretical sensitivity amplification in example 3. A is a one-step PCR method; b is the first round of WOAH nest type PCR; c is WOAH nested PCR second round. M is DNA marker2000,1-11 are 1.83×10 respectively 10 -1.83×10 0 The copies/. Mu.L of the gradient diluted V1 template, 12 refers to the blank.
FIG. 4 is an amplification curve and a standard curve of the fluorescent quantitative PCR assay of example 3. A is a standard curve; b is an amplification curve; NC is a blank.
FIG. 5 shows the results of diagnostic sensitivity amplification in example 4. A is a one-step PCR method; b is the first round of WOAH nest type PCR; c is WOAH nested PCR second round. M is DNA marker2000, 1-10 are 82ng, 8.2ng, 0.82ng, 82pg, 8.2pg, 0.82pg, 82fg, 8.2fg, 0.82fg, 82ag,11 are blank controls.
Description of the sequence Listing:
SEQ ID NO.1: is the main envelope protein VP19 gene sequence of WSSV virus genome (Access No. AF332093);
SEQ ID NO.2: v1 upstream primer in one-step PCR detection method;
SEQ ID NO.3: v1 downstream primer in one-step PCR detection method;
SEQ ID NO.3: an outer primer P1 upstream primer in the WOAH nest PCR detection method;
SEQ ID NO.4: an outer primer P1 downstream primer in the WOAH nest PCR detection method;
SEQ ID NO.5: an inner primer P2 upstream primer in the WOAH nest PCR detection method;
SEQ ID NO.6: the inner primer P2 downstream primer in the WOAH nested PCR detection method.
Detailed Description
The technical scheme of the present invention will be described in detail with reference to specific embodiments. It should be understood that these examples are for the purpose of illustrating the invention only and are not intended to limit the scope of the invention. Other embodiments, which can be obtained by those skilled in the art without making inventive efforts, are within the scope of the present invention based on the following examples.
The experimental methods used in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The applicant obtained the DNA sequence of the WSSV virus genome (Accession No. af 332093) major envelope protein VP19 gene from GenBank, designed specific primers using Oligo 7 biological software, and optimally designed specific primer V1 based on PCR primer design principles (see table 1 below).
TABLE 1 specific PCR primers developed by the present invention
Then, based on the specific primer V1, a one-step PCR detection method for the procambarus clarkia WSSV is established, and the specific implementation comprises the following steps:
step 1, collecting gill tissues of procambarus clarkia, and extracting sample genome DNA (deoxyribonucleic acid) as a template by using a universal animal tissue DNA extraction kit;
step 2, using the extracted genome DNA as a template, and using the PCR primer in the table 1 for amplification, wherein a one-step PCR reaction system is as follows: the reaction volume was 20. Mu.L, 2x Taq MasterMix 10. Mu.L of the DNA template 1. Mu.L, 1. Mu.L of each of the primers V1-F and V1-R, ddH 2 O7 μl; the one-step PCR reaction procedure was: pre-denaturation at 95 ℃ for 4min; denaturation at 95℃for 30s; annealing at 60 ℃ for 30s, extending at 72 ℃ for 30s, and performing 30 cycles; finally, the temperature is 72 ℃ and the extension is carried out for 5min;
step 3, judging the detection result: mu.L of the one-step PCR reaction product was mixed with 1. Mu.L of EB dye and spotted on 2% agarose gel, and the result was observed by a gel imaging system using a gel electrophoresis apparatus for 22min under 130V voltage.
Finally, based on the standardized DNA template, performing sensitivity difference evaluation on the one-step PCR, WOAH nest PCR and fluorescent quantitative PCR detection methods, and carrying out clinical verification of the conventional PCR detection method, wherein the specific implementation comprises the following steps:
step 1, extracting DNA of an experimental sample: collecting gill tissues of procambarus clarkia, and extracting sample genome DNA (deoxyribonucleic acid) as a template by using a universal animal tissue DNA extraction kit;
step 2, preparing a V1 standard plasmid gradient template: sequentially carrying out PCR amplification, gel recovery, connection transformation plating, bacterial liquid PCR verification and Sanger sequencing by using a specific primer V1 to obtain an accurate pMD19T-V1 plasmid, and carrying out 10-fold dilution;
step 3, preparing a P1 standard plasmid gradient template: synthesizing an outer primer P1 and an inner primer P2 of WOAH nest PCR, wherein the primer sequences are shown in SEQ ID NO. 4-7; sequentially carrying out PCR amplification, gel recovery, connection transformation plating, bacterial liquid PCR verification and Sanger sequencing by using an outer primer P1 to obtain an accurate pMD19T-P1 plasmid, and carrying out 10-fold dilution;
step 4, sensitivity theory assessment: performing one-step PCR and fluorescent quantitative PCR by using the standard plasmid pMD19T-V1 gradient template in the step 2, performing WOAH nested PCR by using the standard plasmid pMD19T-P1 gradient template in the step 3, and comparing the lowest detected virus copy numbers of the three methods according to the results;
step 5, sensitivity diagnosis and evaluation: using the positive detection sample in the step 1, diluting by 10 times, respectively performing one-step PCR and WOAH nest PCR detection on the gradient template, and comparing the lowest detection concentration of the two methods according to the result;
step 6, clinical verification: collecting procambarus clarkia samples, extracting sample genome DNA as a template according to the step 1, respectively performing one-step PCR and WOAH nest PCR detection, and comparing the detection rates of the two methods according to the result.
Example 1 one-step PCR detection method establishment
The invention firstly obtains the DNA sequence of the main envelope protein VP19 gene of WSSV virus genome (AF 332093.3) from GenBank, and designs a specific primer V1 by utilizing Oligo 7 biological software. Primers were synthesized by the company and diluted to a PCR concentration of 10. Mu.M.
PCR amplification was performed using the positive control and blank control templates prepared. The PCR reaction system was 20. Mu.L, 2x Taq MasterMix 10. Mu.L of the DNA template, 1. Mu.L of each of the primers V1-F and V1-R, ddH 2 O7. Mu.L. After the annealing temperature is optimized, the running program of the optimal PCR instrument is as follows: 95 ℃ for 4min;95 ℃ for 30s;30 s at 60 ℃;30 s at 72 ℃;30 cycles; and at 72℃for 5min. Gel imaging results showed: the V1 primer was able to amplify the band of interest in the positive WSSV template DNA (FIG. 1).
Positive control: collecting gill tissues of procambarus clarkia artificially infected with WSSV, and extracting sample genome DNA (deoxyribonucleic acid) as a positive DNA template by using a universal animal tissue DNA extraction kit;
blank control: ultrapure water or sterile water is used as a template.
Example 2 one-step PCR detection method specificity analysis
Positive controls, blank controls, four viruses (IHHNV, CMNV, RGV and SVCV) and five bacteria (Aeromonas veronii, aeromonas caviae, citrobacter f were usedReundii, citrobacter braakii, acinetobacter junii) as a template source for PCR amplification reactions. The PCR reaction system was 20. Mu.L, 2x Taq MasterMix 10. Mu.L of the DNA template, 1. Mu.L of each of the primers V1-F and V1-R, ddH 2 O7. Mu.L. The optimal PCR instrument operation program is as follows: 95 ℃ for 4min;95 ℃ for 30s;30 s at 60 ℃;30 s at 72 ℃;30 cycles; and at 72℃for 5min. Gel imaging results showed: the V1 primer was able to specifically amplify the band of interest only in WSSV positive control template DNA (FIG. 2).
Example 3 theoretical sensitivity assessment of one-step PCR detection method
Preparing a standard plasmid template: the one-step PCR primer V1, the WOAH nested PCR outer primer P1 and the inner primer P2 were synthesized by the company and diluted to a PCR reaction concentration of 10. Mu.M. The positive WSSV template DNA was amplified by PCR using V1 and P1, respectively, the amplified products were gel recovered, TA cloned and ligated to pMD19T vector, and the standard plasmids pMD19T-V1 and pMD19T-P1 were determined by Sanger sequencing. The concentration of the standard plasmid pMD19T-V1 was 57.9 ng/. Mu.L, and the corresponding copy concentration was 1.83X 10 in terms of conversion 10 COPies/. Mu.L. Diluted by 10 times ratio to obtain 1.83×10 respectively 0 -1.83×10 10 COPIES/. Mu.L.etc.11 gradient concentration templates. The concentration of the standard plasmid pMD19T-P1 was 107.9 ng/. Mu.L, and the corresponding copy concentration was 2.38X10% by conversion 10 COPies/. Mu.L. Diluted by 10 times ratio to obtain 2.38X10 respectively 0 -2.38×10 10 COPIES/. Mu.L.etc.11 gradient concentration templates.
Theoretical sensitivity evaluation of one-step PCR detection method: the diluted pMD19T-V1 template was used to carry out PCR amplification of primer V1 in a PCR reaction system of 20. Mu.L, wherein 2x Taq MasterMix10. Mu.L of the DNA template, 1. Mu.L of each of the primers V1-F and V1-R, and ddH 2 O7. Mu.L. The optimal PCR instrument operation program is as follows: 95 ℃ for 4min;95 ℃ for 30s;30 s at 60 ℃;30 s at 72 ℃;30 cycles; and at 72℃for 5min. Gel imaging results showed: the lowest detection concentration for one-step PCR detection using primer V1 was 1.83 copy/. Mu.L (FIG. 3A).
Theoretical sensitivity evaluation of WOAH nested PCR detection method: the diluted pMD19T-P1 template is used for carrying out the first round of PCR amplification reaction of the outer primer P1, the PCR reaction system is 20 mu L, wherein 2x Taq MasterMix10 mu L of the outer primer P1 and 1 mu L of the DNA template are used for carrying out the primer1. Mu.L each of the substances P1-F and P1-R, ddH 2 O7. Mu.L. The optimal PCR instrument operation program is as follows: 95 ℃ for 4min;95 ℃ for 30s;57 ℃ for 30s;72 ℃ for 1min;40 cycles; and at 72℃for 5min. The total duration of the PCR amplification reaction was 135min. The second round of PCR amplification reaction of the inner primer P2 was performed using 50-fold dilution of the P1 amplification product as a template, the PCR reaction system was 20. Mu.L, wherein 2xTaq MasterMix 10. Mu.L, 1. Mu.L of the DNA template, 1. Mu.L of each of the primers P2-F and P2-R, ddH 2 O7. Mu.L. The optimal PCR instrument operation program is as follows: 95 ℃ for 4min;95 ℃ for 30s;30 s at 55 ℃;30 s at 72 ℃;40 cycles; and at 72℃for 5min. The total duration of the PCR amplification reaction was 113min. Gel imaging results showed: the lowest detection concentration of the first round of WOAH nested PCR was 2.83×10 3 The lowest detection rate of the second round PCR was 2.83×10, copies/. Mu.L (FIG. 3B) 2 COPIES/. Mu.L (FIG. 3C).
Theoretical sensitivity evaluation of fluorescent quantitative PCR detection method: performing fluorescent quantitative PCR amplification reaction of the primer V1 by using the diluted pMD19T-V1 template, wherein the PCR reaction system is 20 mu L, and the fluorescent quantitative PCR amplification reaction comprisesPremix Ex TaqTMII 10. Mu.L, 1. Mu.L of DNA template, 0.4. Mu.L each of primers V1-F and V1-R, ddH 2 O8.2. Mu.L. The PCR instrument operation program is as follows: 3min at 95 ℃;95 ℃ for 10s; 30s at 60 ℃;40 cycles. The results show that: standard curve of pMD19T-V1 plasmid standard, when Cq<Has a standard S-shaped amplification curve at 35 (correlation coefficient r 2 0.9938, slope-3.0199, amplification efficiency 114%) and a fluorescence signal of 1.83×10 could be detected 0 -1.83×10 10 And a copy of WSSV. The lowest detection concentration for fluorescent quantitative PCR detection using V1 primer was 1.83 copy/. Mu.L (FIG. 4).
In summary, the theoretical sensitivity of the three WSSV detection methods is: one-step PCR = fluorescent quantitative PCR > WOAH nested PCR.
Example 4 evaluation of diagnostic sensitivity of one-step PCR detection method
Preparing a standard detection sample template: the V1 primer is used for carrying out one-step PCR detection on unknown procambarus clarkia clinical samples, positive samples are determined, and the DNA concentration is determined to be 82 ng/. Mu.L. Positive sample DNA was diluted 10-fold, and the gradient concentrations were 82 ng/. Mu.L, 8.2 ng/. Mu.L, 0.82 ng/. Mu.L, 82 pg/. Mu.L, 8.2 pg/. Mu.L, 0.82 pg/. Mu.L, 82 fg/. Mu.L, 8.2 fg/. Mu.L, 0.82 fg/. Mu.L, 82 ag/. Mu.L, respectively.
Diagnostic sensitivity assessment of one-step PCR detection method: the diluted clinical sample template is used for carrying out PCR amplification reaction of the primer V1, the PCR reaction system is 20 mu L, wherein 2x Taq MasterMix10 mu L of the primer V1-F and the primer V1-R are respectively 1 mu L and ddH, and 1 mu L of the DNA template 2 O7. Mu.L. The optimal PCR instrument operation program is as follows: 95 ℃ for 4min;95 ℃ for 30s;30 s at 60 ℃;30 s at 72 ℃;30 cycles; and at 72℃for 5min. Gel imaging results showed: the lowest detection concentration for one-step PCR detection using primer V1 was 0.82 fg/. Mu.L.
Diagnosis sensitivity evaluation of WOAH nested PCR detection method: the first round of PCR amplification of the outer primer P1 was performed with the diluted template of the clinical sample, the PCR reaction system was 20. Mu.L, 2x Taq MasterMix 10. Mu.L, 1. Mu.L of the DNA template, 1. Mu.L of each of the primers P1-F and P1-R, ddH 2 O7. Mu.L. The optimal PCR instrument operation program is as follows: 95 ℃ for 4min;95 ℃ for 30s;57 ℃ for 30s;72 ℃ for 1min;40 cycles; and at 72℃for 5min. The second round of PCR amplification reaction of the inner primer P2 was performed using 50-fold dilution of the P1 amplification product as a template, the PCR reaction system was 20. Mu.L, wherein 2x Taq MasterMix 10. Mu.L, 1. Mu.L of the DNA template, 1. Mu.L of each of the primers P2-F and P2-R, ddH 2 O7. Mu.L. The optimal PCR instrument operation program is as follows: 95 ℃ for 4min;95 ℃ for 30s;30 s at 55 ℃;30 s at 72 ℃;40 cycles; and at 72℃for 5min. Gel imaging results showed: the lowest detection concentration of the first round of WOAH nested PCR was 82 pg/. Mu.L, and the lowest detection rate of the second round of PCR was 0.82 pg/. Mu.L.
In summary, the practical diagnostic sensitivity of one-step PCR is far better than that of WOAH nested PCR, which is 1000 times.
Example 5 one-step PCR detection method clinical sample application
84 samples of procambarus clarkia, which are randomly extracted in clinic, are extracted from gill tissue DNA as templates, and positive control and blank control are simultaneously set. The samples were tested for WSSV by the one-step PCR and WOAH nested PCR methods established in this study, respectively, and 76 positive samples were co-tested (Table 2). The WSSV detection rate of the one-step PCR method is 90.48%; the detection rate of the first round of the WOAH nest PCR method is 70.24%, and the detection rate of the second round can be improved to 82.14%. The results show that in the actual clinical sample detection, the one-step PCR still has higher sensitivity and detection rate than the WOAH nested PCR.
TABLE 2 clinical sample detection results
The foregoing is merely a preferred embodiment of the present application and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present application and are intended to be comprehended within the scope of the present application.
Claims (7)
1. A PCR primer for detecting white spot syndrome virus (White spot syndrome virus, WSSV) has a sequence shown in SEQ ID NO. 2-3, and can amplify 196bp fragment of WSSV virus main envelope protein VP19 gene.
2. The use of the PCR primer of claim 1 in the preparation of a procambarus clarkia white spot syndrome diagnostic kit.
3. A procambarus clarkia white spot syndrome diagnostic kit comprising the PCR primer of claim 1.
4. A kit according to claim 3, further comprising a PCR reaction mixture and ddH 2 O。
5. A one-step PCR method for detecting WSSV for non-diagnostic purposes, comprising the steps of:
1) Extracting genome DNA of a sample to be detected;
2) Amplifying using the PCR primer of claim 1 using the extracted genomic DNA as a template;
3) And (5) detecting the PCR amplification product by agarose gel electrophoresis.
6. The one-step PCR method according to claim 5, wherein: the amplification reaction system is 2x Taq MasterMix10 [ mu ] L, the DNA template is 1 [ mu ] L, and the upstream primer and the downstream primer are respectively 1 [ mu ] L and ddH 2 O 7µL。
7. The one-step PCR method according to claim 5, wherein: the amplification reaction procedure is pre-denaturation at 95 ℃ for 4min; denaturation at 95℃for 30s, annealing at 60℃for 30s, extension at 72℃for 30s, 30 cycles total; finally, the extension is carried out at 72 ℃ for 5min.
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