CN110684866A - Primer pair, kit and method for detecting chicken infectious anemia virus - Google Patents
Primer pair, kit and method for detecting chicken infectious anemia virus Download PDFInfo
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Abstract
The invention discloses a primer pair, a kit and a method for detecting chicken infectious anemia virus. The primer pair comprises CIAV-F70 shown in SEQ ID NO.1 and CIAV-R70 shown in SEQ ID NO.2, and the kit comprises the primer pair, positive standard plasmids, a real-time fluorescence quantitative PCR reagent and ddH2And O, the method takes a sample to be detected as a template, and obtains the content of the chicken infectious anemia virus in the sample to be detected according to a standard curve after performing fluorescence PCR amplification by using the primer pair, so as to realize the detection purpose. The invention designs a primer pair aiming at CIAV, can rapidly detect the CIAV by combining a fluorescence PCR quantitative method, and provides a kit applying the primer pairV research and epidemic prevention.
Description
Technical Field
The invention relates to the technical field of detection of Chicken Infectious Anemia Viruses (CIAV), in particular to a primer pair, a kit and a method for detecting the chicken infectious anemia viruses.
Background
Chicken Infectious Anemia Virus (CIAV) is a unique type of circular single-stranded (negative-stranded) DNA virus, and according to the particle size and its nucleic acid characteristics of CIAV viruses, CIAV was assigned to the family circovirus in the sixth international committee for viral classification (ICTV) in the report on vienna conference, circovirus (circovims), the same family as Porcine Circovirus (PCV) and psittacine disease virus (PBFDV). Because the CIAV has certain difference with other two viruses in terms of genome and antigen structure, in an eighth virus classification report of ICTV in 1999, CIAV is added with a new genus, circovirus (gyrovirus), and is independently classified into the new circovirus genus, and at present, CIAV is the only member of the genus. CIAV is transmitted both horizontally and vertically and is susceptible to all day-old chickens.
At present, the conventional PCR detection is mainly used for diagnosing the chicken infectious anemia, the conventional PCR is the most original technology for detecting the chicken infectious anemia at a molecular level, and the technology has certain accuracy, but the sensitivity is obviously insufficient and the repeatability is poor. Therefore, the method, the kit and the matched primer pair for rapidly detecting the CIAV have great significance.
Disclosure of Invention
The invention aims to provide a primer pair, a kit and a method for detecting chicken infectious anemia virus, which have the characteristics of high accuracy, high sensitivity and good repeatability.
In order to achieve the purpose, the invention adopts the following technical scheme:
a primer pair for detecting chicken infectious anemia viruses comprises CIAV-F70 shown in SEQ ID NO.1 and CIAV-R70 shown in SEQ ID NO. 2.
The invention also provides a kit for detecting the chicken infectious anemia virus, which comprises: the primer pair (CIAV-F70 and CIAV-R70), the positive standard plasmid, SYBR Green Premix Pro Taq real-time fluorescence quantitative PCR reagent and ddH2O; the positive standard plasmid is a recombinant plasmid obtained by connecting a DNA fragment shown by SEQ ID NO.3 with a pMD18-T vector.
The invention also provides a method for detecting the chicken infectious anemia virus, which comprises the following specific steps: taking a sample to be detected as a template, carrying out real-time fluorescent quantitative PCR amplification by using the primer pair to obtain a cycle threshold of the sample to be detected, and substituting the cycle threshold into a standard curve to obtain the content of the chicken infectious anemia virus in the sample to be detected; the standard curve is Y-3.334 lgX +39.08, where Y is the threshold for cycling and X is the copy number of the plasmid. The standard curve is 107、106、105、104And 103The 5 positive plasmids with gradient concentrations of copies/uL are used as templates and are obtained by real-time fluorescent quantitative PCR amplification by using the primer pair (CIAV-F70 and CIAV-R70). According to the standard curve, when the Ct value (cycle threshold) is less than 35, the dissolution curve has a single peak and Tm(temperature of PCR product for opening double chains) is 80.5-81.5 ℃, the sample to be detected is judged to be positive, and the sample to be detected is judged to be negative under other conditions.
Preferably, the reaction system for real-time fluorescent quantitative PCR amplification is as follows: SYBR Green Premix Pro Taq 10. mu. L, ddH2O7 mu L, CIAV-F700.5 mu L, CIAV-R700.5 mu L and template 2 mu L; the reaction procedure of real-time fluorescent quantitative PCR amplification is as follows: at 95 ℃ for 3 min; 95 ℃, 5s, 55 ℃, 15s, 40 cycles; 95 ℃, 30s, 65 ℃, 30s, 95 ℃, 30 s.
Preferably, the positiveThe plasmid was prepared by the following procedure: performing PCR amplification on infectious anemia virus genome by using the primer pair (CIAV-F70 and CIAV-R70), cloning, performing sequencing comparison to confirm that the plasmid is correct, performing plasmid extraction by using a plasmid extraction kit, calculating according to the plasmid concentration to obtain the gene copy number, and diluting to 10 times by using a 10-fold ratio0copies/uL were stored at-20 ℃ until use.
Preferably, the reaction system of the general PCR amplification is: rTaq12.5 mu L, ddH2O9.5. mu. L, CIAV-F700.5. mu. L, CIAV-R700.5. mu.L and template 2. mu.L.
Preferably, the cloning comprises steps of PCR product purification recovery, target fragment ligation and recombinant plasmid transformation.
Preferably, the specific process of this step of purifying and recovering the PCR product is as follows: and adding the PCR product into 1% agarose gel containing Goldview, performing electrophoresis for 30min, cutting a target strip under the irradiation of an ultraviolet lamp, and recovering by using a recovery kit to obtain a PCR purified product.
Preferably, the specific process of the step of connecting the target fragment is as follows: mu.L of the PCR-purified product, 0.25. mu.L of pMD18-T Vector and 2.5. mu.L of 2 XLigase Buffer (ligand Solution I) were mixed well, and then placed in a PCR apparatus to act at 16 ℃ for 4h and stored at-20 ℃ to obtain a Ligation product.
Preferably, the specific process of the step of transforming the recombinant plasmid is as follows: adding the ligation product into competent cells, uniformly mixing, carrying out ice bath for 30min, then carrying out heat shock for 90s at 42 ℃, and carrying out ice bath for 10 min; adding 400 μ L LB liquid culture medium, and culturing at 37 deg.C for 45min to obtain bacterial liquid; then 100 mu L of bacterial liquid is taken and coated on a 1.5% LB agar plate containing Amp, and inverted culture is carried out for 12-16 h under the constant temperature condition of 37 ℃; the Amp concentration is 100 mug/mL.
The invention has the beneficial effects that: the invention designs a primer pair aiming at CIAV, can rapidly detect CIAV by combining a fluorescence PCR quantitative method, and provides a kit applying the primer pair.
Drawings
FIG. 1 is a standard curve chart in example 2 of the present invention;
FIG. 2 is a graph showing the amplification curve after real-time fluorescent quantitative PCR amplification in example 3 of the present invention;
FIG. 3 is a graph showing the amplification curve after the conventional PCR amplification in example 3 of the present invention;
FIG. 4 is a graph showing the amplification curve after real-time fluorescent quantitative PCR amplification in example 4 of the present invention;
FIG. 5 is a graph showing the dissolution profile after real-time fluorescent quantitative PCR amplification in example 4 of the present invention.
Detailed Description
The concept and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments and the accompanying drawings to fully understand the objects, aspects and effects of the present invention.
Example 1:
the inventor selects the whole genome of Guangdong strain JX260426 according to the published CIAV sequence on NCBI and designs a primer pair for detecting the CIAV, which comprises CIAV-F70 shown in SEQ ID NO.1 and CIAV-R70 shown in SEQ ID NO.2, wherein the target size is 70bp, and is shown in Table 1.
TABLE 1
| Code number | Name (R) | Nucleotide sequence (5 '-3') |
| CIAV-F70 | SEQ ID NO.1 | CTTGATTACCACTACTCCCAGCCG |
| CIAV-R70 | SEQ ID NO.2 | CGTCTTGCCATCTTACAGTCTTAT |
And designs a kit for detecting CIAV, which comprises: the primer pair (CIAV-F70 and CIAV-R70), the positive standard plasmid, SYBR Green Premix Pro Taq real-time fluorescence quantitative PCR reagent and ddH2O; the positive standard plasmid is a recombinant plasmid obtained by connecting a DNA fragment shown by SEQ ID NO.3 with a pMD18-T vector.
Example 2:
firstly, preparing a plasmid standard product:
1. PCR amplification and purification of fragments of interest
Viral genomes (templates) were amplified by conventional PCR using the CIAV-F70 and CIAV-R70 primers of example 1, as shown in Table 2
TABLE 2 conventional PCR reaction System
The reaction program is executed as follows: at 95 ℃ for 3 min; 95 ℃, 30s, 55 ℃, 30s, 72 ℃, 30s, 30 cycles; 72 ℃ for 10 min.
After the PCR program is finished, adding all PCR products into 1% agarose gel containing Goldview, performing electrophoresis for 30min, cutting off a target band under the irradiation of an ultraviolet lamp, and performing gel recovery by using a Kangji gel recovery kit. The method comprises the following specific steps:
(1) cutting a single-purpose DNA band from the agarose gel (cutting off redundant parts as much as possible), putting the cut DNA band into a clean centrifuge tube, and weighing and calculating the weight of the gel (recording the weight of the centrifuge tube in advance);
(2) adding 1 volume of Buffer PG to the gel block (e.g., the gel weight is 100mg, the volume can be regarded as 100. mu.L, and so on);
(3) and (3) carrying out warm bath in a water bath at 50 ℃, gently turning the centrifugal tube upside down every 2-3 minutes until the sol solution is yellow to ensure that the gel blocks are fully dissolved. If the undissolved rubber blocks still exist, some sol solution can be added again or the mixture is kept standing for several minutes until the rubber blocks are completely dissolved;
(4) column balancing: adding 200 mu L of Buffer PS (polystyrene) into an adsorption column filled into the collecting pipe, centrifuging at 13000rpm for 1 minute, pouring off waste liquid in the collecting pipe, and putting the adsorption column back into the collecting pipe again;
(5) adding the solution obtained in the step (3) into an adsorption column filled into a collecting pipe, standing for 2 minutes at room temperature, centrifuging for 1 minute at 13000rpm, pouring off waste liquid in the collecting pipe, and putting the adsorption column back into the collecting pipe again;
(6) adding 450 mu L of Buffer PW into the adsorption column, centrifuging at 13000rpm/min for 1 min, pouring off waste liquid in the collecting pipe, and replacing the adsorption column into the collecting pipe again;
(7) centrifuging at 13000rpm for 1 minute, and pouring off waste liquid in the collecting pipe;
(8) the adsorption column was placed in a new 1.5mL centrifuge tube, 50. mu.L LBuffer EB was added dropwise to the middle of the adsorption membrane in suspension and left at room temperature for 2 minutes. 13000rpm for 1 minute, collect DNA solution, at-20 ℃ to store DNA.
2. Ligation of fragments of interest
The ligation of the target fragment was carried out according to the instruction manual of Tarkara pMD18-T Vector, as shown in Table 3, and after mixing, it was exposed to a PCR apparatus at 16 ℃ for 4 hours and stored at-20 ℃.
TABLE 3 ligation reaction System
3. Transformation of recombinant plasmids
(1) Respectively adding the ligation products obtained in the previous step into 50 mu L of competent cells, numbering, and simultaneously establishing positive control of pMD18-T plasmid and competent cell control without adding any plasmid DNA;
(2) gently mixing the ligation product and the competent cells uniformly, immediately carrying out ice bath for 30min, then carrying out heat shock for 90s at 42 ℃, and then immediately carrying out ice bath for 10 min;
(3) adding 400 μ L of fresh LB liquid culture medium into each tube, and culturing in 37 deg.C incubator for 45 min;
(4) 100 mu L of the bacterial liquid was spread on 1.5% (W/V) LB agar plate containing Amp (100. mu.g/mL), and cultured in an inverted state at 37 ℃ for 12-16 hours.
4. Screening and identification of suspicious bacterial liquid
And (3) selecting a single colony which grows well in 500 mu L of LB liquid culture medium containing Ampicillin (AMP), placing the single colony in a constant-temperature shaking table at 37 ℃ for shaking culture for 4-6 h, and taking suspicious bacterial liquid as a template for PCR identification, wherein the suspicious bacterial liquid is shown in Table 4.
TABLE 4 PCR reaction System
After the components are mixed evenly, a PCR program is executed: 94 ℃ for 4 min; 30s at 94 ℃; 30s at 55 ℃; 72 ℃, 30s, 34 cycles; 72 ℃ for 10 min. Then, the bacterial liquid which is identified as positive by PCR is sent to biological engineering company Limited for sequencing.
5. Extraction of Positive plasmids
Carrying out amplification culture on the bacterial liquid with the correct sequencing result, and carrying out plasmid extraction by using a TaKaRa plasmid extraction kit, wherein the method comprises the following specific steps:
(1) and (3) culturing escherichia coli: selecting a single colony from a plate culture medium, inoculating the single colony into 1-4 mL of liquid culture medium containing antibiotics, and culturing at 37 ℃ overnight (the cells are difficult to crack after being cultured for 12-16 hours, and the yield of plasmids is reduced along with the cells cultured for more than 16 hours);
(2) taking 1-4 mL of overnight culture bacterial liquid, centrifuging at 12000rpm for 2 minutes, and removing supernatant;
(3) suspending the bacterial pellet with 250 μ L Solution I (containing RNase A);
(4) adding 250 mu L of Solution II, slightly turning and mixing the mixture up and down for 5-6 times to ensure that the thalli are fully cracked to form a transparent Solution;
(5) adding 350 mu L of Solution III precooled at 4 ℃, slightly turning and mixing the Solution III up and down for 5 to 6 times until compact aggregates are formed, and then standing the aggregates for 2 minutes at room temperature;
(6) centrifuging at 12000rpm for 10min at room temperature, and taking supernatant;
(7) placing Spin Column in the kit on the Collection Tube;
(8) transferring the supernatant obtained in the step (6) into a Spin Column, centrifuging at 12000rpm for 1 minute, and removing the filtrate;
(9) adding 500 μ L of Buffer WA into Spin Column, centrifuging at 12000rpm for 30s, and discarding the filtrate;
(10) adding 700 mu L of Buffer WB into Spin Column, centrifuging at 12000rpm for 30 seconds, and discarding the filtrate;
(11) repeating the operation step (10);
(12) spin Column was replaced on the Collection Tube and centrifuged at 12000rpm for 1 minute to remove the residual wash solution;
(13) placing Spin Column on a new 1.5mL centrifuge tube, adding 50. mu.L of sterilized water or Elution Buffer at the center of the Spin Column membrane, and standing at room temperature for 1 minute;
(14) DNA was eluted by centrifugation at 12000rpm for 1 minute.
6. Preparation of Positive Standard plasmid
Plasmid concentration was measured using a ultramicro fluorescence spectrophotometer and was determined as a function of gene copy number (copies/uL) 6.02 x 1023Concentration of plasmid (ng/uL) 10-9V [ plasmid size (bp) × 660]Calculating the gene copy number, and diluting to 10 times by 100copies/uL were stored at-20 ℃ until use. And (3) taking positive plasmids with any concentration to optimize the primer concentration and the reaction program.
Secondly, constructing a standard curve:
taking any positive plasmid to optimize primer concentration, and then 107、106、105、104And 103The 5 positive plasmids with concentration gradient, i.e. copies/uL, are used as templates for real-time fluorescent quantitative PCR amplification, the reaction system is shown in Table 5, and the reaction program is executed as follows: at 95 ℃ for 3 min; 95 ℃, 5s, 55 ℃, 15s, 40 cycles; 95 ℃, 30s, 65 ℃, 30s, 95 ℃, 30 s. The result is shown in fig. 1, and the standard curve equation is-3.334 lgX + 39.08; coefficient of correlation R20.992; the amplification efficiency E was 99.49% and the results show that there is a good linear relationship between the standards at different concentrations, where the X-axis is the copy number of the plasmid standard and the Y-axis is the cycle threshold, which is in line with the expected results.
Thirdly, detecting a sample to be detected:
taking a sample to be detected as a template, carrying out real-time fluorescence quantitative PCR amplification, wherein a reaction system is shown in Table 5, and the execution reaction program comprises the following steps: at 95 ℃ for 3 min; 95 ℃, 5s, 55 ℃, 15s, 40 cycles; 95 ℃, 30s, 65 ℃, 30s, 95 ℃, 30 s. And when the Ct value is less than 35, a single peak value appears on a dissolution curve and the Tm value is 80.5-81.5 ℃, judging that the sample to be detected is positive, and judging that the sample to be detected is negative under other conditions.
TABLE 5 fluorescent quantitative PCR reaction System
Example 3:
taking the copy number as 106、105、104、103、102、101、100The same real-time fluorescent quantitative PCR amplification as in example 2 was performed using the standard plasmid of (1) as a template, and the same plasmid was subjected to ordinary PCR amplification, thereby determining the lowest copy number of the positive plasmid detected by both the method of the present invention and the ordinary PCR method. The results are shown in FIG. 2 (1-7: 2.7 x 10)6～2.7*100copies/μL；8：ddH2O) and fig. 3 (M: 2000 DNAmarker; 1: ddH2O；2～9：2.7*107～2.7*100copies/. mu.L), the lowest copy number at which the method of the invention can detect positive is 2.7 x 101copies/. mu.L, whereas ordinary PCR can only detect 2.7X 103copies/. mu.L, indicating that the method of the invention is 100 times more sensitive than conventional PCR.
Example 4:
the specificity of the method was verified by real-time fluorescent quantitative PCR amplification of cDNA of Avian Influenza Virus (AIV), ancavir (FAdV), Newcastle Disease Virus (NDV), Avian Reovirus (ARV) using the real-time fluorescent quantitative PCR method of example 2. As a result, as shown in FIGS. 4 and 5 (FIG. 4 is an amplification curve, FIG. 5 is a dissolution curve, wherein 1: CIAV, 2: AIV, 3: NDV, 4: ARV, 5: FAdV-4, 6: ddH2O) shows an amplification curve, AIV also showed an amplification curve, but the Tm value of the dissolution curve of AIV was 78.5 ℃ and thus the detection method was evaluated by combining the amplification curve and the dissolution curve, and was capable of specifically amplifying CIAV with good specificity.
Example 5:
3 batches of plasmids are subjected to 3-batch repeated amplification and 3-batch repeated amplification respectively by adopting the real-time fluorescence quantitative PCR method in the embodiment 2, the change conditions of the Ct value and the Tm value are compared, the stability of the method is verified, and the stability of the method is evaluated by using the intra-batch variation coefficient. The results are shown in Table 6, in which the intra-group coefficient of variation was less than 1.5%, the inter-group coefficient of variation was less than 2.4%, and the reproducibility was good.
TABLE 6 fluorescent quantitative PCR repeatability test results
SEQUENCE LISTING
<110> institute of Buddha science and technology
<120> primer pair, kit and method for detecting chicken infectious anemia virus
<130>2019
<160>3
<170>PatentIn version 3.3
<210>1
<211>24
<212>DNA
<213> Artificial Synthesis
<400>1
cttgattacc actactccca gccg 24
<210>2
<211>24
<212>DNA
<213> Artificial Synthesis
<400>2
cgtcttgcca tcttacagtc ttat 24
<210>3
<211>70
<212>DNA
<213> Artificial Synthesis
<400>3
cttgattacc actactccca gccgaccccg aaccgcaaga aggtgtataa gactgtaaga 60
Claims (10)
1. A primer pair for detecting chicken infectious anemia viruses is characterized by comprising CIAV-F70 shown in SEQ ID NO.1 and CIAV-R70 shown in SEQ ID NO. 2.
2. A kit for detecting chicken infectious anemia virus, comprising: the primer pair, positive standard plasmid, SYBR Green Premix Pro Taq real-time fluorescent quantitative PCR reagent and ddH of claim 12O; the positive standard plasmid is a recombinant plasmid obtained by connecting a DNA fragment shown by SEQ ID NO.3 with a pMD18-T vector.
3. A method for detecting chicken infectious anemia virus is characterized by comprising the following specific steps: using a sample to be detected as a template, carrying out real-time fluorescent quantitative PCR amplification by using the primer pair of claim 1 to obtain a cycle threshold of the sample to be detected, and substituting the cycle threshold into a standard curve to obtain the content of the chicken infectious anemia virus in the sample to be detected; the standard curve is Y-3.334 lgX +39.08, where Y is the threshold for cycling and X is the copy number of the plasmid.
4. The method of claim 3, wherein the reaction system of the real-time fluorescent quantitative PCR amplification is: SYBRGreen Premix Pro Taq 10. mu. L, ddH2O7 mu L, CIAV-F700.5 mu L, CIAV-R700.5 mu L and template 2 mu L, real-time fluorescence quantitative PCR amplificationThe reaction procedure of (1) is as follows: at 95 ℃ for 3 min; 95 ℃, 5s, 55 ℃, 15s, 40 cycles; 95 ℃, 30s, 65 ℃, 30s, 95 ℃, 30 s.
5. The method of claim 4, wherein the standard curve is prepared by fluorescent quantitative PCR amplification of a gradient concentration of positive plasmids prepared by the following steps: using chicken infectious anemia virus genome as a template, using the primer pair of claim 1 to perform common PCR amplification, cloning, performing sequencing comparison to confirm that the plasmid is correct, performing plasmid extraction by using a plasmid extraction kit, calculating according to the plasmid concentration to obtain the gene copy number, and diluting to 10 times by using a 10-fold ratio0copies/uL were stored at-20 ℃ until use.
6. The method of claim 5, wherein the reaction system of the general PCR amplification is: rTaq 12.5. mu. L, ddH2O9.5. mu. L, CIAV-F700.5. mu. L, CIAV-R700.5. mu.L and template 2. mu.L.
7. The method according to claim 5, wherein the cloning comprises the steps of PCR product purification recovery, target fragment ligation and recombinant plasmid transformation.
8. The method according to claim 7, wherein the step of purifying and recovering the PCR product comprises the following specific steps: and adding the PCR product into 1% agarose gel containing Goldview, performing electrophoresis for 30min, cutting a target strip under the irradiation of an ultraviolet lamp, and recovering by using a recovery kit to obtain a PCR purified product.
9. The method according to claim 8, wherein the step of connecting the target segments comprises the following specific processes: mu.L of the PCR-purified product, 0.25. mu.L of pMD18-T Vector and 2.5. mu.L of 2 XLigase Buffer were mixed well and placed in a PCR apparatus at 16 ℃ for 4h and stored at-20 ℃ to obtain a ligation product.
10. The method according to claim 9, wherein the step of transforming the recombinant plasmid comprises the following steps: adding the ligation product into competent cells, uniformly mixing, carrying out ice bath for 30min, then carrying out heat shock for 90s at 42 ℃, and carrying out ice bath for 10 min; adding 400 μ L LB liquid culture medium, and culturing at 37 deg.C for 45min to obtain bacterial liquid; then 100 mu L of bacterial liquid is taken and coated on a 1.5% LB agar plate containing Amp, and inverted culture is carried out for 12-16 h under the constant temperature condition of 37 ℃; the Amp concentration is 100 mug/mL.
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