CN108624716B - seven-PCR primer group for simultaneously detecting seven waterfowl susceptibility viruses and detection method thereof - Google Patents
seven-PCR primer group for simultaneously detecting seven waterfowl susceptibility viruses and detection method thereof Download PDFInfo
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Abstract
The invention discloses a seven-PCR primer group for simultaneously detecting seven waterfowl susceptibility viruses and a detection method thereof, wherein: the primer group sequences are FAdV-F, FAdV-R, DHAV-F, DHAV-R, DEV-F, DEV-R, AIV-F, AIV-R, DTMUV-F, DTMUV-R, NDV-F, NDV-R, GPV-F, GPV-R, AIV-F5 and AIV-R5 respectively. The invention can rapidly identify and diagnose whether the waterfowl is infected with the virus to be detected in the actual production, and can monitor the disease condition of the waterfowl at the same time, thereby providing an effective detection means for large-scale virus monitoring, epidemiological investigation and prevention and control of NDV, AIV, DEV, DHAV, FAdV, DTMUV and GPV clinically. The invention has high detection sensitivity, strong specificity and good repeatability, and has lower cost, rapidness, simplicity, convenience, time saving and labor saving compared with other detection methods.
Description
Technical Field
The invention belongs to the technical field of PCR detection, and particularly relates to a seven-PCR primer group for simultaneously detecting seven waterfowl susceptibility viruses and a detection method thereof.
Background
China is the biggest waterfowl producing country and consuming country in the world, and the waterfowl feeding amount accounts for more than 75 percent of the total amount of the world. With the rapid development of duck breeding in recent years, various factors such as the expansion of breeding scale, the increase of mixed breeding modes, the enhancement of mobility of people and livestock, the reduction of water quality caused by water source pollution and the like create favorable conditions for the spread of viruses, the number of local epidemic viral diseases is obviously increased, new epidemic situations are continuously generated, and serious economic loss is caused to the duck breeding industry. At present, duck-origin Avian Influenza Virus (AIV), Newcastle Disease Virus (NDV), avian adenovirus (FAdV), duck plague virus (DEV), Duck Hepatitis A Virus (DHAV), duck tembusu virus (DTMUV), Goose Parvovirus (GPV) are common susceptible viruses seriously harming duck breeding. The diseases are frequently present in the form of mixed infection clinically, and rapid differential diagnosis is difficult to make by only observing the onset symptoms, autopsy changes, onset characteristics and the like by naked eyes, so that the establishment of a rapid high-throughput differential detection method which can be generally applicable to the virus infection is urgent.
Clinically, virus separation and identification, serological detection, ELISA, immuno-electron microscope and Real-time PCR have been respectively applied to the detection of virus infection, but the traditional methods such as virus separation and identification are time-consuming, serological and ELISA detection are time-consuming and labor-consuming, and are often limited by factors such as freshness of clinical pathological materials, pollution degree or disease course, and the immuno-electron microscope and Real-time PCR have high requirements for equipment and virus purification, and both bring limitations to clinical diagnosis. In clinic, the diseases are often a plurality of mixed infections, and the differential diagnosis is more difficult.
Therefore, how to detect the seven viruses while being capable of being fast, accurate, highly specific and highly sensitive, and not having strict requirements on virus purification, being convenient for operation and saving cost becomes a technical problem to be solved in the field.
Disclosure of Invention
This section is for the purpose of summarizing some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. In this section, as well as in the abstract and the title of the invention of this application, simplifications or omissions may be made to avoid obscuring the purpose of the section, the abstract and the title, and such simplifications or omissions are not intended to limit the scope of the invention.
The present invention has been made in view of the above-mentioned technical drawbacks.
Therefore, as one aspect of the invention, the invention overcomes the defects in the prior art and provides a seven-fold PCR primer group for simultaneously detecting seven waterfowl susceptible viruses.
In order to solve the technical problems, the invention provides the following technical scheme: a seven-PCR primer group for simultaneously detecting seven waterfowl susceptibility viruses is disclosed, wherein: the sequences of the primer groups are respectively FAdV-F, FAdV-R, DHAV-F, DHAV-R, DEV-F, DEV-R, AIV-F, AIV-R, DTMUV-F, DTMUV-R, NDV-F, NDV-R, GPV-F, GPV-R. The sequences of the primer group from the 5 'end to the 3' end are respectively as follows:
FAdV-F：CAACAGCCTCTCGTACCCAG、
FAdV-R：CCGATGTAGTTGGGCCTGAG、
DHAV-F：CTTTCCACTCCCTGCTCCC、
DHAV-R：TTGGCTTCCACATCCTCTTCA、
DEV-F：ATCGCATGTAGACGTTGGTT、
DEV-R：AGACAGCGGTGATGGATGG、
AIV-F：GGCGACTACTACCAACCCA、
AIV-R：CTGCTGTTCCTGCCGATAT、
DTMUV-F：AATCGGTAGTGGCTTTGG、
DTMUV-R：AGTCTGCCGACATGGATAT、
NDV-F:CACCGGCAACCCTATTCTGT、
NDV-R:AGTGCGCCTTCAGTCTTTGA、
GPV-F:TATGTCCTGGGCTCGGCTAC、
GPV-R:AGCTGACACAGGTCCAGGTT；
the seven waterfowl susceptible viruses are duck-origin avian influenza virus, newcastle disease virus, avian adenovirus, duck plague virus, A-type duck hepatitis virus, duck tembusu virus and goose parvovirus.
As another aspect of the invention, the invention overcomes the defects in the prior art and provides a method for simultaneously detecting seven waterfowl susceptible viruses by using a seven-fold PCR primer set.
In order to solve the technical problems, the invention provides the following technical scheme: the method for simultaneously detecting seven waterfowl susceptible viruses by the seven-fold PCR primer set according to claim 1, which comprises the steps of carrying out PCR amplification by using the primer according to claim 1, and judging a positive result according to a specific amplification band of agarose gel electrophoresis after the reaction is finished.
The optimal scheme of the method for simultaneously detecting seven waterfowl susceptible viruses by using the seven PCR primer sets comprises the following steps: the PCR amplification reaction conditions comprise pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30s, annealing at 57-57.5 ℃ for 30s, extension at 72 ℃ for 40s, 35 cycles, and extension at 72 ℃ for 10 min.
The optimal scheme of the method for simultaneously detecting seven waterfowl susceptible viruses by using the seven PCR primer sets comprises the following steps: in the PCR, the concentration of DNA polymerase in the reaction system is 0.05 u/. mu.l.
The optimal scheme of the method for simultaneously detecting seven waterfowl susceptible viruses by using the seven PCR primer sets comprises the following steps: in the PCR, the concentration of dNTP in the reaction system is 0.4 mM.
The optimal scheme of the method for simultaneously detecting seven waterfowl susceptible viruses by using the seven PCR primer sets comprises the following steps: in the PCR reaction system, the final concentrations of the primers are respectively as follows: FAdV-F/R: 0.12. mu. M, DHAV-F/R: 0.12. mu. M, DEV-F/R: 0.16. mu. M, AIV-F/R: 0.24 μ M, DTMUV-F/R: 0.28. mu. M, NDV-F/R: 0.24 μ M, GPV-F/R: 0.24. mu.M.
The optimal scheme of the method for simultaneously detecting seven waterfowl susceptible viruses by using the seven PCR primer sets comprises the following steps: in the PCR, the concentration of magnesium ions in a reaction system is 4 mM.
The optimal scheme of the method for simultaneously detecting seven waterfowl susceptible viruses by using the seven PCR primer sets comprises the following steps: the reaction system of the PCR is a 25-microliter system comprising 2.5 microliter of 10xtaq buffer, 0.25 microliter of DNA polymerase, 1 microliter of dNTP and 25mM Mgcl2 4μl。
The optimal scheme of the method for simultaneously detecting seven waterfowl susceptible viruses by using the seven PCR primer sets comprises the following steps: the DNA detection band size of the avian adenovirus is 102bp, the DNA detection band size of the A-type duck hepatitis virus is 140bp, the DNA detection band size of the duck plague virus is 172bp, the DNA detection band size of the duck source avian influenza virus is 435bp, the DNA detection band size of the duck tembusu virus is 288bp, the DNA detection band size of the goose parvovirus is 516bp, and the DNA detection band size of the Newcastle disease virus is 330 bp.
The invention has the beneficial effects that: compared with the prior art, the m-PCR method provided by the invention can be used for simultaneously detecting seven waterfowl viruses of NDV, AIV, DEV, DHAV, FAdV, DTMUV and GPV, can be used for identifying and diagnosing the seven viruses, and has the advantages of strong detection specificity, high sensitivity, simplicity and rapidness in operation and low cost.
The m-PCR method can simultaneously detect seven waterfowl viruses of NDV, AIV, DEV, DHAV, FAdV, DTMUV and GPV, can quickly identify and diagnose whether waterfowls are infected with the viruses to be detected in actual production, and can monitor the disease condition of the waterfowls, thereby providing an effective detection means for large-scale virus monitoring, epidemiological investigation and prevention and control of the diseases of NDV, AIV, DEV, DHAV, FAdV, DTMUV and GPV clinically. The invention has high detection sensitivity, strong specificity and good repeatability, and has lower cost, rapidness, simplicity, convenience, time saving and labor saving compared with other detection methods.
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In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive exercise. Wherein:
FIG. 1 is a seven-fold PCR electrophoretogram of the present invention.
FIG. 2 is an optimized electrophoretogram of the multiplex PCR enzyme of the present invention.
FIG. 3 is an optimized electrophoretogram of the multiplex PCR dNTPs of the present invention.
FIG. 4 is a comparison of different reaction systems of the multiplex PCR of the present invention.
FIG. 5 is an optimized electrophoretogram of magnesium ion concentration for multiplex PCR according to the present invention.
FIG. 6 is an optimized electrophoresis diagram of the annealing temperature of the multiplex PCR according to the present invention.
FIG. 7 is an optimized electrophoresis diagram of the multiplex PCR primer of the present invention.
FIG. 8 is an optimized electropherogram for multiple PCR cycles according to the invention.
FIG. 9 is an electrophoretogram of the seven-fold PCR sensitivity of the present invention.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with examples are described in detail below.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described and will be readily apparent to those of ordinary skill in the art without departing from the spirit of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.
Furthermore, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
Example 1:
designing and screening primers:
according to the conserved genes of various viruses in GenBank, such as fusion protein gene (F gene) of NDV, matrix protein gene (M gene) of AIV, long specific region gene (UL2 gene) of DEV, nucleocapsid protein gene (VP1 gene) of DHAV, hexon protein gene (H gene) of FAdV, envelope protein gene (E gene) of DTMUV and nucleocapsid protein gene (VP3 gene) of GPV, homology analysis is carried out by using DNAMAN software, specific primers are designed in the conserved regions, 2 or more than 2 primers are designed for each virus for screening, and primers with good specificity, no cross reaction and good amplification efficiency are selected. The specific primers for each virus are shown in Table I.
TABLE-seven pairs of specific primers for detecting seven viruses according to the invention
Example 2:
plasmid standard preparation:
the first step is as follows: primer synthesis
The 7 pairs of virus plasmid construction primers designed by the invention are shown in Table II and synthesized by technical service companies of Shanghai Biotech company.
Standard plasmid primers for construction of seven viruses in Table II
| Name | Sequence(5’-3’) | Targeted gene | Length |
| FAdV-F plasmid | TTACTCCCGACCTGACCAC | H | 949bp |
| FAdV-R plasmid | ATACCCGAACGCTCCGAAT | ||
| DHAV-F plasmid | CTTTCCACTCCCTGCTCCC | VP1 | 140bp |
| DHAV-R plasmid | TTGGCTTCCACATCCTCTTCA | ||
| DEV-F plasmid | ACGATAGGCTCCCGTCTCC | UL2 | 312bp |
| DEV-R plasmid | CAACGCCACGACCTTCCAG | ||
| AIV-F plasmid | GGCGACTACTACCAACCCA | M | 435bp |
| AIV-R plasmid | CTGCTGTTCCTGCCGATAT | ||
| DTMUV-F plasmid | AATCGGTAGTGGCTTTGG | E | 288bp |
| DTMUV-R plasmid | AGTCTGCCGACATGGATAT | ||
| NDV-F plasmid | CACCGGCAACCCTATTCTGT | F | 330bp |
| NDV-R plasmid | AGTGCGCCTTCAGTCTTTGA | ||
| GPV-F plasmid | GTGCCGATGGAGTGGGTAA | VP3 | 917bp |
| GPV-R plasmid | TGATACAGGTCCGGGTTGC |
The second step is that: viral total DNA/RNA extraction
The genomes of the existing strains of seven viruses are respectively extracted according to the instruction of a Viral DNA/RNA Extraction Kit, the RNAs of NDV, AIV, DHAV and DTMUV are reversely transcribed into cDNA according to the instruction, and all the cDNA/DNA are stored at-80 ℃ for later use.
The third step: reverse transcription reaction
Add NDV, AIV, DHAV and DTMUV RNA 1. mu.l each into the RNase-free PCR tube, use Thermo Scientific Reversaid First Strand cDNA Synthesis Kit to perform RT-PCR reaction, wherein 5xBuffer 4. mu.l, 10mM dNTP 2. mu.l, Random Primers 1. mu.l, 20U/. mu.l Ribolock RNase 1. mu.l, 200U/. mu.l Reversaid M-MuLV RTase 1. mu.l, DEPC water 10. mu.l, total 20. mu.l, after the reaction is finished, obtain cDNA.
The fourth step: PCR amplification
50 μ l PCR reaction: 10 × taq buffer 5. mu.l, 5U/ul DNA Polymerase 0.5. mu.l, 10mM dNTP mix 2. mu.l, 25mM Mgcl2And 8 mu l of primers of the viruses synthesized in the first step and 1 mu l of cDNA/DNA obtained in the third step are taken as templates, deionized water is added to make up to 50 mu l, and PCR amplification is respectively carried out. The reaction parameters of the PCR instrument are as follows: pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 74 ℃ for 1min, 35 cycles, and extension at 72 ℃ for 10 min.
The fifth step: preparation of plasmid Standard
And (3) carrying out agarose Gel electrophoresis on the amplification product obtained in the fourth step, recovering a target fragment according to a DNA Gel Extraction Kit operation method, connecting the recovered product to a pMD18-T vector, carrying out competence transformation on DH5 alpha escherichia coli, carrying out plate scratching overnight culture, then selecting a white single colony for identification, and entrusting a recombinant plasmid sequence to Shanghai bio-engineering company for sequence determination. Extracting the recombinant plasmid with correct sequencing by using a plasmid miniprep Kit, quantifying the concentration of the recombinant plasmid by using Nanodrop2000, and performing sterile double-distilled water on the recombinant plasmid according to the proportion of 1x108-1x10-1Plasmid standards were prepared by dilution of copies/μ l in 10-fold gradients.
Example 3:
and (3) verifying the specificity of the primers:
the standard plasmids constructed by seven viruses are respectively used as templates to amplify corresponding target fragments, the total volume of each reaction system is 50 mu l, and the reaction systems comprise 1 mu l of single virus template, 1 mu l of respective upstream and downstream primers, 10xtaq buffer5 mu l, 5U/mu l DNA Polymerase 0.5 mu l, 10mM dNTP mix 2 mu l and 25mM Mgcl2Make up to 50. mu.l with 8. mu.l of water. The reaction program comprises pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 1min, 35 cycles, and final extension at 72 ℃. After the reaction, 8. mu.l of the product was subjected to agarose gelGel electrophoresis analysis, 25. mu.l of product was sent to Shanghai Biotech for sequencing.
Example 4:
optimizing the enzyme dosage:
as shown in FIG. 1, 7 bands in lane 1 are FAdV (102bp), DHAV (140bp), DEV (172bp), DTMUV (288bp), NDV (330bp), AIV (435bp) and GPV (516bp), respectively.
Taq DNA Polymerase is a key factor in PCR reactions, different enzymes have different amplification efficiencies and fidelity, the selection of the enzyme has been optimized in example 2, and the amount of the enzyme is now optimized. Since enzymes are relatively expensive, it is our optimization objective to use the minimum amount to obtain the optimal results, for which we have sought the optimal amount of enzyme, see figure 2. As shown in FIG. 2, the working concentrations of the enzymes in lanes 1-5 were 0.01 u/. mu.l, 0.02 u/. mu.l, 0.03 u/. mu.l, 0.05 u/. mu.l, and 0.07 u/. mu.l. As is clear from FIG. 2, the best reaction results were obtained when the enzyme concentration was 0.05 u/. mu.l.
Example 5:
optimizing the dosage of dNTP:
dNTP in PCR reaction is a general term including dATP, dGTP, dTTP, dCTP and the like, and the main function is that the dNTP needs to be connected with a template strand to be amplified under the action of enzyme by a base complementary principle in the third step of extension of PCR, so that the aim of copying the template strand is fulfilled. In PCR reactions, using low dNTP concentrations, nucleotide misincorporation at non-target sites for priming and extension was reduced, and we searched for optimal dNTP concentrations for this purpose, see FIG. 3. FIG. 3 shows the optimization of multiplex PCR dNTPs of the present invention, lanes 1-4 with working concentrations of 0.1mM, 0.25mM, 0.4mM, 0.55mM dNTPs. As is clear from FIG. 3, the best reaction results were obtained when the concentration of dNTP was 0.4 mM.
Example 6:
optimizing reaction reagents:
FIG. 4 is a comparison of different reaction systems of the multiplex PCR of the present invention, and it can be seen from FIG. 4 that the conventional PCR mix is no longer suitable for the multiplex reaction system when the quintuple PCR is performed; in FIG. 4, lane 1 shows the detection of five viruses using a common PCR mix; lane 2 shows the system shown in table four for detecting five viruses; lanes 3 and 4 are negative controls for lanes 1 and 2, respectively. The ratio of the reaction reagents which we have groped is proved to be superior to that of the PCR mix which is sold on the market.
Therefore, we have found out the proper concentration of magnesium ions, the proper amount of enzyme and dNTP, and see Table four.
TABLE IV multiplex PCR reaction System of the present invention
Example 7:
optimizing the concentration of magnesium ions:
the magnesium ions mainly have the effects that dNTP-Mg2+ interacts with a nucleic acid skeleton and can influence the activity of the Polymerase, the concentration of the magnesium ions is high, the amplification efficiency is high, but the specificity is reduced; the amplification efficiency will decrease but the specificity will increase with low magnesium ion concentration. For this reason we have sought the optimum magnesium ion concentration, see figure 5. In FIG. 5, the magnesium ion concentrations in lanes 1-6 are 1mM, 2mM, 3mM, 4mM, 5mM, 6mM, respectively. The concentration of magnesium ions is preferably 4 mM.
Example 8:
optimizing annealing temperature:
annealing temperature determines PCR specificity and yield: the temperature is high, the specificity is strong, but if the temperature is too high, the primer cannot be firmly combined with the template, and the DNA amplification efficiency is reduced; the yield is high at low temperature, but the mismatch between the primer and the template can be caused by low temperature, and the non-specific product is increased. Since there are 7 pairs of primers in the seven-fold PCR reaction, the respective suitable annealing temperatures are different, and we have searched for the optimal annealing temperatures for the seven-fold PCR reaction, as shown in FIG. 6, the annealing temperatures of lanes 1-6 in FIG. 6 are 51.0 deg.C, 53.1 deg.C, 55.4 deg.C, 57.5 deg.C, 58.9 deg.C, and 61.0 deg.C, respectively. As can be seen from fig. 6, the reaction results were the best when the annealing temperature was 57.5 ℃.
Example 9:
optimizing primers:
meanwhile, it is very difficult to detect seven viruses in one reaction well, the seven pairs of primers often interfere with each other to cause detection failure, hairpin structures and primer dimers are easily formed among the seven pairs of primers, and mismatches easily occur between the primers and DNA, for example, as shown in FIG. 7a, when five-fold PCR is performed to detect five viruses, FAdV, DEV, AIV, NDV and GPV, the primers of GPV can normally work, as shown in the 2439 bp band (the size of the band before optimization) of FIG. 7a, while when two pairs of primers are directly added to perform seven-fold PCR, the same pair of primers of GPV cannot normally work, and GPV cannot be detected, as shown in the 2439 bp band of FIG. 7b, lane 8, and lane 7 is a positive control of lane 8, therefore, seven pairs of primers need to be redesigned, as shown in FIGS. 7c 1 and 2, seven pairs of primers after optimization, seven-fold PCR can simultaneously detect seven viruses (the size of the GPV band after optimization, bp 516), however, the band of AIV (237 bp band size before optimization) was very weak, and thus the detection sensitivity of AIV was significantly reduced. Through continuous repeated design and a large number of experimental verification of the inventor, seven PCR primer pairs capable of detecting seven viruses at the same time with high sensitivity and high specificity are finally determined, as shown in a Lane 2 of figure 7d (an optimized AIV band 435 bp). The seven pairs of primers of the invention do not interfere with each other, and PCR bands are bright, clear and free of miscellaneous bands. FIG. 7a, lane 2, is a quintuple PCR of FAdV, DEV, AIV, NDV, GPV; FIG. 7b shows positive controls in lanes 1-7 for FAdV, DHAV, DEV, AIV, DTMUV, NDV, GPV, respectively, and no GPV virus in lane 8; FIG. 7c, lanes 1 and 2, are seven-fold PCR with optimized GPV primers, but weak AIV bands; FIG. 7d shows that two pairs of newly designed primers for AIV are used in lanes 1 and 2, respectively, lane 2 shows good results, seven bands are FAdV (102bp), DHAV (140bp), DEV (172bp), DTMUV (288bp), NDV (330bp), AIV (435bp), and GPV (516bp), respectively, and lanes 3 and 4 are positive controls for lanes 1 and 2, respectively.
The sample adding amount of the seven PCR 7 pairs of primers is shown in the third table.
TABLE III sample adding amount of seven PCR 7 pairs of primers of the present invention
| Viral primers | Amount of primer to be added | Final concentration | Template sample addition |
| FAdV-H-F/R(10μM) | 0.3μl | 0.12μM | 1μl |
| DHV-VP1-F/R(10μM) | 0.3μl | 0.12μM | 1μl |
| DEV-UL2-F/R(10μM) | 0.4μl | 0.16μM | 1μl |
| AIV-M-F/R(10μM) | 0.6μl | 0.24μM | 1μl |
| DTMUV-E-F/R(10μM) | 0.7μl | 0.28μM | 1μl |
| NDV-F-F/R(10μM) | 0.6μl | 0.24μM | 1μl |
| GPV-VP3-F/R(10μM) | 0.6μl | 0.24μM | 1μl |
Example 10:
and (3) optimizing the number of cycles:
the number of cycles in the PCR reaction is also an influencing factor. If the number of cycles is too small, the amplification is incomplete, and if the number of cycles is too large, the amplification reaction reaches a plateau stage, and the amount of products is not increased, but time is wasted. For this we have searched for the optimum number of cycles, see figure 8. In FIG. 8, the cycle numbers of lanes 1 to 3 are 25c, 30c and 35c, respectively. As can be seen from the figure, 35 cycles are the most preferable.
Example 11:
establishing a multiplex PCR system:
through the optimization, a multiple PCR reaction system is finally established, the reaction system is 25 mu l, seven virus strains and virus plasmids are taken as templates, specific primers of seven viruses are respectively added, and the final concentration is controlled between 0.1 mu M and 0.4 mu M; DNA Polymerase 0.25. mu.l; the annealing temperature is set to 57 ℃; the optimized reaction procedure is as follows: pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30s, annealing at 57 ℃ for 30s, and extension at 72 ℃ for 40s, and after 35 cycles, extension at 72 ℃ for 10 min. The PCR product was analyzed by 1.5% agarose gel electrophoresis, compared to the expected size of the target fragment, and a blank control of deionized water was set.
Example 12:
multiplex PCR specificity detection:
according to the established multiplex PCR method, the plasmid of each virus is used as a template to carry out PCR amplification, each reaction system is 25 mu l, 1 mu l of single virus template is included, 7 mu l of virus primer mixture is contained, and 10x taq buffer (without Mg) is added2+) 2.5. mu.l Taq DNA Polymerase (5U/ul) 0.25. mu.l dNTPmix (10mM each 1. mu.l Mgcl2(25mM) 4. mu.l deionized water make-up 25Mu.l; the reaction procedure is as follows: pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30s, annealing at 57 ℃ for 30s, and extension at 72 ℃ for 40s, and after 35 cycles, extension at 72 ℃ for 10 min. The PCR product was analyzed by 1.5% agarose gel electrophoresis, compared to the expected size of the target fragment, and a blank control of deionized water was set.
Example 13:
multiplex PCR sensitivity evaluation:
extracting the recombinant plasmid with correct sequencing by using a plasmid miniprep Kit, quantifying the concentration of the recombinant plasmid by using Nanodrop2000, and performing sterile double-distilled water on the recombinant plasmid according to the proportion of 1x108copies/μl-1x100Plasmid standards were prepared by dilution of copies/μ l in 10-fold gradients. Mix 7 single templates into multiple templates at equal concentration from 1 × 108copies/tube-1x100The sensitivity of the multiplex PCR method was determined by multiplex PCR with 8 gradients in copies/tube. The results were analyzed by 1.5% agarose gel electrophoresis, and as shown in FIG. 9, the results showed that the sensitivity of the seven-fold PCR reached 103copies/mu l, and the sensitivity reaches the requirement of multiplex PCR detection.
Example 14:
and (3) detecting a clinical sample:
clinical sample detection and virus isolation. The method established by the preliminary application detects 66 clinical samples: samples collected from the surrounding area of Nanjing, Jiangsu, 2017, 4 to 2018, 3 are detected and analyzed for the virus-carrying condition by a multiplex PCR method.
The 66 parts of the grinding fluid of the disease material are respectively used for extracting virus DNA/RNA, and the detection is carried out according to the method of the example 5. The multiple PCR detection result shows that, among 66 specimens, 5 strains of AIV positive virus, 8 strains of NDV virus, 8 strains of DEV10 strains of DHAV virus, 6 strains of FAdV virus, 3 strains of DTMUV virus and 2 strains of GPV virus are identified; DHAV + DEV co-infected 4 strains, DHAV + DTMUV co-infected 2 strains, AIV + NDV co-infected 3 strains, AIV + DEV co-infected 1 strain, NDV + DEV + DHAV co-infected 1 strain, and AIV + DTMUV + NDV co-infected 1 strain.
From the above results, it can be seen that the multiplex PCR detection method for viruses has the characteristics of short time consumption and high sensitivity compared with the virus isolation method, and the positive results of virus isolation are all positive in multiplex PCR detection. The experiment preliminarily proves that the method has feasibility in the aspect of virus detection.
As can be seen from the above examples, the NDV, AIV, DEV, DHAV, FAdV, DTMUV and GPV virus specific primers designed by the present invention have strong specificity, and are negative for detecting viruses except NDV, AIV, DEV, DHAV, FAdV, DTMUV and GPV viruses; the multiple PCR detection method established by batch-to-batch internal experiments has good stability.
In conclusion, compared with the prior art, the m-PCR method provided by the invention can simultaneously detect seven waterfowl viruses of NDV, AIV, DEV, DHAV, FAdV, DTMUV and GPV, can identify and diagnose the seven viruses, and has the advantages of strong detection specificity, high sensitivity, simple and rapid operation and low cost.
The m-PCR method can simultaneously detect seven waterfowl viruses of NDV, AIV, DEV, DHAV, FAdV, DTMUV and GPV, can quickly identify and diagnose whether waterfowls are infected with the viruses to be detected in actual production, and can monitor the disease condition of the waterfowls, thereby providing an effective detection means for large-scale virus monitoring, epidemiological investigation and prevention and control of the diseases of NDV, AIV, DEV, DHAV, FAdV, DTMUV and GPV clinically. The invention has high detection sensitivity, strong specificity and good repeatability, and has lower cost, rapidness, simplicity, convenience, time saving and labor saving compared with other detection methods.
It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.
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Claims (2)
1. A use method of seven PCR primer groups for simultaneously detecting seven waterfowl susceptibility viruses is characterized in that: the method is used for non-diagnosis, the sequences of the primer sets are respectively FAdV-F, FAdV-R, DHAV-F, DHAV-R, DEV-F, DEV-R, AIV-F, AIV-R, DTMUV-F, DTMUV-R, NDV-F, NDV-R, GPV-F, GPV-R, and the sequences of the primer sets from 5 'end to 3' end are respectively:
FAdV-F：CAACAGCCTCTCGTACCCAG、
FAdV-R：CCGATGTAGTTGGGCCTGAG、
DHAV-F：CTTTCCACTCCCTGCTCCC、
DHAV-R：TTGGCTTCCACATCCTCTTCA、
DEV-F：ATCGCATGTAGACGTTGGTT、
DEV-R：AGACAGCGGTGATGGATGG、
AIV-F：GGCGACTACTACCAACCCA、
AIV-R：CTGCTGTTCCTGCCGATAT、
DTMUV-F：AATCGGTAGTGGCTTTGG、
DTMUV-R：AGTCTGCCGACATGGATAT、
NDV-F:CACCGGCAACCCTATTCTGT、
NDV-R:AGTGCGCCTTCAGTCTTTGA、
GPV-F:TATGTCCTGGGCTCGGCTAC、
GPV-R:AGCTGACACAGGTCCAGGTT；
the seven waterfowl susceptible viruses are duck-origin avian influenza virus, newcastle disease virus, avian adenovirus, duck plague virus, A-type duck hepatitis virus, duck tembusu virus and goose parvovirus;
carrying out PCR amplification by using the primer group sequence, and judging a positive result according to a specific amplification strip of agarose gel electrophoresis after the reaction is finished;
performing PCR amplification under the reaction conditions of pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30s, annealing at 57-57.5 ℃ for 30s, extension at 72 ℃ for 40s, performing 35 cycles, and extension at 72 ℃ for 10 min;
in the PCR reaction system, the concentration of dNTP is 0.4mM, and the concentration of magnesium ions is 4 mM;
in the PCR, the concentration of DNA polymerase in a reaction system is 0.05 u/mul;
in the PCR reaction system, the final concentrations of the primers are respectively as follows: FAdV-F1/R1: 0.12. mu. M, DHAV-F2/R2: 0.12. mu. M, DEV-F1/R1: 0.16. mu. M, AIV-F3/R3: 0.24. mu. M, DTMUV-F1/R1: 0.28. mu. M, NDV-F2/R2: 0.24. mu. M, GPV-F5/R5: 0.24 μ M; the reaction system of the PCR is a 25-microliter system comprising 2.5 microliter of 10xtaqbuffer, 0.25 microliter of DNA polymerase, 1 microliter of dNTP and 25mM MgCl24 μl。
2. The method for simultaneously detecting seven waterfowl susceptible viruses by using the seven-fold PCR primer set according to claim 1, wherein the seven PCR primer set comprises the following steps: the DNA detection band size of the avian adenovirus is 102bp, the DNA detection band size of the A-type duck hepatitis virus is 140bp, the DNA detection band size of the duck plague virus is 172bp, the DNA detection band size of the duck source avian influenza virus is 435bp, the DNA detection band size of the duck tembusu virus is 288bp, the DNA detection band size of the goose parvovirus is 516bp, and the DNA detection band size of the Newcastle disease virus is 330 bp.
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