CN110777219A - Triple PCR detection primer group, kit and method for chicken parvovirus and H9 subtype avian influenza virus - Google Patents
Triple PCR detection primer group, kit and method for chicken parvovirus and H9 subtype avian influenza virus Download PDFInfo
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Abstract
The invention discloses a triple PCR detection primer group, a kit and a method for chicken parvovirus and H9 subtype avian influenza virus, which designs specific primers for the chicken parvovirus and the H9 subtype avian influenza virus, obtains a primer combination with better amplification effect by performing test screening on different primer combinations, then continuously optimizes the primer concentration, the proportion among the primers, the annealing temperature and the amplification time in a reaction system, and finally establishes a method for simultaneously detecting the chicken parvovirus and the H9 subtype avian influenza virus. The primer and the detection method can simultaneously determine the mixed infection and single infection condition of the chicken parvovirus and the H9 subtype avian influenza virus in a sample through one-time PCR reaction, and the method has the advantages of strong specificity, high sensitivity, rapidness, simplicity, convenience, easy operation and the like, is very suitable for application and popularization in basic units, and provides technical support for monitoring and prevention and control of the chicken parvovirus and the H9 subtype avian influenza virus.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and relates to a triple PCR detection primer group, a triple PCR detection kit and a triple PCR detection method for chicken parvovirus and H9 subtype avian influenza virus.
Background
Chicken parvovirus (ChPV) is one of the important viruses that cause intestinal diseases in chicken flocks and is clinically characterized by diarrhea, mental depression, growth retardation, increase in feed conversion ratio and the like. ChPV mainly affects chicks and mainly causes sick chicken diarrhea and dysplasia, the virus is ubiquitous in certain chicken flocks, and the ChPV DNA can be detected in the serum of the chicken flocks with the dysplasia and the short syndrome. Epidemiological investigation shows that the infection rate of commercial broilers is higher, and the number of laying hens or breeding hens is lower. Studies in recent years have shown that ChPV is ubiquitous in chicken flocks in many countries around the world, and in recent years reports have been found successively in the united states and brazil in north america, poland hungary in eastern europe, korea in crohn's disease and asia, and in china, among other areas.
Avian Influenza Virus (AIV) is a member of the influenza a genus of the orthomyxoviridae family. AIV can be divided into 16 different HA subtypes (H1-H16) and 9 different NA subtypes (N1-N9) according to the difference between surface antigens of Hemagglutinin (HA) and Neuraminidase (NA). AIV can also be classified into highly pathogenic AIV (hpaiv) and low pathogenic AIV (lpaiv) according to the degree of its pathogenicity to chickens. AIV subtype H9 was first discovered in the United states since 1966 and has now caused significant economic losses to the poultry industry worldwide. Research shows that H9 subtype AIV is easy to be mixed with other pathogens (viruses, bacteria and the like) to infect, thereby causing more serious loss to poultry industry in China; when the novel avian influenza virus is mixed with other different subtypes of AIV for infection, gene fragment recombination is easy to occur among strains, some unpredictable novel avian influenza viruses are generated, and then the novel avian influenza viruses can cause pandemics, and part of reports from 2013 also show that the novel avian influenza virus can provide internal genes for highly pathogenic subtypes of AIV such as H5N6 and H7N 9. The infection of human with AIV of subtype H9N2 has also been ongoing since the first discovery in Guangdong of 1998 of human infections with AIV of subtype H9N 2. Therefore, the H9 subtype AIV has important significance for poultry cultivation and human health and hygiene.
The chicken parvovirus is difficult to multiply in large quantity in normal chick embryo and cell culture, so that the diagnosis of the chicken parvovirus by using methods such as virus separation, electron microscope observation and the like is difficult.
The traditional avian influenza virus detection method is characterized in that the virus is subjected to serological method identification after being subjected to separation culture, the culture period is long, more standard positive serum is used, but cross immune protection reactions of different degrees exist among different subtype serum of the avian influenza virus, the accuracy of the result is limited, so that the avian influenza virus is detected by adopting a molecular biological method at present more frequently. The molecular biological method, particularly the PCR detection technology, has the advantages of rapidness, simplicity, convenience, accuracy and the like, and is widely applied in the diagnosis and large-scale monitoring process of the avian influenza. Since AIV has similar clinical symptoms after infecting animals and the phenomenon of mixed infection also exists commonly, the AIV is difficult to identify and diagnose accurately in time in daily diagnosis. In recent years, with the development of molecular biology technology, the application of multiple PCR technology is more and more, the multiple PCR has the advantage of simultaneously detecting the mixed infection of a plurality of pathogens, the defect that the time consumption for detecting mixed infection samples one by the conventional single PCR is overcome, and the continuous improvement and development progress of the multiple PCR diagnosis technology are overcome, and the technology is mature to be applied to the diagnosis of mixed infection of a plurality of poultry diseases and other animal pathogens
Disclosure of Invention
The invention aims to provide a triple PCR detection primer group, a kit and a method for chicken parvovirus and H9 subtype avian influenza virus.
In order to realize the purpose of the invention, the technical scheme of the invention is as follows: the invention provides a primer group capable of detecting chicken parvovirus and H9 subtype avian influenza virus through triple PCR, which comprises a primer M-F, a primer M-R, a primer H9-F, a primer H9-R, a primer ChPV-F and a primer ChPV-R.
The sequence of the primer M-F is shown as SEQ.ID.NO.1, the sequence of the primer M-R is shown as SEQ.ID.NO.2, the sequence of the primer H9-F is shown as SEQ.ID.NO.3, the sequence of the primer H9-R is shown as SEQ.ID.NO.4, the sequence of the primer ChPV-F is shown as SEQ.ID.NO.5, and the sequence of the primer ChPV-R is shown as SEQ.ID.NO. 6.
The reagent or the kit containing the primer group belongs to the protection scope of the invention.
The invention provides a method for detecting chicken parvovirus and H9 subtype avian influenza virus by triple PCR (polymerase chain reaction) by using the primer group.
The method further comprises the following steps:
(1) extracting cDNA and DNA of a sample to be detected;
(2) establishing a triple PCR reaction system, and carrying out triple PCR reaction by using the primer group;
(3) and detecting the PCR reaction product by agarose gel electrophoresis, wherein the sample has chicken parvovirus when a 551bp band is amplified, the sample has H9 subtype avian influenza virus when 378bp and 249bp bands are amplified, and the sample has other subtype avian influenza viruses when only the 249bp band but not the 378bp band is amplified.
Wherein, the PCR reaction system in the step (2) is as follows: 2 XPCR Mix 12.5. mu.L, sample to be tested cDNA and DNA mixture template 2. mu.L, primers H9-F and H9-R (25 pmol/. mu.L) each added 0.5. mu.L, primers ChPV-F and ChPV-R (25 pmol/. mu.L) each added 0.8. mu.L, primers M-F and M-R (25 pmol/. mu.L) each added 0.8. mu.L, and finally made up to 25. mu.L with ultra pure water.
Wherein, the triple PCR reaction conditions in the step (2) are as follows: 5min at 95 ℃; 35 cycles of 95 ℃ for 30s, 56 ℃ for 45s and 72 ℃ for 45s are carried out; then further extension at 72 ℃ for 10 min.
Wherein, 1 μ L of cDNA and 1 μ L of DNA of a sample to be tested are mixed to obtain a mixture template.
The invention provides application of the primer group or a kit containing the primer group in detection of chicken parvovirus and H9 subtype avian influenza virus.
The invention has the advantages of
The invention designs specific primers aiming at chicken parvovirus and H9 subtype avian influenza virus, obtains a primer combination with better amplification effect by testing and screening different primer combinations, then continuously optimizes the primer concentration, the proportion among the primers, the annealing temperature and the amplification time in a reaction system, and finally establishes a method for simultaneously detecting the chicken parvovirus and the H9 subtype avian influenza virus. The primer and the detection method can simultaneously determine the mixed infection and single infection condition of the chicken parvovirus and the H9 subtype avian influenza virus in a sample through one-time PCR reaction, and the method has the advantages of strong specificity, high sensitivity, rapidness, simplicity, convenience, easy operation and the like, is very suitable for application and popularization in basic units, and provides technical support for monitoring and prevention and control of the chicken parvovirus and the H9 subtype avian influenza virus.
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FIG. 1 shows the results of a specificity test;
FIG. 2 shows the results of the sensitivity test;
FIG. 3 shows the results of a part of clinical samples.
Detailed Description
The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 primer design and Synthesis
According to M gene sequences of different subtype avian influenza viruses, HA gene sequences of H9 subtype AIV and specific conserved regions of ChPV NS genes, 3 pairs of specific primers for amplifying the M genes, the H9 subtype AIV HA genes and the ChPV NS genes are designed and screened by verification, wherein the specific primers comprise a primer M-F, a primer M-R, a primer H9-F, a primer H9-R, a primer ChPV-F and a primer ChPV-R. The sequence of the primer M-F is shown as SEQ.ID.NO.1, the sequence of the primer M-R is shown as SEQ.ID.NO.2, the sequence of the primer H9-F is shown as SEQ.ID.NO.3, the sequence of the primer H9-R is shown as SEQ.ID.NO.4, the sequence of the primer ChPV-F is shown as SEQ.ID.NO.5, and the sequence of the primer ChPV-R is shown as SEQ.ID.NO. 6. Primers were synthesized by the guangzhou division, siemmer.
Example 2 establishment and optimization of triple PCR reaction System and specificity test
1. Strain: AIV strains (H1N2, H3N2, H6N2, H9N2, H9N6, NDV, IBV, ARV, ILTV, ChPV, Adv4, RTV, aMPV, CAV, AHEV, and MDV) were stored by the Guangxi veterinary biotech focus laboratory; the cDNA template for AIV strain (H7N2) was complimentary to American Pennsylvania State university; AIV strain (H5N1) cDNA template was offered by the united states connecticut state university; other AIV strains (H2N3, H8N4, H10N3 and H11N3) or cDNA templates were each gifted by hong kong university;
2. main reagents and instruments: PCR instruments were purchased from Bio-Rad Laboratories, Inc., USA; a micro-spectrophotometer is available from Thermo corporation, usa; 2 XPCR Super Mix, DNA/RNA co-extraction kit, small-amount plasmid extraction kit, gel recovery kit, competent cells, PCR product quick connection carrier and DNA Marker are purchased from Beijing all-type gold biotechnology limited; M-MLV, random primers, dNTPs, and RNA inhibitors were purchased from Baozhi (Beijing) Ltd; other reagents were purchased from commercial companies;
3. extraction of viral RNA/DNA and cDNA Synthesis: the DNA/RNA of AIV, NDV, IBV, ARV, ILTV, ChPV, Adv4, RTV, aMPV, CAV, AHEV and MDV used in this study was extracted with reference to the nucleic acid extraction kit using the instructions and the extracted nucleic acid template was dissolved in 33. mu.L DEPC water. Synthesizing cDNA by the RNA template according to the specification of the reverse transcription kit, and storing all the cDNA and the DNA template at the low temperature of-30 ℃ for later use;
4. establishing and optimizing a triple PCR reaction system: a25. mu.L PCR reaction was set up: 2 XPCR Mix12.5 uL, ChPV DNA and H9 subtype AIV cDNA template each 1 uL, primer ChPV-F, ChPV-R, H9-F, H9-R, M-F and M-R (25 pmol/. mu.L) each 0.1-1.0 uL, set up 10 test gradients in total, each gradient increases by 0.1 uL, carry on the optimization of three pairs of primer combination concentration, finally complement to 25 uL with RNA-free;
finally, the optimal reaction system of the triple PCR is determined as follows: : 2 XPCR Mix12.5 uL, sample cDNA and DNA mixture template 2 uL to be tested, primers H9-F and H9-R (25 pmol/. mu.L) each added 0.5 uL, primers ChPV-F and ChPV-R (25 pmol/. mu.L) each added 0.8 uL, primers M-F and M-R (25 pmol/. mu.L) each added 0.8 uL, finally make up to 25 uL with ultra pure water; the optimal reaction conditions are as follows: 5min at 95 ℃; 35 cycles of 95 ℃ for 30s, 56 ℃ for 45s and 72 ℃ for 45s are carried out; then further extension at 72 ℃ for 10 min.
5. The triple PCR detection method established above is used to detect and verify the specificity of the established method according to the detection of cDNA/DNA of H1N2, H2N3, H3N2, H5N1, H6N2, H7N2, H8N4, H9N2, H9N6, H10N3, H11N3, NDV, IBV, ARV, ILTV, ChPV, Adv4, RTV, aMPV, CAV, AHEV and MDV. The PCR reaction product was electrophoresed through agarose gel, the electrophoresis image is shown in FIG. 1, and the lanes in FIG. 1 are respectively: dl2000 DNA Marker; H9N2+ ChPV; ChPV + H6N 2; H9N 6; ChPV; H6N 2; H1N 2; H2N 3; H3N 2; H5N 2; H7N 2; H8N 4; H10N 3; H11N 3; 14. chicken infectious anemia virus; 15. avian group I adenovirus type 4; 16. avian rotavirus; 17. avian metapneumovirus; 18. avian hepatitis e virus 19 infectious bronchitis virus of chicken; 20. avian reovirus; 21. infectious laryngotracheitis virus of chicken; 22. newcastle disease virus of chicken; 23. marek's virus; 24. and (5) negative control.
As can be seen from FIG. 1, 3 specific bands were detected from H9 and ChPV mixed infection samples by the established triple PCR method, which are 551bp (ChPV), 249bp (AIV M gene general purpose) and 378bp (H9 subtype), respectively; only 2 specific bands appear in the PCR detection results of H9N2 and H9N6 subtype AIV, and the sizes of the fragments are 378bp and 249bp respectively; only 1 specific band appears on the ChPV detection result, and the fragment size is 551 bp; 1 specific 249bp band appears for other subtype AIV, and no band is amplified by other common avian pathogens, so that the method has good specificity.
Example 3 sensitivity test
1. Preparation of a standard substance: reference [ Li Dan, Xiezui, Song De Gui, etc. ] establishment of double PCR detection method for subtype H9 and H6 avian influenza virus [ J]Chinese veterinary medicine 2016,43(12):3101-3106.]The method comprises the steps of carrying out PCR amplification by using primers of ChPV NS gene, AIV M gene and H9N2 HA full-length gene and using cDNA and DNA of H9N2 and ChPV as templates respectively to obtain full-length target fragments of the HA, M gene and NS gene, and linking the three gene fragments to a pMD18-T vector respectively. The recombinant vectors containing the correct sequences of the HA gene M gene and ChPV NS gene fragments of H9 subtype AIV were named H9-T, M-T and NS-T, respectively. Extracting plasmids of H9-T, M-T and NS-T by a plasmid extraction kit, determining the nucleic acid concentration by a trace nucleic acid detector, calculating corresponding copy numbers according to the molecular mass and the nucleic acid concentration, mixing the copy numbers of H9-T, M-T, NS-T and the like, and diluting by 10 times to obtain the plasmids of H9-T, M-T and NS-T with the DNA concentration of 5 multiplied by 10
9～5×10
1Copies/. mu.L of standard.
2. Sensitivity test the optimized triple PCR detection method is used for H9-T, M-T and NS-T plasmid concentration of 5 x 10
7Copies/. mu.L to 5X 10
2The sensitivity of the method was tested by specific amplification of samples in copies/. mu.L. The PCR product was electrophoresed through agarose gel, the electrophoresis image is shown in FIG. 2, and the lanes in FIG. 2 are: dna standard DL 2000; 1.5X 10
7Copy/. mu.L; 2.5X 10
6Copy/. mu.L; 3.5X 10
5Copy/. mu.L; 4.5X 10
4Copy/. mu.L; 5.5X 10
3Copy/. mu.L; 6.5X 10
2Copy/. mu.L; 7. and (5) negative control. As can be seen from FIG. 2, the concentration is 5X 10
7～5×10
4Copy/mul AIV M gene, H9 subtype AIV HA gene and ChPV NS gene plasmid have 3 obvious specific amplification bands, the size of the segment is 249bp, 378bp and 551 bp; for concentration equal to or less than 5X 10
3No amplification band was found in each of the copy/. mu.L of AIV M gene, H9 subtype AIV HA gene and ChPV NS gene plasmids. It can be seen that the method can detect 5 × 10 at the lowest
4The copy/mu L of AIV M gene, H9 subtype AIV HA gene and ChPV NS gene plasmid shows that the method HAs high detection sensitivity.
Example 4 clinical sample testing
1. Detecting a sample: 130 parts of chicken oral cavity and cloaca swab samples are collected in the Nanning live poultry market, DNA and RNA of the samples are extracted, and the cDNA is obtained by reverse transcription of the RNA;
2. PCR reaction system and reaction conditions:
the reaction system is as follows: 2 XPCR Mix12.5 uL, sample to be tested cDNA and DNA (mixture, 1 uL each) template 2 uL, primers H9-F and H9-R (25 pmol/. mu.L) each added 0.5 uL, primers ChPV-F and ChPV-R (25 pmol/. mu.L) each added 0.8 uL, primers M-F and M-R (25 pmol/. mu.L) each added 0.8 uL, finally make up to 25 uL with ultra pure water;
the reaction conditions are as follows: 5min at 95 ℃; 35 cycles of 95 ℃ for 30s, 56 ℃ for 45s and 72 ℃ for 45s are carried out; then extending for 10min at 72 ℃;
3. the PCR reaction products are detected by agarose gel electrophoresis, and the detection result shows that 2 samples can simultaneously amplify target bands of 378bp, 249bp and 551bp, which are H9 subtype AIV and ChPV mixed infection positive, 6 samples can simultaneously amplify target bands of 378bp and 249bp, which are single H9 subtype AIV positive, 13 samples can only amplify target bands of 551bp, which are single ChPV infection positive, 3 samples can only amplify target bands of 249bp, which are H3 and H6 subtype AIV infection except H9 subtype; the results of the partial clinical sample tests are shown in FIG. 3, samples 19, 23 and 24 are positive for ChPV, and samples 7 and 12 are positive for AIV of H9 subtype; 10 is positive for mixed infection of H9 subtype AIV and ChPV; 11 is positive for other subtypes of AIV; the remainder were negative. The detection result of the sample is consistent with the separation and identification of the classical chick embryo virus and the sequencing results of the HA gene and the M gene of the chick embryo virus by 100 percent.
Sequence listing
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Claims (7)
1. A triple PCR detection primer group for chicken parvovirus and H9 subtype avian influenza virus comprises a primer M-F, a primer M-R, a primer H9-F, a primer H9-R, a primer ChPV-F and a primer ChPV-R;
the sequence of the primer M-F is shown as SEQ.ID.NO.1, the sequence of the primer M-R is shown as SEQ.ID.NO.2, the sequence of the primer H9-F is shown as SEQ.ID.NO.3, the sequence of the primer H9-R is shown as SEQ.ID.NO.4, the sequence of the primer ChPV-F is shown as SEQ.ID.NO.5, and the sequence of the primer ChPV-R is shown as SEQ.ID.NO. 6.
2. A reagent or a kit comprising the primer set according to claim 1.
3. A method for detecting chicken parvovirus and H9 subtype avian influenza virus by using triple PCR, which is characterized in that the primer used by the triple PCR is the primer group of claim 1.
4. A method according to claim 3, comprising the steps of:
(1) extracting cDNA and DNA of a sample to be detected;
(2) establishing a triple PCR reaction system, and carrying out triple PCR reaction by using the primer group;
(3) and detecting the PCR reaction product by agarose gel electrophoresis, wherein the sample has chicken parvovirus when a 551bp band is amplified, the sample has H9 subtype avian influenza virus when 378bp and 249bp bands are amplified, and the sample has other subtype avian influenza viruses when only the 249bp band but not the 378bp band is amplified.
5. The method of claim 4, wherein: the PCR reaction system in the step (2) is as follows: 2 XPCR Mix12.5. mu.L, 2. mu.L of the cDNA and DNA mixed template of the sample to be tested, 0.5. mu.L of each of the primers H9-F and H9-R (25 pmol/. mu.L), 0.8. mu.L of each of the primers ChPV-F and ChPV-R (25 pmol/. mu.L), 0.8. mu.L of each of the primers M-F and M-R (25 pmol/. mu.L), and finally, 25. mu.L of the mixture is made up with ultrapure water.
6. The method of claim 4, wherein: the triple PCR reaction conditions in the step (2) are as follows: 5min at 95 ℃; 35 cycles of 95 ℃ for 30s, 56 ℃ for 45s and 72 ℃ for 45s are carried out; then further extension at 72 ℃ for 10 min.
7. The primer group of claim 1 or the kit containing the primer group is applied to detection of chicken parvovirus and H9 subtype avian influenza virus.
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