CN117737298A - Multiplex PCR primer, detection reagent, method and application for identifying avian infectious bronchitis viruses with different genotypes simultaneously - Google Patents
Multiplex PCR primer, detection reagent, method and application for identifying avian infectious bronchitis viruses with different genotypes simultaneously Download PDFInfo
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Abstract
The invention discloses a multiplex PCR primer, a detection reagent, a method and application for simultaneously identifying avian infectious bronchitis viruses with different genotypes. The multiplex PCR primer constructed by the invention can identify the dominant epidemic strains QX type, GVI-1 type and common vaccine strains of the avian infectious bronchitis virus through one-time PCR operation. And the whole operation process is simple, one-time PCR can identify one or more genotypes contained in the sample to be detected, so that the detection cost is greatly reduced, and the detection efficiency is improved.
Description
Technical Field
The invention relates to the field of virus specificity detection, in particular to a multiplex PCR primer, a detection reagent, a method and application for simultaneously identifying avian infectious bronchitis viruses with different genotypes.
Background
Avian infectious bronchitis virus (Infectious bronchitis virus, IBV) is highly pathogenic and susceptible to flocks, and its transmission causes serious economic losses to the world's poultry industry. In 1937, the first separation of American science into IBV,1972, occurred in the first instance IB, which was found in Guangdong province. IBV can infect chickens of different varieties and sexes and all ages of days, and chickens of 1 to 6 weeks age are most susceptible to disease, focusing on disease in winter and spring. The sick chickens infected with IBV behave differently and include respiratory, digestive, genital and kidney diseases. IBV infection is particularly serious for laying hens, the earlier the infection is, the larger the infection is, the chicken infection mainly causes permanent damage to reproductive systems, the chicken becomes a fake hen after adult, the egg quality is reduced, the laying rate is reduced by 10-30%, the chicken flocks have no egg laying peak, and the cost is increased, so that the production loss is caused. IBV belongs to gamma coronavirus, has high mutation rate, lacks pertinence in vaccine use in culture production, and has large difference between common vaccine strains and epidemic strains and weak cross protection. Therefore, although the disease has been reported in China for decades, the prevention and control of the disease are still not ideal nowadays.
The epidemic situation of IBV is complex, and dominant epidemic strains of different chicken farms in different areas are different. IBVs are divided into 6 genotypes, including GI through VI, where GI contains 27 lineages and GII through VI each have 1 lineages. Relevant researches show that the current epidemic type of chicken IBV in various provinces in China is 5 genotypes, the QX (GI-19, the most dominant epidemic type in the last 10 years) accounts for 66.5%, the GVI-1 (most novel) accounts for 27.7%, and the two types are the current dominant types.
However, there is currently no easy detection method for distinguishing between the dominant epidemic strain QX type, the GVI-1 type and the common vaccine strain genotype.
Disclosure of Invention
Aiming at the prior art, the invention aims at providing a multiplex PCR primer, a detection reagent, a method and application capable of rapidly realizing the distinguishing detection of dominant epidemic strains and common vaccine strains of IBV viruses and simultaneously identifying avian infectious bronchitis viruses of different genotypes aiming at the defects of detection of IBV viruses of different genotypes in the prior art.
In order to achieve the aim, the invention provides a multiplex PCR primer for identifying avian infectious bronchitis viruses with different genotypes at the same time, which comprises a first primer pair with nucleotide sequences shown as SEQ ID No. 1 and SEQ ID No. 2, a second primer pair with nucleotide sequences shown as SEQ ID No. 3 and SEQ ID No. 4, a third primer pair with nucleotide sequences shown as SEQ ID No. 5 and SEQ ID No. 6, a fourth primer pair with nucleotide sequences shown as SEQ ID No. 7 and SEQ ID No. 8, and a fifth primer pair with nucleotide sequences shown as SEQ ID No. 9 and SEQ ID No. 10.
The invention also provides a detection reagent for identifying different genotypes of avian infectious bronchitis viruses simultaneously, wherein the detection reagent comprises the multiplex PCR primer.
The invention also provides a method for identifying avian infectious bronchitis viruses with different genotypes by using the non-disease diagnosis purpose, and the multiplex PCR primer and/or the detection reagent are adopted.
Preferably, the method comprises:
s100, obtaining a nucleic acid sample of a sample to be detected;
s200, performing multiplex PCR amplification on the nucleic acid sample by using the multiplex PCR primer and/or the detection reagent;
s300, performing agarose gel electrophoresis detection on the amplification product obtained in the step S200;
s400, judging the sample to be tested according to the bands appearing in the obtained agarose gel electrophoresis diagram.
Preferably, in step S200, the concentration of each primer is 0.1 to 0.5. Mu. Mol/L, and most preferably 0.1. Mu. Mol/L, relative to the total volume of the 25. Mu.L PCR amplification system.
Preferably, in step S200, the PCR amplification process is performed at an annealing temperature of 48-60℃and a cycle number of 20-35. Further preferably, the annealing temperature is 58℃and the number of cycles is 35.
Preferably, in step S400, if the length of the DNA fragment appearing in the lane is 268bp, it is determined that the sample to be tested contains IBV Mass type;
the length of the DNA fragment appearing in the lane is 168bp, and the sample to be detected is judged to contain IBV QX type;
the length of the DNA fragment appearing in the lanes is 537bp, and the sample to be detected is judged to contain IBV G VI-1;
the length of the DNA fragment appearing in the lane is 642bp, and the sample to be detected is judged to contain IBV 4/91 type;
and if the length of the DNA fragment appearing in the lane is 440bp, judging that the sample to be detected contains IBV LDT3.
The invention also provides application of the multiplex PCR primer in distinguishing avian infectious bronchitis viruses with different genotypes, wherein the avian infectious bronchitis viruses comprise IBV QX type, IBV G VI-1 type, IBV Mass type, IBV 4/91 type and IBV LDT3 type.
Preferably, the detection limit of the multiplex PCR primer pair IBV QX type is 1 copy/mu L, and the detection limit of the multiplex PCR primer pair IBV GVI-1 type is 10 2 Copy/. Mu.L, detection limits for IBV Mass type, IBV LDT3 type and IBV 4/91 type are 10 3 Copy/. Mu.L.
Through the technical scheme, the multiplex PCR primer constructed by the invention can identify the dominant epidemic strains QX type, the GVI-1 type and the common vaccine strains of the avian infectious bronchitis virus through one-time PCR operation. In addition, the whole operation process is simple, one-time PCR can identify one or more genotypes contained in the sample to be detected, the detection cost is greatly reduced, the detection efficiency is improved, a foundation is laid for systematically exploring the epidemic rule of IBV and scientifically guiding the selection of targeted vaccines, and the breeding benefit of laying hens is improved.
Drawings
The accompanying drawings are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate the invention and together with the description serve to explain, without limitation, the invention. In the drawings:
FIG. 1 is an agarose gel electrophoresis chart obtained in verification example 1;
FIG. 2 is an agarose gel electrophoresis chart obtained in verification example 2;
FIGS. 3A-3D are agarose gel electrophoresis patterns obtained in example 1;
FIGS. 4A-4D are agarose gel electrophoresis patterns obtained in example 2;
FIGS. 5A-5D are agarose gel electrophoresis patterns obtained in example 3;
FIG. 6 is an agarose gel electrophoresis chart obtained in example 4;
FIG. 7 is an agarose gel electrophoresis chart obtained in verification example 3;
FIGS. 8A to 8F are agarose gel electrophoresis patterns obtained in example 5;
FIG. 9 is an agarose gel electrophoresis chart obtained in application example 1;
FIGS. 10A to 10G are agarose gel electrophoresis patterns obtained in application example 2.
Detailed Description
The following describes specific embodiments of the present invention in detail. It should be understood that the detailed description and specific examples, while indicating and illustrating the invention, are not intended to limit the invention.
The technical scheme of the invention is described in detail by specific examples.
Wherein, the IBV live vaccine LaSota strain +QXL87 strain, shi-Zhang Wei FNO-E55 (793/B or 4/91) strain, xin-zhuan-Ning LDT3-A strain, wei-zhuan-ling VG/GA+H2120 strain, infectious Bursal Disease Virus (IBDV) vaccine strain B87 strain and Newcastle Disease Virus (NDV) vaccine strain La Sota strain are purchased and stored in the laboratory; the IBV QX type, IBV 4/91 type and G VI-1 type wild strains applied in the experiment are presented to Shandong province academy of agricultural sciences; the H9N 2C/SH/F/98 strain is donated to the university of Yangzhou veterinary medical college; MDRV HNF strain is donated to the national academy of agricultural sciences of Fujian province. It should be noted that the technical scheme of the present invention is effective for IBV Mass type viruses, and the specific embodiment of the present invention adopts H120 type therein. However, the present invention is not limited thereto, and other IBV Mass type viruses as will be understood by those skilled in the art can be identified by the present invention.
Viral RNA Extraction Kit DNAMaroker is available from TaKaRa; 2 XTaq mix was purchased from Beijing kang as century biotechnology Co., ltd; reverse transcription kits were purchased from nuozhen biotechnology, inc.
Preparation example
IBV H120 (i.e. Williams VG/GA+H2 strain, denoted as 1#), IBV 4/91 (i.e. Shi Zhang Wei FNO-E55, denoted as 2#), IBV LDT3-A (i.e. Xinzhuangning LDT3-A strain, denoted as 3#), IBV QXL87 (i.e. IBV live vaccine LaSota+QXL87 strain, denoted as 4#), IBDV B87 (denoted as 5#), and NDV La Sota (denoted as 6#) vaccine dilutions were each 200. Mu.L, IBV QX wild strain (denoted as 7#), IBV 4/91 wild strain (denoted as 8#), IBV G VI-1 wild strain (denoted as 9#), H9N 2C/SH/F/98 (denoted as 10#) and MDRV strain provided by the Fujian university veterinary academy were each 200. Mu.L) were extracted from Shando agricultural sciences, and stored as a total cDNA kit by reverse transcription at a temperature of 200. Mu.L, respectively.
The primer sequences of the first primer pair, the second primer pair, the third primer pair, the fourth primer pair and the fifth primer pair are specifically as follows:
SEQ ID No:1：AAGTGCGGCTTCCATAGC
SEQ ID No:2：TGATCACGTGCAATCATGC
SEQ ID No:3：GTGGTAGGTTAGCTTGTCAG
SEQ ID No:4：ACACAAGCAATACGCACTC
SEQ ID No:5：GGAGGTCCTTTGCTGTGT
SEQ ID No:6：TAGCCAAACCTGCGTCTGCT
SEQ ID No:7：GAAACAACTCACGTTACGGGTGC
SEQ ID No:8：GTATTGCGTTAGCTCCCCTCT
SEQ ID No:9：TTCATATAATGGTCCTCGCGC
SEQ ID No:10：GAGGCTGAGAACCTGTTGCATT
example 1
The cDNAs of the four vaccine strains # 5, # 1, # 2 and # 3 in the preparation example and the strain # 9 were mixed in equal volumes to obtain a mixed template. Multiplex PCR amplification is performed on the mixed templates using the multiplex PCR primers of the present invention (including the first primer pair, the second primer pair, the third primer pair, the fourth primer pair, and the fifth primer pair).
The PCR reaction system was 25. Mu.L: the total amount of the mixed primers was 2.5. Mu.L (0.25. Mu.L for each primer), 12.5. Mu.L for 2 XTaq mix enzyme, 2. Mu.L for each viral template cDNA, and the balance water.
The PCR reaction conditions were: pre-denaturation at 94℃for 4min, denaturation at 94℃for 30s, annealing at 48-54℃for 45s, extension at 72℃for 1min, and final extension at 72℃for 10min, the reaction was repeated for 20-35 cycles.
Wherein the concentration of each primer was 0.1. Mu. Mol/L with respect to 25. Mu.L of the total reaction system. In all of the following examples and preparations, the concentration of the primer was also relative to the total reaction system of the PCR reaction.
The agarose gel electrophoresis patterns obtained are shown in FIGS. 3A-3D. Wherein, in fig. 3A, the annealing temperature is 48 ℃, and the reaction is performed for 20 cycles; in fig. 3B, the annealing temperature is 50 ℃, and the reaction is performed for 25 cycles; in fig. 3C, the annealing temperature is 52 ℃, and the reaction is performed for 30 cycles; in fig. 3D, the annealing temperature was 54 ℃, and the reaction was performed for 35 cycles.
Example 2
The preparation was carried out in the same manner as in example 1 except that the concentration of each primer was 0.2. Mu. Mol/L.
The agarose gel electrophoresis patterns obtained are shown in FIGS. 4A to 4D. Wherein, in fig. 4A, the annealing temperature is 48 ℃, and the reaction is performed for 20 cycles; in fig. 4B, the annealing temperature is 50 ℃, and the reaction is performed for 25 cycles; in fig. 4C, the annealing temperature is 52 ℃, and the reaction is performed for 30 cycles; in fig. 4D, the annealing temperature was 54 ℃, and the reaction was performed for 35 cycles.
Example 3
The preparation was carried out in the same manner as in example 1 except that the concentration of each primer was 0.4. Mu. Mol/L.
The agarose gel electrophoresis patterns obtained are shown in FIGS. 5A to 5D. In FIG. 5A, the annealing temperature is 48℃and the reaction is performed for 20 cycles; in fig. 5B, the annealing temperature is 50 ℃, and the reaction is performed for 25 cycles; in fig. 5C, the annealing temperature is 52 ℃, and the reaction is performed for 30 cycles; in fig. 5D, the annealing temperature was 54 ℃, and the reaction was performed for 35 cycles.
Example 4
Preparation was performed as in example 1, except that the annealing temperature of the strip 49 was 56℃and the cycle number was 30; the annealing temperature of the strip 50 was 56℃and the number of cycles was 35; the annealing temperature of the strip 51 was 58℃and the number of cycles was 30; the annealing temperature of the strip 52 was 58 ℃ and the cycle number was 35; the annealing temperature of the strip 53 is 60 ℃ and the cycle number is 30; the annealing temperature of the strip 54 was 60℃and the number of cycles was 35. The agarose gel electrophoresis pattern obtained is shown in FIG. 6.
Example 5
The cDNAs of preparation examples # 1, # 2, # 3, # 4 and # 9 were amplified, respectively (specific methods can be referred to the specific procedures in verification example 1 below), to obtain respective corresponding amplified products. The obtained amplified products were each taken at 5. Mu.L, detected and purified by 2% agarose gel electrophoresis, cloned into pUC-SP vectors, and sequenced and identified, and the correct recombinant plasmids were identified as plasmid standards, named pUC-H120 (corresponding to # 1), pUC-4/91 (corresponding to # 2), pUC-LDT3 (corresponding to # 3), pUC-GVI-1 (corresponding to # 9) and pUC-QX (corresponding to # 4), respectively. The recombinant plasmid standard is respectively subjected to concentration calculation by a spectrophotometer to calculate copy number (copy/mu L), diluted by 10 times, and the final concentration is respectively selected to be 10 6 Copy/. Mu.L-10 0 The copy/mu L plasmid standard is used as a template, and the optimized multiplex PCR method is used for amplification respectively to evaluate the sensitivity of the multiplex PCR method to a single plasmid standard; mixing 5 plasmid standard substances subjected to 10-time ratio dilution after measurement and conversion in equal volume, and selecting the final concentration as 10 6 Copy/. Mu.L-10 0 The copy/. Mu.L of the mixed plasmid standard is used as a template, and the sensitivity of the multiple PCR method to the mixed plasmid standard is obtained by utilizing the optimized multiple PCR method for amplification. The results obtained are shown in FIGS. 8A-8F.
Verification example 1
cDNA of four vaccine strains # 4, # 1, # 2, # 3 and # 9 strains in the preparation examples were used as templates. Carrying out single PCR amplification on the cDNA of the No. 4 vaccine strain by adopting a first primer pair shown in SEQ ID No. 1 and SEQ ID No. 2; adopts the sequences shown as SEQ ID No. 5 and SEQ ID No. 6The third primer pair shown performs single PCR amplification on cDNA of the 1# vaccine strain; carrying out single PCR amplification on cDNA of the 2# vaccine strain by adopting a fourth primer pair shown as SEQ ID No. 7 and SEQ ID No. 8; carrying out single PCR amplification on cDNA of the 3# vaccine strain by adopting a fifth primer pair shown as SEQ ID No. 9 and SEQ ID No. 10; the cDNA of strain 9# was amplified by single PCR using a second primer pair as shown in SEQ ID No. 3 and SEQ ID No. 4. Corresponding target fragments are obtained after amplification, and ddH is arranged at the same time 2 O is a negative control group.
The PCR reaction system was 25. Mu.L: each of the upstream and downstream primers was 0.5. Mu.L, and each of the 2 XTaq mix enzyme was 12.5. Mu.L, and each of the viral template cDNAs was 2. Mu.L, and ddH was added 2 O was made up to 25. Mu.L.
Each of the above single PCR reaction procedures was set up according to the following conditions: pre-denaturation at 94℃for 4min, denaturation at 94℃for 30s, annealing at 52℃for 45s, extension at 72℃for 1min, and final extension at 72℃for 10min, 30 cycles of reaction. The corresponding agarose gel electrophoresis patterns are shown in fig. 1A-1E, wherein fig. 1A corresponds to # 4, fig. 1B corresponds to # 1, fig. 1C corresponds to # 3, fig. 1D corresponds to # 9, and fig. 1E corresponds to # 2.
Verification example 2
The cDNAs of the four vaccine strains # 4, # 1, # 2 and # 3 in the preparation example and the strain # 9 were mixed in equal volumes to obtain a mixed template, and the mixed template was subjected to single PCR amplification by using five primer pairs according to the method of verification example 1.
Wherein the reaction system was 25. Mu.L: the amount of each of the upstream and downstream primers was 0.5. Mu.L, 12.5. Mu.L of 2 XTaq mix enzyme, and 11.5. Mu.L of 5 kinds of viral cDNA mixture template (ensuring that the amount of template was sufficient).
The reaction conditions were as in verification example 1. The agarose gel electrophoresis pattern obtained is shown in FIG. 2.
Verification example 3
Multiplex PCR was performed according to the PCR system and the PCR conditions in the strip 52 of example 4, and the agarose gel electrophoresis patterns obtained in the preparation examples 1#, 3#, 9#, 7# and 8# and 6#, 10#, 5# and 11# were respectively amplified as shown in FIG. 7. Wherein, from left to right, the 1 st band is a molecular weight mark, and the 2 nd to 10 th bands sequentially correspond to the 7# in the preparation example, the 1# in the preparation example, the 8# in the preparation example, the 3# in the preparation example, the 9# in the preparation example, the 6# in the preparation example, the 10# in the preparation example, the 5# in the preparation example, and the 11 th band in the preparation exampleThe band is negative control ddH 2 O。
Application example 1
IBV H120 (i.e. 1#), IBV LDT3-A (i.e. 3#) vaccine strain and IBV 4/91 (i.e. 8#), IBV GVI-1 (i.e. 9#), IBV QX (i.e. 7#) strain were used for 100EID per virus 50 After two, three or four kinds of random mixing and five kinds of mixing, inoculating 9-day-old SPF chick embryos (n=3) through allantoic cavities respectively, collecting embryo bodies and allantoic fluid respectively after 72 hours, shearing and grinding the embryo bodies, adding PBS (phosphate buffer solution) to prepare homogenate, repeatedly freezing and thawing for 3 times, centrifuging at 5000r/min for 10 minutes, and collecting supernatant as a mixed infection sample. The total RNA of the genome of the supernatant and allantoic fluid after the embryo treatment is extracted by the method of the preparation example, the total RNA is reversely transcribed into cDNA and then detected by using the established multiplex PCR method, and meanwhile, the chicken embryo which is not infected by virus is taken as a negative control. The agarose gel electrophoresis pattern obtained is shown in FIG. 9. Wherein, strip 1 is a 1# and 7# artificial mixed infectious chick embryo sample, strip 2 is a 1# and 3# artificial mixed infectious chick embryo sample, strip 3 is a 8# and 9# artificial mixed infectious chick embryo sample, strip 4 is a 7# and 8# artificial mixed infectious chick embryo sample, strip 5 is a 3# and 9# artificial mixed infectious chick embryo sample, strip 6 is a 7#, 1# and 8# artificial mixed infectious chick embryo sample, strip 7 is a 8#, 3# and 9# artificial mixed infectious chick embryo sample, strip 8 is a 7#, 1#, 3# and 8# artificial mixed infectious chick embryo sample, strip 9 is a 1#, 3#, 8# and 9# artificial mixed infectious chick embryo sample, strip 10 is a 7#, 1#, 3#, 9# and 8# artificial mixed infectious chick embryo sample, and 11 is an uninfected chick embryo sample.
Application example 2
Grinding 6 parts (A-F) of suspected IBV infection pathogenesis laying hen tissue disease materials collected from Jiangsu region into homogenate, adding a proper amount of PBS into 122 parts of mixed sample of laying hen throat and anus double-swab to prepare homogenate, extracting total RNA according to the method of preparation example, reversely transcribing the total RNA into cDNA, respectively detecting by using an established multiple PCR method and single PCR, comparing the detection results of the two methods, and simultaneously setting ddH 2 O is a negative control. Agarose gel electrophoresis obtained after single PCR detection of 6 parts of suspected IBV infection disease layer tissue disease material is shown as 10A-10F (in FIGS. 10A-10F, the lanes in each figure are from left to right: the first lane is molecular weight marker, and the second lane is corresponding to the sampleThe primer used is the first primer pair, the primer used corresponding to the third lane is the second primer pair, the primer used corresponding to the fourth lane is the third primer pair, the primer used corresponding to the fifth lane is the fourth primer pair, and the primer used corresponding to the sixth lane is the fifth primer pair), and the agarose gel electrophoresis diagram obtained after multiplex PCR detection (the primer is a mixed primer comprising the first primer pair, the second primer pair, the third primer pair, the fourth primer pair and the fifth primer pair) is shown in FIG. 10G. The results obtained for 122 parts of the mixed sample of the laying hen throat and anus double swab are shown in table 1. In fig. 10G, the sample of the band 31 corresponds to fig. 10A, the sample of the band 32 corresponds to fig. 10B, the sample of the band 33 corresponds to fig. 10C, the sample of the band 34 corresponds to fig. 10D, the sample of the band 35 corresponds to fig. 10E, the sample of the band 36 corresponds to fig. 10F, and the band 37 is ddH 2 O negative control, band M is molecular weight marker.
TABLE 1
As can be seen from Table 1, 6 layers of layer tissue disease materials which are collected from different layer farms and are suspected of being infected by IBV are detected by adopting a single PCR method and a multiple PCR method respectively, and the results show that a sample A, B, C, D, F is positive to QX type IBV, E is negative to IBV, and the single PCR detection result is consistent with the multiple PCR detection result.
As can be seen from FIG. 1, the target gene fragments of the expected sizes can be specifically amplified after PCR amplification of a single cDNA template using each primer pair.
As can be seen from FIG. 2, after PCR amplification of the cDNA mixture template using each primer pair, each primer pair can similarly amplify only one target band consistent with the expected size.
Therefore, it was confirmed that the five primer pairs had no cross-reaction between 5 strains, respectively, and were highly specific.
As can be seen from FIGS. 3A-3D, 4A-4D, 5A-5D and 6, the multiplex PCR primer of the invention can effectively amplify corresponding target fragments under various PCR reaction conditions when simultaneously amplifying IBV mixed templates with different genotypes. In addition, the target band was obtained with higher clarity when the primer concentration was 0.1. Mu. Mol/L, the annealing temperature was 58℃and the number of cycles was 35 under the condition that the reaction system was 25. Mu.L.
As can be seen from FIG. 7, the specific target bands were amplified only for the IBV virus No. 1, no. 3, no. 9, no. 7 and No. 8, while the other viruses No. 6, no. 10, no. 5 and No. 11 and the control were not seen in any bands, which further confirmed that the multiplex PCR primers and methods of the present invention have good specificity.
As can be seen from FIGS. 8A-8F, the amplified 5 gene sequences are identical to the target gene sequences of the viruses, and 5 recombinant plasmids pUC-QX, pUC-H120, pUC-LDT3, pUC-GVI-1 and pUC-4/91 are correctly constructed. Each of the PCR products was subjected to 10-fold ratio (10 6 Copy/. Mu.L-10 0 Copy/. Mu.L) of the five plasmids after dilution, the results showed that the detection limits of the method were 10 for pUC-QX, pUC-H120, pUC-LDT3, pUC-GVI-1 and pUC-4/91, respectively 3 Copy/. Mu.L, 10 1 Copy/. Mu.L, 10 3 Copy/. Mu.L, 10 0 Copy/. Mu.L and 10 2 Copy/. Mu.L. In the case of 5 mixed plasmid standards, the lower limit of pUC-QX detection by the method is 10 0 Copy/. Mu.L, pUC-GVI-1 had a lower limit of detection of 10 2 Copy/. Mu.L, lower detection limits for pUC-H120, pUC-LDT3 and pUC-4/91 are 10 3 Copy/. Mu.L, the method is highly sensitive. In FIG. 8, the band 1 corresponds to 10 6 Copy/. Mu.L, band 2 corresponds to 10 5 Copy/. Mu.L, band 3 corresponds to 10 4 Copy/. Mu.L, band 4 corresponds to 10 3 Copy/. Mu.L, band 5 corresponds to 10 2 Copy/. Mu.L, lane 6 corresponds to 10 copies/. Mu.L and lane 7 corresponds to 1 copy/. Mu.L.
As can be seen from FIG. 9, the established multiplex PCR method is adopted to detect the artificial mixed infection of the five IBV viruses or the artificial random mixed infection of two, three and four strains of chicken embryo samples, the uninfected chicken embryo samples are set as negative controls, the artificial infected samples have expected amplified bands, the negative controls have no amplified bands, the multiplex PCR primers established in the invention are further proved not to be affected mutually, and when the IBV virus types which can be identified by the invention are detected simultaneously, the virus types can be accurately determined by one PCR.
As can be seen from fig. 10A-10G, the single PCR and multiple PCR methods are adopted to detect 6 parts of layer chicken tissue disease materials with suspected IBV infection disease collected from different layer chicken fields, and the results show that sample A, B, C, D, F is QX-positive IBV, E is IBV-negative, and the single PCR and multiple PCR detection results are consistent.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.
In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.
Moreover, any combination of the various embodiments of the invention can be made without departing from the spirit of the invention, which should also be considered as disclosed herein.
Claims (9)
1. A multiplex PCR primer for identifying avian infectious bronchitis viruses with different genotypes simultaneously is characterized by comprising a first primer pair with nucleotide sequences shown as SEQ ID No. 1 and SEQ ID No. 2, a second primer pair with nucleotide sequences shown as SEQ ID No. 3 and SEQ ID No. 4, a third primer pair with nucleotide sequences shown as SEQ ID No. 5 and SEQ ID No. 6, a fourth primer pair with nucleotide sequences shown as SEQ ID No. 7 and SEQ ID No. 8, and a fifth primer pair with nucleotide sequences shown as SEQ ID No. 9 and SEQ ID No. 10.
2. A detection reagent for simultaneously identifying avian infectious bronchitis viruses of different genotypes, wherein the detection reagent comprises the multiplex PCR primer as defined in claim 1.
3. A method for identifying avian infectious bronchitis viruses of different genotypes for non-disease diagnostic purposes, characterized in that multiplex PCR primers as defined in claim 1 and/or detection reagents as defined in claim 2 are used.
4. A method according to claim 3, characterized in that the method comprises:
s100, obtaining a nucleic acid sample of a sample to be detected;
s200, performing multiplex PCR amplification on the nucleic acid sample by using the multiplex PCR primer and/or the detection reagent;
s300, performing agarose gel electrophoresis detection on the amplification product obtained in the step S200;
s400, judging the sample to be tested according to the bands appearing in the obtained agarose gel electrophoresis diagram.
5. The method according to claim 4, wherein in step S200, the concentration of each primer is 0.1 to 0.5. Mu. Mol/L, respectively, relative to the total volume of the PCR amplification system of 25. Mu.L.
6. The method according to claim 4 or 5, wherein in step S200, the annealing temperature is 48-60℃and the cycle number is 20-35 during PCR amplification.
7. The method according to claim 4 or 5, wherein in step S400, the presence of a DNA fragment of 168bp in the lane is determined to be IBV QX-type in the sample to be tested;
the length of the DNA fragment appearing in the lane is 268bp, and the sample to be detected is judged to contain IBV Mass type;
the length of the DNA fragment appearing in the lanes is 537bp, and the sample to be detected is judged to contain IBV G VI-1;
the length of the DNA fragment appearing in the lane is 642bp, and the sample to be detected is judged to contain IBV 4/91 type;
and if the length of the DNA fragment appearing in the lane is 440bp, judging that the sample to be detected contains IBV LDT3.
8. Use of multiplex PCR primers according to claim 1 for differentiating avian infectious bronchitis viruses of different genotypes, including IBV QX, IBV G VI-1, IBV Mass, IBV 4/91 and IBV LDT3.
9. The use according to claim 8, wherein the multiplex PCR primer has a lower limit of detection of IBV QX type of 1 copy/. Mu.L and a lower limit of detection of IBV GVI-1 type of 10 2 Copy/. Mu.L, detection limits for IBV Mass type, IBV LDT3 type and IBV 4/91 type are 10 3 Copy/. Mu.L.
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