Primer group, kit and method for detecting virus mixed infection
阅读说明：本技术 一种检测病毒混合感染的引物组、试剂盒及其方法 (Primer group, kit and method for detecting virus mixed infection ) 是由 周祖涛 杨荣坤 肖运才 胡思顺 李自力 刘梅 许青荣 崔卫涛 毕丁仁 于 2019-12-03 设计创作，主要内容包括：本公开公开了一种检测病毒混合感染的引物组、试剂盒及其方法,属于生物技术领域。所述引物组包括：第一引物对、第二引物对、第三引物对、第四引物对、第五引物对、第六引物对和通用引物对,所述第一引物对包括：第一正向引物和第一反向引物,所述第一正向引物的序列如序列表中SEQ ID NO:1所示,所述第一反向引物的序列如序列表中SEQ ID NO:2所示。本公开提供的引物组能够检测禽白血病病毒以及由禽白血病病毒作为原发性病原引发H5/H9 AIV、NDV或IBV 4病原,有利于提高对禽白血病病毒以及由以禽白血病病毒作为原发性病原引发H5/H9 AIV、NDV或IBV 4病原的诊断结果的准确性及防控方案的有效性。(The invention discloses a primer group, a kit and a method for detecting virus mixed infection, and belongs to the technical field of biology. The primer group comprises: the primer set comprises a first primer pair, a second primer pair, a third primer pair, a fourth primer pair, a fifth primer pair, a sixth primer pair and a universal primer pair, wherein the first primer pair comprises: the primer comprises a first forward primer and a first reverse primer, wherein the sequence of the first forward primer is shown as SEQ ID NO. 1 in a sequence table, and the sequence of the first reverse primer is shown as SEQ ID NO. 2 in the sequence table. The primer group provided by the disclosure can detect avian leukemia virus and H5/H9AIV, NDV or IBV 4 pathogen triggered by the avian leukemia virus as a primary pathogen, and is beneficial to improving the accuracy of diagnosis results and the effectiveness of a prevention and control scheme of the avian leukemia virus and H5/H9AIV, NDV or IBV 4 pathogen triggered by the avian leukemia virus as a primary pathogen.)
1. A primer set for detecting virus mixed infection, wherein the primer set comprises: a first primer pair, a second primer pair, a third primer pair, a fourth primer pair, a fifth primer pair, a sixth primer pair and a universal primer pair,
the first primer pair comprises: a first forward primer and a first reverse primer, wherein the sequence of the first forward primer is shown as SEQ ID NO. 1 in the sequence table, the sequence of the first reverse primer is shown as SEQ ID NO. 2 in the sequence table,
the second primer pair comprises: a second forward primer and a second reverse primer, wherein the sequence of the second forward primer is shown as SEQ ID NO. 3 in the sequence table, the sequence of the second reverse primer is shown as SEQ ID NO. 4 in the sequence table,
the third primer pair comprises: a third forward primer and a third reverse primer, wherein the sequence of the third forward primer is shown as SEQ ID NO. 5 in the sequence table, the sequence of the third reverse primer is shown as SEQ ID NO. 6 in the sequence table,
the fourth primer pair comprises: a fourth forward primer and a fourth reverse primer, wherein the sequence of the fourth forward primer is shown as SEQ ID NO. 7 in the sequence table, the sequence of the fourth reverse primer is shown as SEQ ID NO. 8 in the sequence table,
the fifth primer pair comprises: a fifth forward primer and a fifth reverse primer, wherein the sequence of the fifth forward primer is shown as SEQ ID NO. 9 in the sequence table, and the sequence of the fifth reverse primer is shown as SEQ ID NO. 10 in the sequence table;
the sixth primer pair includes: a sixth forward primer and a sixth reverse primer, wherein the sequence of the sixth forward primer is shown as SEQ ID NO. 11 in the sequence table, and the sequence of the sixth reverse primer is shown as SEQ ID NO. 12 in the sequence table;
the universal primer pair comprises: a universal forward primer and a universal reverse primer.
2. The primer group of claim 1, wherein the sequence of the universal forward primer is shown as SEQ ID NO. 13 in the sequence Listing, and the sequence of the universal reverse primer is shown as SEQ ID NO. 14 in the sequence Listing.
3. A kit for detecting a viral mixed infection, comprising the primer set according to claim 1 or 2.
4. The kit of claim 3, wherein the final concentrations of the first forward primer, the first reverse primer, the fifth forward primer and the fifth reverse primer are all 1 μ M/L, the final concentrations of the second forward primer and the second reverse primer, the third forward primer and the third reverse primer are all 1.25 μ M/L, the final concentrations of the fourth forward primer and the fourth reverse primer are all 0.75 μ M/L, the final concentrations of the sixth forward primer and the sixth reverse primer are all 1.5 μ M/L, and the final concentrations of the forward universal primer and the reverse universal primer are all 20 μ M/L.
5. The kit of claim 3, further comprising: positive control sample, negative control sample, EX Taq polymerase and RNase Free dH2O。
6. The kit of claim 5, wherein EX Taq polymerase is present at a final concentration of 5U/μ L.
7. The kit of claim 5, wherein the positive control sample comprises: the template is a positive standard plasmid containing an HA gene of H5 subtype avian influenza virus, a positive standard plasmid containing an HA gene of H9 subtype avian influenza virus, a positive standard plasmid containing an F gene of Newcastle disease virus, a positive standard plasmid containing an N gene of avian infectious bronchitis virus, a positive standard plasmid containing a gp85 gene of B subtype avian leukemia virus and a positive standard plasmid containing a gp85 gene of J subtype avian leukemia virus.
8. The kit of claim 5, wherein the negative control sample is pMD18-T unloaded plasmid.
9. A method for detecting a viral mixed infection using the primer set of claim 1, comprising:
extracting the RNA of the sample to be detected,
reverse transcribing the RNA to cDNA, the cDNA serving as a template;
carrying out amplification reaction on the template by adopting the primer group to obtain an amplification product;
analyzing the amplified product for virus by capillary electrophoresis.
10. The method of claim 9, wherein the amplification reaction comprises a first stage and a second stage, and wherein the first stage of the amplification procedure comprises, for each cycle: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 57 ℃ for 45s, and extension at 72 ℃ for 30s for 10 cycles;
the second phase of the amplification procedure comprises for each cycle: denaturation at 94 ℃ for 30s, annealing at 50 ℃ for 45s, and extension at 72 ℃ for 45s for 20 cycles; final extension: extension at 72 ℃ for 10 min.
Technical Field
The disclosure relates to the field of biotechnology, and in particular relates to a primer group, a kit and a method for detecting virus mixed infection.
Background
The Avian leukosis is a vertically-transmitted disease of poultry caused by Avian leukosis virus (Avian L eukosis Viruses, A L V), which can cause the production performance of the poultry to be reduced, the immunity to be inhibited, multiple organ tissue tumors and the like, and has great harm to the poultry industry.
In clinical diagnosis, the conventional detection method mainly aims at H5/H9AIV, NDV or IBV 4 pathogen, but ignores the condition that the avian leukemia virus is used as a primary pathogen to trigger H5/H9AIV, NDV or IBV 4 pathogen, thereby influencing the accuracy of a diagnosis result and the effectiveness of a prevention and control scheme.
Disclosure of Invention
In order to solve the problems of the prior art, the embodiments of the present disclosure provide a primer set, a kit and a method for detecting virus mixed infection. The technical scheme is as follows:
in one aspect, the present disclosure provides a primer set for detecting virus mixed infection, the primer set comprising: a first primer pair, a second primer pair, a third primer pair, a fourth primer pair, a fifth primer pair, a sixth primer pair and a universal primer pair,
the first primer pair comprises: a first forward primer and a first reverse primer, wherein the sequence of the first forward primer is shown as SEQ ID NO. 1 in the sequence table, the sequence of the first reverse primer is shown as SEQ ID NO. 2 in the sequence table,
the second primer pair comprises: a second forward primer and a second reverse primer, wherein the sequence of the second forward primer is shown as SEQ ID NO. 3 in the sequence table, the sequence of the second reverse primer is shown as SEQ ID NO. 4 in the sequence table,
the third primer pair comprises: a third forward primer and a third reverse primer, wherein the sequence of the third forward primer is shown as SEQ ID NO. 5 in the sequence table, the sequence of the third reverse primer is shown as SEQ ID NO. 6 in the sequence table,
the fourth primer pair comprises: a fourth forward primer and a fourth reverse primer, wherein the sequence of the fourth forward primer is shown as SEQ ID NO. 7 in the sequence table, the sequence of the fourth reverse primer is shown as SEQ ID NO. 8 in the sequence table,
the fifth primer pair comprises: a fifth forward primer and a fifth reverse primer, wherein the sequence of the fifth forward primer is shown as SEQ ID NO. 9 in the sequence table, and the sequence of the fifth reverse primer is shown as SEQ ID NO. 10 in the sequence table;
the sixth primer pair includes: a sixth forward primer and a sixth reverse primer, wherein the sequence of the sixth forward primer is shown as SEQ ID NO. 11 in the sequence table, and the sequence of the sixth reverse primer is shown as SEQ ID NO. 12 in the sequence table;
the universal primer pair comprises: a universal forward primer and a universal reverse primer.
Specifically, the sequence of the universal forward primer is shown as SEQ ID NO. 13 in the sequence table, and the sequence of the universal reverse primer is shown as SEQ ID NO. 14 in the sequence table.
In another aspect, the present disclosure provides a kit for detecting virus mixed infection, wherein the kit comprises the primer set.
Specifically, the final concentrations of the first forward primer, the first reverse primer, the fifth forward primer and the fifth reverse primer are all 1 mu M/L, the final concentrations of the second forward primer, the second reverse primer, the third forward primer and the third reverse primer are all 1.25 mu M/L, the final concentrations of the fourth forward primer and the fourth reverse primer are all 0.75 mu M/L, the final concentrations of the sixth forward primer and the sixth reverse primer are all 1.5 mu M/L, and the final concentrations of the forward universal primer and the reverse universal primer are all 20 mu M/L.
Specifically, the kit further comprises: positive control sample, negative control sample, EX Taq polymerase and RNaseFreedH2O。
Further, the final concentration of EX Taq polymerase was 5U/. mu. L.
Further, the positive control sample comprises: the template is a positive standard plasmid containing an HA gene of H5 subtype avian influenza virus, a positive standard plasmid containing an HA gene of H9 subtype avian influenza virus, a positive standard plasmid containing an F gene of Newcastle disease virus, a positive standard plasmid containing an N gene of avian infectious bronchitis virus, a positive standard plasmid containing a gp85 gene of B subtype avian leukemia virus and a positive standard plasmid containing a gp85 gene of J subtype avian leukemia virus.
Further, the negative control sample was pMD18-T unloaded plasmid.
In another aspect, the embodiment of the present disclosure provides a method for detecting virus mixed infection by using the primer set described above, where the method includes:
extracting the RNA of the sample to be detected,
reverse transcribing the RNA to cDNA, the cDNA serving as a template;
carrying out amplification reaction on the template and the primer group to obtain an amplification product;
analyzing the amplified product for virus by capillary electrophoresis.
Specifically, the amplification reaction comprises a first stage and a second stage, and the amplification procedure of the first stage comprises for each cycle: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 57 ℃ for 45s, and extension at 72 ℃ for 30s for 10 cycles;
the second phase of the amplification procedure comprises for each cycle: denaturation at 94 ℃ for 30s, annealing at 50 ℃ for 45s, and extension at 72 ℃ for 45s for 20 cycles; final extension: extension at 72 ℃ for 10 min.
The technical scheme provided by the embodiment of the disclosure has the following beneficial effects: the primer group provided by the disclosure can detect avian leukemia virus and H5/H9AIV, NDV or IBV 4 pathogen triggered by the avian leukemia virus as a primary pathogen, and is favorable for the accuracy of diagnosis results and the effectiveness of a prevention and control scheme of the avian leukemia virus and H5/H9AIV, NDV or IBV 4 pathogen triggered by the avian leukemia virus as a primary pathogen. The kit can comprehensively detect the avian leukemia virus and H5/H9AIV, NDV or IBV 4 pathogen caused by taking the avian leukemia virus as a primary pathogen, thereby obtaining an accurate detection result. The detection method combines multiple PCR amplification with capillary electrophoresis, utilizes the characteristics of double primers, adopts two-step amplification, effectively avoids the preference problem in the conventional multiple PCR amplification, and improves the amplification efficiency; the method is combined with capillary electrophoresis, is more environment-friendly and safer than common gel electrophoresis, avoids the use of toxic and harmful nucleic acid dye, improves the resolution ratio, can distinguish target fragments with a difference of more than 7bp, and improves the detection accuracy.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present disclosure, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present disclosure, and it is obvious for those skilled in the art to obtain other drawings based on the drawings without creative efforts.
FIG. 1 is a diagram of capillary electrophoresis analysis provided in the third embodiment of the present disclosure;
FIG. 2 is a diagram of capillary electrophoresis analysis of simulated ND and AIV-H5 mixed infection provided in example III of the present disclosure;
FIG. 3 is a diagram of capillary electrophoresis analysis of simulated AIV-H9, ND and IB mixed infection provided in example III of the present disclosure.
Detailed Description
To make the objects, technical solutions and advantages of the present disclosure more apparent, embodiments of the present disclosure will be described in detail with reference to the accompanying drawings.
The experimental methods used in the examples of the present disclosure are all routine experimental methods unless otherwise specified.
Reagents and instruments used in the examples of the present disclosure and the like are commercially available in general unless otherwise specified.