Triple PCR detection primer group, kit and method for chicken parvovirus and H9 subtype avian influenza virus
阅读说明：本技术 鸡细小病毒与h9亚型禽流感病毒三重pcr检测引物组、试剂盒及方法 (Triple PCR detection primer group, kit and method for chicken parvovirus and H9 subtype avian influenza virus ) 是由 谢芝勋 李丹 李孟 谢丽基 罗思思 张艳芳 张民秀 黄娇玲 范晴 王盛 谢志勤 于 2019-10-31 设计创作，主要内容包括：本发明公开了鸡细小病毒与H9亚型禽流感病毒三重PCR检测引物组、试剂盒及方法,设计了针对鸡细小病毒与H9亚型禽流感病毒的特异性引物,经过对不同引物组合进行试验筛选得到扩增效果较好的一个引物组合,然后继续对反应体系中引物浓度、引物之间的比例、退火温度和扩增时间进行优化,最终建立起可同时检测鸡细小病毒与H9亚型禽流感病毒的方法。本发明的引物和检测方法通过一次PCR反应便可同时确定样品中鸡细小病毒与H9亚型禽流感病毒的混合感染及单一感染情况,该方法同时具有特异性强、灵敏度高、快速简便及易于操作等优点,十分适于在基层单位应用推广,为鸡细小病毒与H9亚型禽流感病毒的监测及其防控提供技术支撑。(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.)
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.
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
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
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.
Drawings
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.
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