PCR (polymerase chain reaction) primer pair for identifying H3 subtype avian influenza virus and application thereof

A bird flu virus and primer pair technology, applied in the field of PCR primer pairs, achieves the effects of simple operation, high specificity, and easy promotion

Inactive Publication Date: 2013-06-05
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no PCR primer and method for detection of H3 subtype avian influenza virus, which is used for epidemiological monitoring and diagnosis of influenza virus

Method used

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  • PCR (polymerase chain reaction) primer pair for identifying H3 subtype avian influenza virus and application thereof
  • PCR (polymerase chain reaction) primer pair for identifying H3 subtype avian influenza virus and application thereof
  • PCR (polymerase chain reaction) primer pair for identifying H3 subtype avian influenza virus and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Embodiment 1, PCR primer pair design

[0019] According to the highly conserved region gene design of the H3 subtype avian influenza virus HA gene primer pair H3V: upstream primer H3-622F: 5'-TTCACCACCCGAGCACAA-3' (sequence number 1 sequence); downstream primer H3-837R: 5'-GGGCGATTAGGTTTCCATTA -3' (Sequence Listing Sequence 2); the annealing temperature is 51° C.; the target sequence of the primer pair H3V is the 622-639 and 818-837 of Genbank EU492531.

Embodiment 2

[0020] Embodiment 2, the specific detection of PCR primer pair

[0021] 1. Total RNA extraction and quality control

[0022] A / Duck / BeiJing / 33 / 2004, A / Duck / BeiJing / 40 / 2004, A / Duck / BeiJing / 44 / 2004, A / Duck / BeiJing / 56 / 2005, A / Duck / BeiJing / 56 / 2005, A / Duck / BeiJing / 59 / 2005 or A / Duck / BeiJing / 61 / 2005 (Pu, J., Liu, Q.F., Xia, Y.J., Fan, Y.L., Brown, E.G., Tian, ​​F.L. and Liu, J.H., 2009. Genetic analysis of H3 subtype influenza viruses isolated from domestic ducks in northern China during 2004-2005. Virus Genes 38, 136-42.) Chicken embryo allantoic fluid and H5N1 subtype avian influenza virus A similar to H3 subtype avian influenza virus infection / Chicken / Huabei / 202 / 2010, H5N1 subtype avian influenza virus A / tree sparrow / Jiangsu / 1 / 2008 (Liu, Q., Ma, J., Kou, Z., Pu, J., Lei, F. , Li, T. and Liu, J., 2010b.Characterization of a highly pathogenic avian influenza H5N1clade 2.3.4virus isolated from a tree sparrow.Virus Res 147, 25-9.), H5N1 subtype avian influenza virus A / Duck / Huabe...

Embodiment 3

[0032] Embodiment 3, the sensitivity detection of PCR primer pair

[0033] 1. Total RNA extraction and preparation of each dilution

[0034] The allantoic fluid of chicken embryos infected with H3 subtype avian influenza virus A / Duck / Beijing / 40 / 2004 was used for virus EID by Reed-Muench method 50 The determination of this H3 subtype avian influenza virus in the allantoic fluid of chicken embryos was obtained. According to the virus content, the chicken embryo allantoic fluid was diluted to a virus concentration of 10 with sterile PBS. 6 EID 50 / 100μl (such as the virus concentration of chicken embryo allantoic fluid is 10 7 EID 50 / 100μl, then perform 10-fold dilution (10 7 / 10 6 =10)), and then perform 10-fold dilution on this basis, in each dilution, the virus content is 10 5 EID 50 / 100μl, 10 4 EID 50 / 100μl, 10 3 EID 50 / 100μl, 10 2 EID 50 / 100μl, 10 1 EID 50 / 100μl, 10 0 EID 50 / 100μl, 10 -1 EID 50 / 100μl, 10 -2 EID 50 / 100μl. Determination of virus...

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Abstract

The invention discloses a PCR (polymerase chain reaction) primer pair for identifying H3 subtype avian influenza virus and an application thereof. The PCR primer pair provided by the invention comprises two single-stranded DNAs (deoxyribonucleic acids), and the two single-stranded DNAs are the single-stranded DNA as shown in sequence 1 in a sequence table and the single-stranded DNA as shown in sequence 2 in the sequence table. The primer pair provided by the invention has high specificity when used for detecting the H3 subtype avian influenza virus, the sensitivity can reach1EID50 / 100 mu l, and the coincidence rate is 100% in comparison with the identification results obtained by conventional experimental methods, such as virus isolation, hemagglutination inhibition test and the like; and the primer pair can be used for detecting not only buccal swabs and cloacal swabs of sick poultry but also allantoic fluid of the sick poultry, the operation is simple, the popularization is easy, the basic level operation and the application are convenient, and the primer pair can become a useful detection tool for the diagnosis and the epidemiological investigation of the H3 subtype avian influenza virus disease.

Description

technical field [0001] The invention relates to a pair of PCR primers for identifying H3 subtype avian influenza virus and its application. Background technique [0002] Avian influenza is a viral infectious disease of poultry caused by avian influenza virus. H3 subtype avian influenza virus is one of the main subtypes prevalent in waterfowl; in recent years, it has been found that chickens in most parts of my country have seropositive antibodies to H3 subtype influenza virus, and H3 subtype avian influenza has been isolated from chickens The report of the virus shows that the H3 subtype avian influenza virus has infected chickens and entered the chicken flock; in addition, the epidemiological investigation shows that the H3 subtype avian influenza virus and the main epidemic subtypes (H5 and H9 subtypes) in the chicken flock Viruses have undergone gene exchange to generate new influenza viruses and cause disease in chickens; moreover, H3 subtype avian influenza viruses were ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
Inventor 蒲娟王金亮唐庆冬包静楠孙洪磊刘金华
Owner CHINA AGRI UNIV
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