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Primer pair for identifying newcastle disease virus and multi-subtype avian influenza virus and application thereof

An avian influenza virus, Newcastle disease virus technology, applied in the direction of microorganisms, microorganism-based methods, biochemical equipment and methods, etc., to achieve the effect of strong specificity, high sensitivity and low cost

Inactive Publication Date: 2012-01-18
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 multiplex RT-PCR method capable of simultaneously detecting Newcastle disease and H3, H5, H9 subtype avian influenza viruses at home and abroad

Method used

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  • Primer pair for identifying newcastle disease virus and multi-subtype avian influenza virus and application thereof
  • Primer pair for identifying newcastle disease virus and multi-subtype avian influenza virus and application thereof
  • Primer pair for identifying newcastle disease virus and multi-subtype avian influenza virus and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1. PCR primer pair design

[0061] Using the latest sequences of Newcastle disease virus and avian influenza virus in GenBank, four pairs of primers were designed for the H3 subtype, H5 subtype and H9 subtype avian influenza virus HA gene and Newcastle disease virus F gene as follows:

[0062] The primer pair NDV designed with the F gene of Newcastle disease virus as the target gene:

[0063] Upstream primer NDV-275F: 5'-TCACTCCTCTTGGCGACTC-3' (sequence of sequence 1 in sequence listing);

[0064] Downstream primer NDV-556R: 5'-CAAACTGCTGCATCTTCC-3' (sequence of Sequence Listing 2);

[0065] The primer pair H3V with the HA gene of H3 subtype avian influenza virus as the target gene:

[0066] Upstream primer H3-622F: 5'-TTCACCACCCGAGCACAA-3' (sequence of sequence 3 in sequence listing);

[0067] Downstream primer H3-837R: 5'-GGGCGATTAGGTTTCCATTA-3' (sequence of sequence table sequence 4);

[0068] The primer pair H5V with the HA gene of H5 subtype avian influenza virus as the...

Embodiment 2

[0079] Example 2. Specific detection of PCR primer pairs

[0080] 1. The specific detection of primers for NDV

[0081] 1. Total RNA extraction and quality control

[0082] Take the Newcastle disease virus F48E9, Chicken / Henan / 2009, Chicken / ShangDong / 2010, Chicken / Hebei / 2010, Duck / ShangDong / 2011 or HG / Beijing / 2009 (Rui, Z., Juan, P., Jingliang, S., Jixun, Z., Xiaoting, W., Shouping, Z., Xiaojiao, L. and Guozhong, Z., 2010a. Phylogenetic characterization of Newcastle disease virus isolatedin the mainland of China during 2001-2009. Vet Microbiol 141, 246-57.) chicken embryo allantoic fluid and H4N6 subtype avian influenza virus A / Duck / ShangDong / 1 / 2010 similar to Newcastle disease virus, H6N1 subtype avian influenza virus A / Duck / Beijing / 1 / 2003, Infectious bronchitis virus (IBV), infectious bursal bursal virus (IBDV) or reovirus (REO) chicken embryo allantoic fluid and negative chicken embryo allantoic fluid, respectively, to extract total RNA (the method is the same as in Example 4 )...

Embodiment 3

[0128] Example 3. Sensitivity detection of PCR primer pairs

[0129] 1. Sensitivity detection of primers to NDV

[0130] 1. Extraction of total RNA and preparation of each dilution

[0131] Take the chicken embryo allantoic fluid infected with Newcastle disease virus HG / Beijing / 2009 and use the Reed-Muench method for virus EID 50 To obtain the toxic content of this Newcastle disease virus in chicken embryo allantoic fluid. According to the virus content, the chicken embryo allantoic fluid was diluted with sterile PBS to a virus concentration of 10 6 EID 50 / 100μl (for example, the virus concentration of chicken embryo allantoic fluid is 10 7 EID 50 / 100μl, then a 10-fold dilution (10 7 / 10 6 =10)), and then perform a 10-fold dilution on this basis, and the virus content in each dilution 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. Determine the virus concentration at 10...

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Abstract

The invention discloses a primer pair for identifying newcastle disease virus and multi-subtype avian influenza virus and an application thereof. The PCR primer pair composition provided by the invention comprises primer pair NDV, primer pair H3V, primer pair H5V, and primer pair H9V; the PCR primer pair composition is applicable to the preparation of reagents for the diagnosis or auxiliary diagnosis of newcastle disease and avian influenza, or to the preparation of reagents for the identification or auxiliary identification of newcastle disease virus and multi-subtype avian influenza virus. When the PCR primer pair composition provided by the invention is used to simultaneously detect newcastle disease virus, H3, H5 and H9-subtype avian influenza virus, the specificity is strong; the sensitivity is 1EID50 / 100 microliters, 1EID50 / 100 microliters, 1*10-2EID50 / 100 microliters, and 1EID50 / 100 microliters respectively; compared with identification results of routine test methods such as virus isolation and hemagglutination inhibition tests, the results obtained by using the PCR primer pair composition of the invention has a coincidence rate of up to 100%; the PCR primer pair composition provided by the invention is applicable to the development of corresponding multiplex RT-PCR kits for the clinical diagnosis and epidemiological control of newcastle disease and avian influenza.

Description

Technical field [0001] The invention relates to a primer pair for identifying Newcastle disease virus and multi-subtype avian influenza virus and its application. Background technique [0002] Avian influenza and Newcastle disease are two viral infectious diseases caused by avian influenza virus and Newcastle disease virus respectively. The two kinds of infectious diseases have rapid onset, rapid spread, and high incidence, which can cause a fatal blow to the breeding industry. The subtypes of avian influenza viruses circulating in chickens in my country are mainly H5 and H9 subtypes. Influenza viruses are very prone to genetic mutations, leading to changes in antigenicity. Therefore, although my country has adopted comprehensive immunization measures, H5 and H9 influenza viruses still occur repeatedly in chickens. Variations in the genetic and antigenic characteristics of the H5 and H9 subtypes of avian influenza viruses have led to the failure of conventional serological met...

Claims

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Application Information

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