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
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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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