Primer combination for identifying ChPV (Chicken Parvovirus) and ARV (Avian Reoviruses) and application thereof
A technology for avian reovirus and chicken parvovirus, which is applied to microorganisms, recombinant DNA technology, microorganism-based methods, etc., can solve the problems of lack of rapid differential diagnosis, low sensitivity, and long time consumption.
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[0095] Example 1. Primer design
[0096] A large number of sequence analysis and comparisons have obtained several primers for the identification of chicken parvovirus and several primers for the identification of avian reovirus. Perform preliminary experiments on each primer to compare the sensitivity and specificity, and finally obtain a pair of primers for identifying chicken parvovirus and a pair of primers for identifying avian reovirus.
[0097] The specific primer pair used to identify chicken parvovirus consists of the following two primers (5’→3’):
[0098] ChPV-F (sequence 1 of the sequence listing): CGAAAGAAGAGGAACCCACC;
[0099] ChPV-R (sequence 2 of the sequence listing): TCTGGCTCGTCTGGTAATC;
[0100] The specific primer pair used to identify avian reovirus consists of the following two primers (5’→3’):
[0101] ARV-F (sequence 3 of the sequence listing): TCTATGAACGGCTGACCAA;
[0102] ARV-R (sequence 4 of the sequence listing): CACTAAGTGGAGGCGAAAA.
[0103] The primer pair us...
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[0106] Example 2. Optimization of double PCR reaction conditions
[0107] 1. Template preparation
[0108] 1. Extract the genomic DNA of chicken parvovirus to obtain sample A.
[0109] 2. Extract the total RNA of avian reovirus and reverse transcribed it into cDNA to obtain sample B.
[0110] 3. Mix sample A and sample B to obtain a mixed sample.
[0111] 2. Optimization of primer concentration
[0112] Take the mixed sample obtained in step 1 as a template, and use the primer combination prepared in Example 1 to perform double PCR.
[0113] Double PCR reaction system (25.0μL): contains 2×PCRMix12.5μL, the mixed sample obtained in step 1 is 2.0μL (in the 2.0μL mixed sample, the content of the chicken parvovirus genomic DNA is 0.58ng, and the avian reovirus CDNA content of 0.53ng), primer pair I and primer pair II, and finally use ddH 2 Make up O to 25.0 μL.
[0114] According to the concentration of primer pair I and primer pair II in the reaction system, 9 different reaction systems are ...
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[0145] Example 3. Specificity
[0146] 1. Extract the genomic DNA of the sample to be tested. The samples to be tested are: chicken parvovirus (ChPV), Marek virus (MDV), and infectious laryngotracheitis virus (ILTV).
[0147] 2. Extract the total RNA of the sample to be tested and reverse transcribed into cDNA. The samples to be tested are: avian reovirus (ARV), chicken Newcastle disease virus (NDV), H9 subtype avian influenza virus (AIVH9), and infectious bronchitis virus (IBV).
[0148] 3. Using each genomic DNA sample obtained in step 1, each cDNA sample obtained in step 2, and the mixed sample obtained in step 1 of Example 2 as templates, the primer combination prepared in Example 1 is used to perform double PCR.
[0149] Double PCR reaction system (25.0μL): contains 2×PCRMix12.5μL, template 2.0μL, primer pair I and primer pair II, and finally ddH 2 Make up O to 25.0 μL. In the double PCR reaction system, the concentration of ChPV-F and ChPV-R are both 0.4pmol / μL, and the concen...
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