Method for detection of carp spring viremia virus based on liquid chip
A viremia and virus technology is applied in the field of detection of carp spring viremia virus based on liquid phase chips, which can solve the problems of cross-contamination, cumbersome operation, harm to human body and environment, etc., and achieve short time required, no pollution to the environment, good specific effect
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Embodiment 1
[0037] Embodiment 1, the design of specific primer pair and specific probe
[0038] A specific primer pair and a specific probe for amplifying the coding gene of the glycoprotein of carp spring viremia virus are designed through a large number of sequence alignments and comparisons of amplification effects.
[0039] The specific primer pair is as follows (the target sequence is 153bp):
[0040] F1 (sequence 1 of the sequence listing): 5'-TTGGGATGACTGGGAGTT-3';
[0041] R1 (SEQ ID NO: 2 of the Sequence Listing): 5'-GGGATAATATCGGCTTGG-3'.
[0042] The nucleotide sequence of the specific probe (sequence 3 in the sequence listing) is as follows:
[0043] 5'-TGTATATAAAGGAAAGGATGGGAAG-3'.
Embodiment 2
[0044] Example 2, application of specific primer pairs to aid in the identification of carp spring viremia virus
[0045] Carp spring viremia virus, infectious hematopoietic necrosis virus, grass carp hemorrhagic disease virus, viral hemorrhagic sepsis virus and infectious pancreatic necrosis virus were used as the tested viruses to carry out the following experiments:
[0046] 1. Use the RNA extraction kit to extract the total RNA of the virus to be tested.
[0047] 2. Using the total RNA obtained in step 1 as a template, using a primer pair composed of F1 and R1, and using an RT-PCR kit, perform RT-PCR amplification on a gradient PCR amplification instrument to obtain RT-PCR amplification products.
[0048] RT-PCR amplification reaction system: In a 0.2mL PCR reaction tube, add 10×RT-PCR buffer 2.5μL, dNTP (each 2.5mmol / L) 2.0μL, 10pmol / μL F1 1μL, 10pmol / μL R1 1μL , Inhibiter 0.5μL, AMV XL 0.5μL, AMV Taq (5U / μL) 0.25μL, 25mmol / L MgCl 2 5.0 μL, 5.0 μL total RNA (about 300 ...
Embodiment 3
[0052] Example 3, application of primer probe composition to assist in the identification of carp spring viremia virus
[0053] 1. Preparation of primers and probes
[0054] Prepare primers and probes as follows:
[0055] F2: 5'-biotin-TTGGGATGACTGGGAGTT-3';
[0056] R2: 5'-biotin-GGGATAATATCGGCTTGG-3'.
[0057] Probe T1: 5'-NH 2 -TGTATATAAAGGAAAGGATGGGAAG-3'.
[0058] F2 is biotinylated at the 5' end of F1, and R2 is biotinylated at the 5' end of R1. Probe T1 is obtained by amination modification at the 5' end of the single-stranded DNA fragment shown in Sequence 3 of the sequence listing.
[0059] 2. Establishment of liquid phase chip detection system
[0060] 1. Use the RNA extraction kit to extract the total RNA of carp spring viremia virus.
[0061] 2. Using the total RNA obtained in step 1 as a template, using a primer pair composed of F2 and R2, and using an RT-PCR kit, perform RT-PCR amplification on a gradient PCR amplification instrument to obtain RT-PCR ampli...
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