Nucleic acid for rapidly performing typing detection on avian metapneumovirus subgroup with RT-PCR and application of nucleic acid
An avian metapneumovirus and RT-PCR technology, which is applied to RT-PCR rapid typing and detection of nucleic acid and application fields of avian metapneumovirus subtypes, can solve the problem that indirect enzyme-linked immunosorbent reaction cannot be used as a diagnostic basis, and fluorescent antibodies are not suitable for Large-scale application, unsuitable for large-scale application, etc., to avoid false positive and false negative results, save operation time, and improve detection efficiency
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Embodiment 1
[0042] The design of embodiment 1 primer
[0043] The present invention adopts reverse transcription polymerase chain reaction (RT-PCR) to detect, and in RT-PCR, an RNA strand is reverse transcribed into complementary DNA, and then DNA amplification is performed by PCR using this as a template. This technology is more sensitive and faster than conventional virus isolation in detecting aMPV, and it is more affordable than fluorescent quantitative RT-PCR.
[0044] In the present invention, the G gene and M gene of avian metapneumovirus are selected as the target genes for detection, because the G gene and M gene are highly conserved within subtypes and have strong specificity between subtypes.
[0045] Using reverse transcription polymerase chain reaction (RT-PCR), avian metapneumovirus is typed, detected and identified, and can detect four subtypes simultaneously in the same sample, and the design of its primer pair is the success of the present invention The key is that there...
Embodiment 2
[0050] Example 2 RT-PCR rapid typing detection
[0051] The method for detecting four subtypes of avian metapneumovirus rapidly and simultaneously genotyped by RT-PCR specifically comprises the following steps:
[0052] (1) Prepare the RNA of the test sample;
[0053] (2) Perform one-step RT-PCR amplification on the RNA sample in step (1): use 20 μL for the reaction system. In a 20 μL reaction system, including: primer pair 1 for avian metapneumovirus type A, primer pair 2 for avian metapneumovirus type B, primer pair 3 for avian metapneumovirus type C, and primer pair for avian metapneumovirus type D The concentration of each primer in 4 can be 10pmol, the dosage is 0.5μL, 5×Buffer 4μL, 2.5mM dNTPs 1.5μL, 5U / μL mixed enzyme (reverse transcriptase and DNA polymerase) 1μL, sample RNA 2μL, dd h 2 O 10.5 μL. All of the above were added into a 0.2mL reaction tube and fully uniform. Then put it into the PCR machine and perform the following reactions: reverse transcription at ...
Embodiment 3
[0064] The detection of embodiment 3 sensitivity
[0065] The RNA quality control product transcribed in vitro of aMPV / C subtype [only the sequence of SEQ ID No.11] is serially diluted by 10 times, detected by the method in Example 2, and analyzed by 1.0% agarose gel electrophoresis , to detect its sensitivity. Its electropherogram is as figure 1 As shown, where M: 2000bp DNAMarker, spotting 1-10 sequence 3.6×10 3 , 3.6×10 2 , 3.6×10, 3.6×10 0 , 3.6×10 -1 , 3.6×10 -2 , 3.6×10 -3 , 3.6×10 -4 , 3.6×10 -5 , 3.6×10 -6 ng / μL, water. The results show that the initial concentration of aMPV / C in vitro transcribed RNA is 3639.4ng / μL. After 10-fold dilution, it is diluted to 1000 times, and the concentration is 3.6ng / μL, which can be detected well, which proves that the sensitivity of the detection method is very good ( See figure 1 shown).
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