Primer, probe and method for detecting mouse pneumonia virus
A pneumonia virus, mouse technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve problems such as short detection time, achieve simple and fast operation, improve sensitivity, and good linear relationship. Effect
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Embodiment 1
[0038] The design of embodiment 1 mouse pneumonia virus special primer and probe
[0039] The full-length genome sequences of PVM-15 and PVM-J3666 were found from genbank, and their genbank numbers are AY729016 and AY743909, respectively. Sequence comparison showed that most of the PVM sequences of the two strains were homologous. Determine the conserved homologous sequence: the nucleotide sequence shown in SEQ ID NO:4.
[0040] Design special primers and probe sequences for real-time fluorescent quantitative PCR detection for the found conserved homologous sequence region, and the amplified target fragment is 97 bases in size. Wherein the preferred primer and probe sequences are:
[0041] Upstream primer QPVM-97bp-F: 5'-CAGAGAGGTGGCTTGATTTGCT-3', ie the nucleotide sequence shown in SEQ ID NO: 1;
[0042] Downstream primer QPVM-97bp-R: 5'-TCATTGCAGATCCTGATGAAGTTC-3', ie the nucleotide sequence shown in SEQ ID NO: 2;
[0043] The probe QPVM-97bp-P:
[0044] 5'-TTCCAGCCGAGC...
Embodiment 2
[0047] The preparation of embodiment 2 positive RNA standard items
[0048] First prepare the pMD18-T vector containing the nucleotide sequence shown in the homologous sequence SEQ ID NO: 4, use it as the vector to amplify a 589bp DNA fragment, recover it for in vitro transcription reaction, digest the DNA template and purify the RNA after the reaction , quantified as a positive RNA standard.
[0049] 1. Preparation of templates for in vitro transcription
[0050] The nucleotide sequence shown in SEQ ID NO: 4 was synthesized and inserted into the pMD18-T vector (synthesized by Invitrogen), and named pMD18T-PVM. Using pMD18T-PVM as a template, use the upstream primer PVM-589bp-F: 5'-CAAGGCGATTAAGTTGGGT-3' and the downstream primer PVM-589bp-R: 5'-CAGGAAACAGCTATGACCATG-3' to amplify a DNA fragment of 589bp , recovered by agarose gel electrophoresis, used as a template for in vitro transcription.
[0051] 2. In vitro transcription to prepare a large amount of RNA
[0052] Pre...
Embodiment 3
[0064] Embodiment 3 Establishment of real-time fluorescent quantitative PCR amplification method
[0065] 1. Real-time fluorescence quantitative PCR template preparation - total RNA extraction:
[0066] According to the instructions for use, the sample RNA was extracted with Trizol / Trizol LS kit (provided by Invitrogen), the specific method is as follows:
[0067] 1) Add an appropriate amount of TRIzol / / Trizol LS to the sample (if it is a tissue that needs to be ground and homogenized), mix well, and let stand at room temperature for 5 minutes;
[0068] 2) Add 200 μl of chloroform: isoamyl alcohol (24:1) solution, mix vigorously with vortex for 10 seconds, leave at room temperature for 5 minutes, and centrifuge at 15,000 rpm at 4°C for 15 minutes;
[0069] 3) Take the supernatant, add an equal volume of isopropanol to precipitate, and centrifuge at 15,000 rpm at 4°C for 15 min;
[0070] 4) Remove the supernatant, add pre-cooled 75% ethanol (volume ratio 1:1) to wash the RNA ...
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