Duplex fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection reagent for Nipah virus and porcine reproductive and respiratory syndrome virus (PRRSV) as well as preparation method and application of duplex fluorescent RT-PCR detection reagent
A technology of RT-PCR and respiratory syndrome, applied in the direction of biochemical equipment and methods, recombinant DNA technology, microbial determination/inspection, etc.
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[0048] The double fluorescent RT-PCR detection reagent for Nipah virus and porcine reproductive and respiratory syndrome virus and its preparation method and application of the present invention comprise the following steps.
[0049] The first step is to prepare the positive control substance (RNA) of Nipah virus M gene, and the process includes:
[0050] (1) On the NCBI website, perform BLAST comparison on the M gene sequence of Nipah virus, and select a conserved sequence for the preparation of the M gene positive control, as shown in SEQ ID NO.4.
[0051] (2) Design and synthesize 4 primers based on the sequence of SEQ ID NO.4, the primer sequences are as follows:
[0052] NIPHA-M-F1:5'-CCTGGAACAACAGTTGTGAGATCAGCCGAGTAGCAGCTGTGTTGCAGCCTTCTGTTCC-3
[0053] NIPHA-M-R1:5'-GGAACAGAAGGCTGCAACACAGCTGCTACTCGGCTGATCTCACAACTGTTGTTCCAGG-3
[0054] NIPHA-M-F2:5'-GATGGACATCAATCCTTGGCTCAACAGATTGACCTGGAACAACAGTTGTGAGATC-3'
[0055] NIPHA-M-R2:5'-GAAGACATCATCATAGATCATGAACTCTCTTGGAACAGA...
Embodiment 1
[0103] Example 1 Preparation of Nipah virus M gene positive control
[0104] 1. Selection of reference gene sequence
[0105] BLAST was performed on the Nipah virus M gene sequence (GenBank accession number: AJ564621.1), and a conservative and suitable sequence for designing fluorescent RT-PCR primers and probes was selected as a reference sequence for preparing Nipah virus M gene positive control (RNA) , as shown in SEQ ID NO.1.
[0106] 2. Design and synthesis of amplification primers
[0107] Based on the above sequences, 4 PCR amplification primers were designed and synthesized, in which NIPHA-M-F1 and NIPHA-M-R1 sequences were completely complementary and used as templates, and NIPHA-M-F2 and NIPHA-M-R2 were respectively compatible with Partially overlapping with the NIPHA-M-R1 sequence, the synthesized sequence is as follows:
[0108] NIPHA-M-F1:5'-CCTGGAACAACAGTTGTGAGATCAGCCGAGTAGCAGCTGTGTTGCAGCCTTCTGTTCC-3
[0109] NIPHA-M-R1:5'-GGAACAGAAGGCTGCAACACAGCTGCTACTCGGCTG...
Embodiment 2
[0125] Example 2 Preparation of Porcine Reproductive and Respiratory Syndrome Virus NSP2 Gene Positive Control (RNA)
[0126] 1. Selection of reference gene sequence
[0127] BLAST was performed on the porcine reproductive and respiratory syndrome virus NSP2 gene sequence (GenBank accession number: JX087437), and a conservative and suitable sequence for designing fluorescent RT-PCR primers and probes was selected as a positive control for porcine reproductive and respiratory syndrome virus NSP2 gene (RNA) reference sequence, as shown in SEQ ID NO.1.
[0128] 2. Design and synthesis of amplification primers
[0129] Based on the above sequences, 4 PCR amplification primers were designed and synthesized, in which PRRSV-NSP2-F1 and PRRSV-NSP2-R1 sequences were completely complementary and used as templates, and PRRSV-NSP2-F2 and PRRSV-NSP2-R2 were respectively combined with PRRSV-NSP2-F1 and
[0130] The sequences of PRRSV-NSP2-R1 partially overlap, and the synthesized sequenc...
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