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Duplex fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection reagent for nipah virus and swine influenza virus (SIV) as well as preparation method and application of duplex fluorescent RT-PCR detection reagent

A technology of RT-PCR and swine influenza virus, applied in the field of double fluorescent RT-PCR detection reagent and its preparation

Inactive Publication Date: 2015-07-01
ANIMAL & PLANT & FOOD INSPECTION CENT OF TIANJIN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the fluorescent RT-PCR method and public patents for detecting single Nipah virus and swine influenza virus have been reported, and the dual fluorescent quantitative RT-PCR detection reagent capable of simultaneously detecting Nipah virus and swine influenza virus and its preparation method and application , also rarely reported

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  • Duplex fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection reagent for nipah virus and swine influenza virus (SIV) as well as preparation method and application of duplex fluorescent RT-PCR detection reagent
  • Duplex fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection reagent for nipah virus and swine influenza virus (SIV) as well as preparation method and application of duplex fluorescent RT-PCR detection reagent
  • Duplex fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection reagent for nipah virus and swine influenza virus (SIV) as well as preparation method and application of duplex fluorescent RT-PCR detection reagent

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preparation example Construction

[0054] The double fluorescent RT-PCR detection reagent of Nipah virus and classical swine fever virus and its preparation method and application of the present invention comprise the following steps.

[0055] The first step is to prepare the positive control substance (RNA) of Nipah virus M gene, and the process includes:

[0056] (1) On the NCBI website, the Nipah virus M gene sequence was compared by BLAST, and a conserved sequence was selected for the preparation of the Nipah virus M gene positive control, as shown in SEQ ID NO.4.

[0057] (2) Design and synthesize 4 primers based on the sequence of SEQ ID NO.4, the primer sequences are as follows:

[0058] NIPHA-M-F1:5'-CCTGGAACAACAGTTGTGAGATCAGCCGAGTAGCAGCTGTGTTGCAGCCTTCTGTTCC-3

[0059] NIPHA-M-R1:5'-GGAACAGAAGGCTGCAACACAGCTGCTACTCGGCTGATCTCACAACTGTTGTTCCAGG-3

[0060] NIPHA-M-F2:5'-GATGGACATCAATCCTTGGCTCAACAGATTGACCTGGAACAACAGTTGTGAGATC-3'

[0061] NIPHA-M-R2:5'-GAAGACATCATCATAGATCATGAACTCTCTTGGAACAGAAGGCTGCAACACAGCT...

Embodiment 1

[0110] The preparation of embodiment 1 Nipah virus M gene positive control

[0111] 1. Selection of reference gene sequence

[0112] 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.

[0113] 2. Design and synthesis of amplification primers

[0114] 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:

[0115] NIPHA-M-F1:5'-CCTGGAACAACAGTTGTGAGATCAGCCGAGTAGCAGCTGTGTTGCAGCCTTCTGTTCC-3

[0116] NIPHA-M-R1:5'-GGAACAGAAGGCTGCAACACAGCTGCTAC...

Embodiment 2

[0132]The preparation of embodiment 2 swine influenza virus M gene positive control (RNA)

[0133] 1. Selection of reference gene sequence

[0134] Carry out BLAST to swine influenza virus M gene sequence (GenBank accession number: KF986907), choose a section of conservative and suitably design the sequence of fluorescent RT-PCR primer and probe as the reference A) of preparation swine influenza virus M gene positive control (RNA) Reference is shown in SEQ ID NO.1.

[0135] 2. Design and synthesis of amplification primers

[0136] Based on the above sequences, 4 PCR amplification primers were designed and synthesized, in which SIV-M-F1 and SIV-M-R1 sequences were completely complementary and used as templates, and SIV-M-F2 and SIV-M-R2 were respectively compatible with SIV-M-F1 Partially overlapping with the SIV-M-R1 sequence, the synthesized sequence is as follows:

[0137] SIV-M-F1:5-GTGCCTGAGTCCATGAGGGAAGAATATCAGCAGGAACAGCAGAGTGCTGTGGATGTTG-3

[0138] SIV-M-R1:5-CAACATC...

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Abstract

The invention discloses a duplex fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection reagent for Nipah virus and swine influenza virus (SIV) as well as a preparation method and application of the duplex fluorescent RT-PCR detection reagent. Two sets of specific primers and Taqman probes which are respectively used for an M gene of the Nipah virus and an M virogene of the SIV as well as positive control are designed and synthesized; a duplex fluorescent RT-PCR detection system with the advantages of quickness, simplicity, convenience, high specificity and high sensitivity is established by using the two sets of primers and probes; the duplex fluorescent RT-PCR detection system can be used for simultaneously detecting nucleic acids of the Nipah virus and the SIV quickly, accurately, specifically, safely, simply and conveniently from a detected sample within 3 to 4 hours, and simultaneously detecting nucleic acids of trace Nipah virus and the SIV from a domestic pig and a correlated sample of the domestic pig.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a double fluorescent RT-PCR detection reagent for Nipah virus and swine influenza virus, a preparation method and application thereof. Background technique [0002] Nipah virus is the pathogen of Nipah disease, a zoonotic disease that seriously endangers the health of humans and pigs. The clinical manifestations are characterized by nervous system and respiratory system diseases, and the clinical symptoms of pigs of different ages are also different. Humans are mainly infected through direct contact between the wound and the secretions and excreta of infected pigs, and can also be infected through the air, resulting in morbidity and death. Therefore, Nipah virus-infected pigs are an important source of infection for human Nipah disease. Swine influenza is an acute, zoonotic respiratory infectious disease caused by type A influenza virus in pigs or humans. Since Nipah di...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11C12Q1/70
CPCC12Q1/701C12Q1/686C12Q2600/16C12Q2537/143C12Q2561/101
Inventor 赵祥平王建华董志珍肖妍王玉玲赵丹张俊哲王乃福陈小金陈本龙
Owner ANIMAL & PLANT & FOOD INSPECTION CENT OF TIANJIN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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