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Duplex fluorescent RT-PCR detection reagent for Nipah virus and hog cholera virus as well as preparation method and application thereof

A RT-PCR, Nipah virus technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial assay/inspection, etc.

Inactive Publication Date: 2015-07-08
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 open patents for detecting single Nipah virus and swine fever virus have been reported, and the double fluorescent RT-PCR detection reagent and its preparation method and application that can detect Nipah virus and swine fever virus simultaneously, also rarely reported

Method used

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  • Duplex fluorescent RT-PCR detection reagent for Nipah virus and hog cholera virus as well as preparation method and application thereof
  • Duplex fluorescent RT-PCR detection reagent for Nipah virus and hog cholera virus as well as preparation method and application thereof
  • Duplex fluorescent RT-PCR detection reagent for Nipah virus and hog cholera virus as well as preparation method and application thereof

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preparation example Construction

[0056] 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.

[0057] The first step is to prepare the positive control substance (RNA) of Nipah virus M gene, and the process includes:

[0058] (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.

[0059] (2) Design and synthesize 4 primers based on the sequence of SEQ ID NO.4, the primer sequences are as follows:

[0060] NIPHA-M-F1:5'-CCTGGAACAACAGTTGTGAGATCAGCCGAGTAGCAGCTGTGTTGCAGCCTTCTGTTCC-3

[0061] NIPHA-M-R1:5'-GGAACAGAAGGCTGCAACACAGCTGCTACTCGGCTGATCTCACAACTGTTGTTCCAGG-3

[0062] NIPHA-M-F2:5'-GATGGACATCAATCCTTGGCTCAACAGATTGACCTGGAACAACAGTTGTGAGATC-3'

[0063] NIPHA-M-R2:5'-GAAGACATCATCATAGATCATGAACTCTCTTGGAACAGAAGGCTGCAACACAGCTG-3'

[...

Embodiment 1

[0111] The preparation of embodiment 1 Nipah virus M gene positive control

[0112] 1. Selection of reference gene sequence

[0113] 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.

[0114] 2. Design and synthesis of amplification primers

[0115] 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:

[0116] NIPHA-M-F1:5'-CCTGGAACAACAGTTGTGAGATCAGCCGAGTAGCAGCTGTGTTGCAGCCTTCTGTTCC-3

[0117] NIPHA-M-R1:5'-GGAACAGAAGGCTGCAACACAGCTGCTAC...

Embodiment 2

[0133] The preparation of embodiment 2 swine fever virus 5'UTR gene positive control (RNA)

[0134] 1. Selection of reference gene sequence

[0135] Perform BLAST on the 5'UTR gene sequence of classical swine fever virus (GenBank accession number: AY259122), and select a sequence that is conservative and suitable for designing fluorescent RT-PCR primers and probes as the preparation of the positive control (RNA) of the 5'UTR gene of classical swine fever virus Refer to A) as shown in SEQ ID NO.1.

[0136] 2. Design and synthesis of amplification primers

[0137] Based on the above sequences, four PCR amplification primers were designed and synthesized, in which CSFV-F1 and CSFV--R1 sequences were completely complementary and used as templates, and CSFV-F2 and CSFV--R2 partially overlapped with CSFV-F1 and CSFV-R1 sequences, respectively. The resulting sequence is as follows:

[0138] CSFV-F2:5'-CTGGGTGGTCTAAGTCCTGAGTACAGGACAGTCGTCAGTAGTTCGACGTGAGC-3'

[0139] CSFV-F1:5'-GT...

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Abstract

The invention discloses a duplex fluorescent RT-PCR detection reagent for nipah virus and hog cholera virus as well as a preparation method and an application thereof. Two sets of specific primers for a nipah virus M gene and a hog cholera virus 5' UTR gene as well as a Taqman probe and positive control are designed and synthesized respectively, and a duplex fluorescent RT-PCR detection system which is quick, simple, convenient, strong in specificity and high in sensitivity is established by utilizing the two sets of primers and the probe, so that nucleic acids of the nipah virus and the hog cholera virus can be quickly, accurately, specifically, safely, simply and conveniently detected from a detected sample within 3-4 hours, and the detection reagent can be used for simultaneously detecting trace nucleic acids of the nipah virus and the hog cholera virus in a hog and a relevant sample thereof.

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 fever virus, a preparation method and application thereof. Background technique [0002] Nipah virus (NiV) is a new virus identified in 1999 that seriously harms domestic pigs and humans. It can cause nervous system and respiratory diseases in humans and pigs. Classical Swine Fever (CSF) is an acute, febrile, and highly contagious infectious disease of pigs caused by CSFV. It presents acute symptoms according to the virulence of the prevailing virus strains and the immune status of pigs. Type, subacute type, mild type and other clinical symptoms. Since Nipah disease and classical swine fever in pigs have similar clinical manifestations, it is not easy to distinguish them based on clinical symptoms alone. Therefore, the differential detection of Nipah virus and classical swine fever virus in clinicall...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11C12Q1/70
CPCC12Q1/701C12Q1/686C12Q2600/16
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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