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Nipah virus detection kit and dedicated primer of same

A detection kit and Nipah virus technology, applied in biochemical equipment and methods, microbe measurement/inspection, DNA/RNA fragments, etc., can solve problems such as indistinguishability, achieve simple operation, broad market prospects, suitable for The effect of promoting the application on a large scale

Active Publication Date: 2018-12-18
WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Electron microscopy cannot distinguish Nipah virus from Hendra virus or other paramyxoviruses
RT-PCR and Real-time RT-PCR techniques are sensitive methods for rapid detection of Nipah virus, however, it is difficult to implement these methods in small laboratories and field testing due to the need for expensive thermal cycling equipment

Method used

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  • Nipah virus detection kit and dedicated primer of same
  • Nipah virus detection kit and dedicated primer of same
  • Nipah virus detection kit and dedicated primer of same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1. Design of primers for detecting Nipah virus

[0065] 1. Selection of conservative target sequence

[0066] Download the full-length N gene sequence of all Nipah virus genotypes (including genotype M and genotype B) from the NCBI Nucleotide database (each N gene is 1599bp in length) (n=35, n is the number of selected N genes) ), and selected 3 representative strains of 2 genotypes-Malaysia representative strain M, Bangladesh representative strain B, and Thailand isolate representative strain T. The full-length N gene sequences of the 3 representative strains refer to the Nipah virus genome accession numbers respectively They are: KY425646.1, JN808862.1 and KT163255.1. Through multiple sequence alignment and homology analysis, the conservative target sequences of the N genes of each strain are obtained, such as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID respectively. NO: shown in 3.

[0067] 2. Primer design

[0068] According to the three conserved target gene sequences...

Embodiment 2

[0073] Example 2. Establishment of detection methods for all genotypes of Nipah virus Stand up

[0074] The three sets of primers obtained in Example 1 were used to perform LAMP detection on the full-length N gene plasmid of Nipah virus to obtain the best reaction system and reaction conditions.

[0075] Synthesize the full-length N genes of three representative strains of Nipah virus (KY425646.1, JN808862.1 and KT163255.1), respectively, insert the pUC57 plasmid (plasmid from the Wuhan Synthesis Department of Kinco Biology) to obtain the complete set of three Nipah virus The long N gene plasmids were named pUC57-NiV-MN (representative strain in Malaysia), pUC57-NiV-BN (representative strain in Bangladesh) and pUC57-NiV-TN (representative strain in Thailand).

[0076] 1. Determination of the best detection primer

[0077] Under the same reaction system, three sets of candidate primers-NiV-1, NiV-2 and NiV-3 were used to react to the N gene positive plasmid (pUC57-NiV) of the represen...

Embodiment 3

[0101] Example 3. Specificity and sensitivity experiment

[0102] 1. Specificity

[0103] 1. LAMP detection specificity: the N gene plasmid pUC57-NiV-BN of the representative strain of Nipah virus in Bangladesh, the N gene plasmid pUC57-NiV-MN of the representative strain of Nipah virus Malaysia, and the N gene plasmid of the representative strain of Nipah virus Thailand pUC57- NiV-TN, hepatitis C virus HCV genome full-length plasmid (Chinese People's Liberation Army Disease Control and Prevention Institute), hepatitis B virus HBV genome full-length plasmid (Chinese People's Liberation Army Disease Prevention and Control Institute), human acquired immunodeficiency virus HIV genome Full-length plasmid (Chinese People's Liberation Army Disease Control and Prevention Institute), SARS coronavirus envelope protein S1 C-terminal deletion 19 AA plasmid pCAGGS-SARS-S1Δ-19 (Chinese People's Liberation Army Disease Control and Prevention Institute), Middle East Respiratory Syndrome Virus ME...

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Abstract

The invention discloses a Nipah virus detection kit and a dedicated primer of same, wherein the primer is used for performing RT-LAMP detection on the Nipah virus or LAMP detection on the genome cDNAof the Nipah virus. The primer is designed on the basis of specific conserved target sequences of the Nipah virus, which are represented as the SEQ ID No.1-3. The primer or detection kit in the invention can detect the Nipah virus under an isothermal condition quickly, conveniently, high-effectively, high-specifically and high-sensitively without use of complex instruments. The kit can be used forperforming the RT-LAMP detection on the genome RNA of the Nipah virus.

Description

Technical field [0001] The invention belongs to a method for molecular biology detection of viruses in the field of biotechnology, and particularly relates to a special primer for the detection of Nipah virus reverse transcription-loop-mediated isothermal nucleic acid amplification (RT-LAMP), a detection kit containing the primer, And the application of the primer and the detection kit in the detection of Nipah virus. Background technique [0002] Nipah virus (NiV) belongs to the Paramyxoviridae (Paramyxoviridae) and Hennipaviruses (Henipavirus). It is classified as Biosafety level 4 (Biosafety level 4, BSL) due to its relatively new discovery and high pathogenicity. -4) Viruses can cause acute severe respiratory diseases and encephalitis in humans, with a fatality rate of 40%-70%. Nipah virus is a single-stranded negative-strand RNA virus, and its N gene is highly conserved. It is often used for RT-PCR diagnosis of Nipah virus. Nipah virus has two typical genotypes: genotype M...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/6844C12Q1/701C12Q2521/107C12Q2531/119
Inventor 刘翟陈全姣马莉萍
Owner WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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