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Primers used for amplifying nucleotide segment of nucleocapsid protein gene of H5N1 influenza virus and detection method

An influenza virus and nucleotide technology, applied in the medical field, can solve problems such as high requirements for primers and probes, quantitative detection of unsuitable viruses, non-specific amplification, etc., and achieve small errors, good intervals, and good repeatability Effect

Active Publication Date: 2013-03-20
INST OF LAB ANIMAL SCI CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are two quantitative detection methods for influenza virus, one is the Taqman probe method, which has good specificity, but the probe is relatively expensive, the requirements for primers and probes are very high, and the detection cost for a large number of samples is too high; One is the common SYBR Green quantitative PCR, which has lower technical cost and higher sensitivity than the former. Although the specificity is not as good as the probe method, it can meet the detection requirements of influenza virus.
The World Health Organization (WHO) recommends suitable primers for the detection of H5N1 by the probe method and the dye method. Doubtful, but when the dye method is used to detect H5N1 in ferret and monkey tissues, there is non-specific amplification, which is not suitable for quantitative detection of viruses in ferret and monkey tissues

Method used

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  • Primers used for amplifying nucleotide segment of nucleocapsid protein gene of H5N1 influenza virus and detection method
  • Primers used for amplifying nucleotide segment of nucleocapsid protein gene of H5N1 influenza virus and detection method
  • Primers used for amplifying nucleotide segment of nucleocapsid protein gene of H5N1 influenza virus and detection method

Examples

Experimental program
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Effect test

Embodiment 1

[0031] Utilize the primer set having the upstream primer of the nucleotide sequence shown in SEQ ID NO:1 and the downstream primer with the nucleotide sequence shown in SEQ ID NO:2, take H5N1 influenza virus standard virus strain (SZ406H) as template According to the reaction system of the Power SYBR Green PCR Master Mix (ABI, 4367659) kit, quantitative amplification was carried out on a stepone 2.0 quantitative PCR instrument. Amplification curve such as figure 1 As shown, the melting curve is as figure 2 As shown, the standard curve is as image 3 shown.

[0032] Depend on figure 1 As shown, the overall parallelism of the amplification curve is good; when the template is subjected to 10-fold ratio gradient dilution, the interval of each concentration is good; the repeatability of the parallel experiment of each concentration is good. Depend on figure 2 As shown, the melting curve has a single peak, no primer peak or miscellaneous peak occurs, and the peak symmetry a...

Embodiment 2

[0036] Roche, Cat.12033674001; Ambion, AM1912; BioMIGA; Qiagen, cat.74106; Trizol invitrogen, Cat.No.15596-026 5 kits were used to extract the total RNA in the lung and intestinal tissue samples of ferrets and monkeys to be tested .

[0037] The total RNA extracted from the lung and intestinal tissues of ferrets and monkeys was reverse-transcribed with the SuperScript III First-Strand Synthesis System kit from Invitrogen to obtain cDNA.

[0038] See Table 1 for the primers recommended by WHO to detect the NP gene of H5N1 influenza virus in ferret or / and monkey tissues by common PCR and dye-based quantitative PCR.

[0039] Table 1: Primers recommended by WHO for general PCR and dye-based quantitative PCR

[0040]

[0041] The primer set designed for the H5N1 (SZ406H) NP gene (Genebank accession number: 133711835) consists of an upstream primer with a nucleotide sequence shown in SEQ ID NO: 1 and an upstream primer with a nucleotide sequence shown in SEQ ID NO: 2 Downstream...

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Abstract

The invention discloses a primer pair used for amplifying the nucleotide segment of the nucleocapsid protein (NP) gene of H5N1 influenza virus. The primer pair is characterized in that the primer pair consists of a forward primer with the nucleotide sequence shown in the SEQ ID NO:1 and a reverse primer with the nucleotide sequence shown in the SEQ ID NO:2. The invention also provides an application of the primer pair in the detection of H5N1 influenza virus in the tissue of ferret or / and monkey and a method for detecting the NP gene of the H5N1 influenza virus by utilizing the primer pair. By adopting the primer pair and amplification method, the nonspecific interference of the animal genome can be suppressed, the specificity can be good and the sensitivity can be high; and the primer pair and amplification method are easy to popularize.

Description

technical field [0001] The invention relates to the field of medical technology, in particular to primers for amplifying NP gene nucleotide fragments of H5N1 influenza virus. Background technique [0002] Pandemic influenza (influenza for short) is a severe infectious disease caused by influenza virus, which is seriously harmful to humans and livestock and poultry. healthy. There are three types of influenza virus, A, B, and C, among which the influenza epidemic caused by type A is the most widespread and severe. Influenza virus particles are composed of three layers, the outermost layer is two protruding surface antigens, namely hemagglutinin (HA) and neuraminidase (NA), the middle layer is composed of a layer of lipid body and a layer of membrane protein (MP ), the core layer is the RNA gene and the protein combined with it, that is, nucleoprotein (NP). Among them, hemagglutinin (HA) and neuraminidase (NA) are the main basis for the division of influenza A virus subtypes...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12Q1/70C12Q1/68
Inventor 占玲俊鲍琳琳李枫棣吕琦许黎黎秦川
Owner INST OF LAB ANIMAL SCI CHINESE ACAD OF MEDICAL SCI
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