Nanometer immunogold labeling probe for detecting peste des petits ruminants virus (PPRV) and detection kit

A PPR and probe technology, applied in the field of molecular biology, can solve the problems of false positives, limited resolution, primer-dimer interference, etc., and achieve the effects of simple operation, good repeatability and high sensitivity

Inactive Publication Date: 2012-08-29
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Abstract
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Problems solved by technology

However, the resolving power of agarose gel electrophoresis is limited, and in the case of low amplification yield, it may not be possible to display
In addition, for short fragments with amplified fragments below 250bp, they are also susceptible to interference from primer dimers, resulting in false positives
Another new method for detecting the nucleic acid of the virus is the loop-mediated isothermal amplification assay (LAMP), but at present, the technology is not yet mature, and it is prone to false negatives.

Method used

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  • Nanometer immunogold labeling probe for detecting peste des petits ruminants virus (PPRV) and detection kit
  • Nanometer immunogold labeling probe for detecting peste des petits ruminants virus (PPRV) and detection kit
  • Nanometer immunogold labeling probe for detecting peste des petits ruminants virus (PPRV) and detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Embodiment 1 detects the determination of the PPRV target sequence, the design and synthesis of primers and probes

[0060] Refer to the gene sequences of PPRV Nigeria75 / 1 (GenBank: X74443.2) and other strains (HQ197753.1, EU267274.1, AJ849636.2, FJ905304.1, AJ563705.1) published by NCBI, which are conserved in the PPRV N gene The target nucleic acid fragment for detection of Peste des Petits Ruminants virus was found in the region, and its nucleotide sequence is shown in SEQ ID NO.1. Design specific primers to amplify it according to the target sequence:

[0061] PPRV-N115F: 5'-TCATATTTTGACCCGGCCTATTTCC-3' (SEQ ID NO. 2), PPRV-N115R: 5'-GTTTGGCTTCCTCTGCTGTGATAC-3'

[0062] (SEQ ID NO. 3).

[0063] Two specific probe sequences: P1-B: 5′-Biotin-CTCGGACAGGAGATGGT CAGAAGAT-3′ (SEQ ID NO.4), P2-G: 5′-GGTCAGCTCTGTAATC GCGGC(A)10SH-3′ (SEQ ID NO. .5).

[0064] Extract the total RNA of the PPR virus, reverse transcription PCR to obtain a 115bp fragment of the highly conser...

Embodiment 2

[0065] The preparation of embodiment 2 nano-gold labeled nucleic acid probes

[0066] 1. Preparation and characterization of gold nanoparticles

[0067] According to the method of Zhao Lifan et al. (Zhao Lifan, Li Baisheng, Hei Xiaohan, et al. Silver-stained enhanced nano-gold probe technology for detecting trace amounts of nucleic acid. Chinese Journal of Biochemistry and Molecular Biology. 2006 (11): 919-923.), prepare Nano gold solution with particle diameter of 13-15nm. Store at 4°C protected from light. The morphology and size of gold nanoparticles were observed by transmission electron microscope (accelerating voltage 120KV). According to Georganopoulou et al. (Georganopoulou, D.G., Chang, L., Nam, J.M., et al. Nanoparticle-based detection in cerebral spinal fluid of a soluble pathogenic biomarker for Alzheimer's disease.Proc Natl Acad Sci USA.2005, 102( 7): 2273-2276.) The method reported to calculate the concentration of nano gold solution. by the formula

[0068]...

Embodiment 3

[0075] Example 3 Establishment of Nano-gold Labeled Nucleic Acid Probe Detection Method for Peste des Petits Ruminants Virus

[0076] 1. Hybridization reaction

[0077] Take 10 μl of 1 nmol / L target nucleic acid solution prepared in Example 1, add 20 μl of hybridization buffer (buffer composition is 20 mmol / LPB, 0.3 mol / L NaCl, 0.01% SDS), denature at 95 ° C for 5 min, and place on ice for 5 min . Add 20 μl 100nmol / L biotinylated probe P1-B, the sequence is

[0078] P1-B: 5′-Biotin-CTCGGACAGGAGATGGTCAGAAGAT-3′, react at 42°C for 15 minutes. Next, 10 μl of the nano-gold nucleic acid probe prepared in Example 2 was added, and incubated at 42° C. for 15 minutes. The hybridization product was added to a streptavidin-coated microtiter plate (10 μg / ml streptavidin, 100 μl per well was coated). Place in a humid box and incubate at 37°C for 30min. Wash 3 times with PBST and pat dry. Add 100 μl silver staining solution (1% (w / v) gelatin: 0.52mol / L hydroquinone: pH 3.5 citrate buf...

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Abstract

The invention provides a nanometer immunogold labeling probe for detecting a peste des petits ruminants virus (PPRV) and a detection kit. Two specific oligonucleotide probes are designed by aiming at a highly conserved region of an N gene of the PPRV. The nucleotide sequence of the 5' modified biotin of one of the probes and the nucleotide sequence of the 3' modified sulfydryl of the other probe are respectively shown by SEQ ID NO.2 and SEQ ID NO.3. The invention also provides a method for detecting the nucleic acid of the PPRV by utilizing the nanometer immunogold labeling oligonucleotide probe. The sulfhydrylated probes are respectively connected onto nanometer gold particles through Au-S bonds; both ends of the targeted nucleic acid are respectively bonded with the two probes to form a hybrid compound, and the targeted nucleic acid is quantitatively detected through bonding the hybrid compound with a solid-phase carrier and performing silver staining on an amplifying signal. According to the detection method provided by the invention, the minimum concentration of the nucleic acid reaches 10fmol/L. The method is high in sensitivity and strong in specificity. A novel method is provided for clinically detecting the PPRV.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a target nucleic acid for detecting Peste des Petits Ruminants virus, a primer for amplifying the target nucleic acid, a method for detecting the target nucleic acid with a nano-gold-labeled nucleic acid probe, and a detection kit. Background technique [0002] Peste des petits ruminants (PPR) is a severe and contagious infectious disease caused by Peste des petits ruminants virus (PPRV). The main infected animals are small ruminants, among which goats are highly susceptible. PPRV is a member of the genus Morbillivirus in the Paramyxoviridae family. There are 4 groups and 1 serotype of the virus. The PPRV genome is a single-stranded negative-strand non-segmented RNA, with 6 genes, N-P-M-F-H-L, in sequence from 3′ to 5′ end, encoding 6 structural proteins (N, P, M, F, H, L) and 2 nonstructural proteins. Proteins (C and V). At present, the methods for detecting PPRV mainly incl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
Inventor 李刚陶春爱邱文英史利军
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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