Triple-fluorescence PCR (Polymerase Chain Reaction) detection method and kit for Nosema bombycis (Nb), Bombyx mori Nuclear Polyhedrosis Virus (BmNPV) and Bombyx mori Densovirus (BmDNV), as well as primers and probes

A technology for karyotype polyhedrosis and microsporidia, which is applied in the field of biological detection technology, can solve the problems of unstable serological detection, low specificity and sensitivity, and inability to simultaneously differentiate and diagnose.

Inactive Publication Date: 2013-09-04
ZHEJIANG ACAD OF SCI & TECH FOR INSPECTION & QUARANTINE
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AI Technical Summary

Problems solved by technology

[0004] Microscopes are widely used in the detection of silkworm sporozoites, but their specificity and sensitivity are not high, and their detection speed and efficiency are low; while the detection of silkworm nuclear polyhedrosis virus and silkworm densovirus requires an electron microscope, and the detection speed is relatively low. and low efficiency, and cannot be differentially diagnosed at the same time
The serological identification method has the characteristics of strong specificity and high sensitivity, but there are relatively high cross-reactions and non-specific reactions, and silkworm sporozoites are granular antigens, and serological detection is unstable

Method used

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  • Triple-fluorescence PCR (Polymerase Chain Reaction) detection method and kit for Nosema bombycis (Nb), Bombyx mori Nuclear Polyhedrosis Virus (BmNPV) and Bombyx mori Densovirus (BmDNV), as well as primers and probes
  • Triple-fluorescence PCR (Polymerase Chain Reaction) detection method and kit for Nosema bombycis (Nb), Bombyx mori Nuclear Polyhedrosis Virus (BmNPV) and Bombyx mori Densovirus (BmDNV), as well as primers and probes
  • Triple-fluorescence PCR (Polymerase Chain Reaction) detection method and kit for Nosema bombycis (Nb), Bombyx mori Nuclear Polyhedrosis Virus (BmNPV) and Bombyx mori Densovirus (BmDNV), as well as primers and probes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Design of primers and probes

[0044] Based on the highly species-specific microsporidia small subunit ribosomal RNA gene sequence of the silkworm ( Nosema bombycis small subunit ribosomal RNA, FJ854545 (gene sequence number)), silkworm nuclear polyhedrosis virus DNA polymerase gene sequence ( Bombyx mori nucleopolyhedrovirus gene for DNA polymerase, D16231) and the hypothetical structural protein gene sequence of the silkworm densovirus Zhenjiang strain ( Bombyx mori densovirus isolate Zhenjiang putative structural protein gene, AY236978), using bioinformatics methods based on primer design principles, and using Primer Express design software, designed several sets of specific primers and fluorescent probes for amplifying the above gene fragments. The designed primers and probes were verified by BLAST (http: / / blast.ncbi.nlm.nih.gov / ) to ensure the high specificity of the primers. Furthermore, DNAStar software was used to verify whether primer-dimers we...

Embodiment 2

[0048] Example 2: Sample DNA Extraction

[0049] Select 0.2 g of the silkworm material infected with the above three pathogens, break it by glass bead crushing method, 6000 r / min, 20 s×6 times, use the DNA extraction kit (Promega FF3750) to carry out DNA extraction according to the instructions. The extracted DNA was dissolved in 20 μl of water.

Embodiment 3

[0050] Example 3: Triple fluorescent PCR amplification reaction and fluorescence compensation reaction

[0051] Simultaneous detection of three samples by triple fluorescent PCR amplification method. The sequences of primers and probes are shown in Table 1. The triple fluorescent PCR reaction system was as follows: 10 μl Premix Ex Taq (Takara), 0.13 μl of each primer (10 pmol / μl), 0.27 μl of each probe (10 pmol / μl), Take 4 μl of the DNA extracted above (the DNA content of Nb, BmNPV and BmDNV is 2.5 ng / μl, respectively), and make up the insufficient volume to 20 μl with water. The fluorescence quantitative PCR instrument lightcycle 480 (Roche) was used for the reaction, and the reaction program was (1) 95°C, 10 sec; (2) 95°C, 5 sec; 60°C, 23 sec; 40 cycles. Call the corresponding fluorescence compensation program, perform fluorescence compensation, and judge the results. It is generally believed that when the Ct value is less than or equal to 35, the result is positive, whe...

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Abstract

The invention belongs to the technical field of bioinstrumentation, and relates to a triple-fluorescence PCR (Polymerase Chain Reaction) detection method and kit for Nosema bombycis (Nb), Bombyx mori Nuclear Polyhedrosis Virus (BmNPV) and Bombyx mori Densovirus (BmDNV), as well as primers and probes. According to the invention, the primers and the probes for the Nb, the BmNPV and the BmDNV are respectively designed, and different fluorescence signals are marked on the three probes, so that the Nb, the BmNPV and the BmDNV can be synchronously detected by triple-fluorescence-quantitation PCR under the condition of optimizing a PCR reaction system. According to the invention, the synchronous extraction of DNA (deoxyribonucleic acid) can be realized, and the synchronous detection based on fluorescence PCR can be performed, so that the detection method is simplified greatly; and through a program of florescence compensation, false positive due to intersection of the fluorescence signals is eliminated effectively. The method can be used for quickly, peculiarly and sensitively verifying and detecting the Nb, the BmNPV and the BmDNV, and improving the early detection rate, thereby effectively preventing disease transmission and reducing the economic loss.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and relates to Microsporidia silkworm ( Nosema bombycis , Nb), Bombyx mori nuclear polyhedrosis virus ( Bombyx mori Nuclear Polyhedrosis Virus, BmNPV) and Bombyx mori densovirus ( Bombyx mori Densovirus, BmDNV) triple fluorescent quantitative PCR detection method and kit, primers and probes. Background technique [0002] Microsporidium silkworm ( Nosema bombycis , Nb), Bombyx mori nuclear polyhedrosis virus ( Bombyx mori Nuclear Polyhedrosis Virus, BmNPV) and Bombyx mori densovirus ( Bombyx mori Densovirus, BmDNV), are the pathogens that cause microparticle disease, nuclear polyhedrosis and densovirus of silkworm respectively, all of which can cause huge economic losses in the world's sericulture, and may even be mixed infection at the same time. Due to the lack of effective control methods for the above-mentioned silkworm diseases, early detection and identification of pa...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12Q1/04C12N15/11G01N21/64
Inventor 何永强吴珊帅江冰王素华张晓峰黎昊雁鲁兴萌李孝军余招锋许赢升戴云通
Owner ZHEJIANG ACAD OF SCI & TECH FOR INSPECTION & QUARANTINE
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