Nosema bombycis SYBT Green fluorescence quantitative PCR (Polymerase Chain Reaction) detection method as well as kit and specific primer thereof

A microsporidia, fluorescence quantitative technology, applied in the direction of microbial determination/inspection, fluorescence/phosphorescence, biochemical equipment and methods, to avoid missed detection and false detection, enhanced sensitivity and specificity, good repeatability and stability sexual effect

Inactive Publication Date: 2011-01-05
ZHEJIANG ACAD OF SCI & TECH FOR INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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This method is innovative to a certain extent, but it is ...

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  • Nosema bombycis SYBT Green fluorescence quantitative PCR (Polymerase Chain Reaction) detection method as well as kit and specific primer thereof
  • Nosema bombycis SYBT Green fluorescence quantitative PCR (Polymerase Chain Reaction) detection method as well as kit and specific primer thereof
  • Nosema bombycis SYBT Green fluorescence quantitative PCR (Polymerase Chain Reaction) detection method as well as kit and specific primer thereof

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Embodiment 1

[0033] Example 1: Specimen collection

[0034] Applicable specimen types include samples such as silkworms, silkworm eggs and feces. Take an appropriate amount of suspected disease materials such as silkworms, silkworm eggs or feces from the scene, seal them, label them, and send them for inspection as soon as possible or in a short-term refrigerated storage.

Embodiment 2

[0035] Embodiment 2: the extraction of silk microsporidia DNA

[0036] Take the above specimen and add appropriate amount of normal saline to grind, filter the homogenate with gauze, collect the filtrate, and use differential centrifugation (500rpm, centrifuge for 2 minutes, discard the precipitate, then centrifuge the supernatant at 5000rpm for 10 minutes), discard the supernatant, and divide Centrifuge quickly until the precipitate is off-white, suspend it with an appropriate amount of normal saline, and obtain a crude extract of Nb spores. Take 0.5ml (10 8 individual / ml) Microsporidia suspension, centrifuge at 5000rpm for 5 minutes, remove the supernatant, keep the spores, add 0.2mol / L KOH 0.25ml at the same time, mix thoroughly, induce at 27℃ for 1 hour, centrifuge at 5000rpm for 5 minutes, and remove clear. Resuspend with 0.5ml of normal saline and place at 25°C for 30 minutes. Centrifuge at 5000rpm for 5 minutes, add 50μl of nucleic acid extraction solution to the pre...

Embodiment 3

[0037] Embodiment 3: the amplification of silk microsporidia DNA

[0038] Take out the Nb-PCR MIX from the kit, melt at room temperature and shake to mix, then centrifuge at 10,000rpm for 10 seconds. Each Nb-PCR MIX detection unit consists of 10 μl 2×SYBR Premix Ex Taq, 0.4 μl ROX ReferenceDyeII, 0.4 μl upstream primer (10 μM), 0.4 μl downstream primer (10 μM), 6.8 μl ddH 2 O composition. The sequence of the upstream primer is as follows: 5'-GGAAAGAAGAAAGGCCCAAG-3', and the sequence of the downstream primer is as follows: 5'-ATCTTCCAGCGTCGTTGATT-3'. 2×SYBR Premix Ex Taq and ROX Reference Dye II in the reaction system used to detect microsporidia above were provided by TaKaRa Bioengineering (Dalian) Co., Ltd.

[0039] Each test reaction system was prepared as follows: 18 μl of Nb-PCR MIX was placed in a clean 0.2ml PCR tube, and 2 μl of processed sample (extracted DNA) or positive standard or negative standard was added to the set PCR reaction tube, 10,000 Centrifuge briefly...

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Abstract

The invention relates to a detection method of nosema bombycis (Nb), in particular to a nosema bombycis SYBR Green fluorescence quantitative PCR (Polymerase Chain Reaction) detection method as well as a kit and a specific primer thereof. The nosema bombycis SYBR Green fluorescence quantitative PCR detection method comprises the following steps of: (1) collecting samples; (2) extracting the DNA of nosema bombycis; (3) detecting the samples by using the SYBR Green fluorescence quantitative PCR amplification method; and (4) analyzing the corresponding samples according to the fluorescence intensity of the reaction after the amplification reaction is finished so as to judge whether nosema bombycis exists in the collected samples, and performing accurate quantitation to nosema bombycis so as to realize the real-time quick detection of nosema bombycis. Because the specific amplification primer and the SYBR Green Buffer system are introduced in the method, the sensitivity and the specificity of nosema bombycis detection can be furthest enhanced, and miss detection and mistake detection are avoided.

Description

technical field [0001] The invention relates to a detection method for Nosema bombycis (Nb), in particular to a SYBR Green fluorescent quantitative PCR detection method for Nosema bombycis, its kit and specific primers. Background technique [0002] Pebrine is a devastating disease in sericulture, and the pathogenic microorganism causing the disease is Nosema bombycis (Nb for short). Because of its special mode of ovum infection, it causes serious harm to sericulture production, especially silkworm egg production and cannot be cured for a long time. France, Italy and other major European sericulture countries once caused the destruction of the silk industry due to the outbreak of the disease. In recent years, millions of species of various species have been eliminated in my country due to particulate disease every year, and the direct economic loss has reached hundreds of millions. Yuan Zhiju. The widespread prevalence of this disease not only causes serious economic losses...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/06G01N21/64C12N15/11
Inventor 何永强吴姗鲁兴萌王素华帅江冰董强张晓峰徐国群许赢升黎昊雁陈胜
Owner ZHEJIANG ACAD OF SCI & TECH FOR INSPECTION & QUARANTINE
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