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Reagent for detecting byssochlamys fulva, real-time fluorescent PCR kit and detection method

A silk mold and real-time fluorescence technology, applied in the biological field, can solve the problems of long detection process cycle, cumbersome and time-consuming operation, fat ear bursting, etc., achieve real-time monitoring of PCR amplification, avoid cross-contamination, and reduce false positive rate Effect

Inactive Publication Date: 2017-01-04
ZHEJIANG INST OF QUALITY INSPECTION SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Fruit juices contaminated by heat-resistant molds are prone to spoilage, stratification, and even bursting during storage, resulting in product quality problems
[0003] Pure Chlamydomonas flavus is currently mainly identified through traditional cultivation, separation and biochemical methods. The entire detection process is long, cumbersome and time-consuming, and cannot meet the rapid monitoring of product quality in the production process of enterprises, and the rapid and accurate detection of fruit juice products during import and export. needs

Method used

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  • Reagent for detecting byssochlamys fulva, real-time fluorescent PCR kit and detection method
  • Reagent for detecting byssochlamys fulva, real-time fluorescent PCR kit and detection method

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Effect test

Embodiment 1

[0034] Example 1: Specific primers, fluorescent probes

[0035] 1. Materials:

[0036] PCR reaction buffer (10×) (Mg 2+ free), dNTP, DNA polymerase, MgCl 2 All purchased from Takara company;

[0037] ROX dye was purchased from Shanghai Site Biotechnology Co., Ltd.

[0038] ABI7300 real-time fluorescent PCR instrument is a product of Applied Biosystems in the United States.

[0039] 2. Primer and probe design and synthesis:

[0040] Using the pure Beta tubulin gene sequence as a template, use PrimerExpress TM (V3.0, American ABI Company) software analyzes primers and TaqMan probe sites, from which the best combination is selected:

[0041] Upstream primer: 5′-TTGGAAGGTCTATGAGATGAGGAAT-3′

[0042] Downstream primer: 5′-GCGGAGCGTCTTATTGCAAT-3′

[0043] Fluorescent probe: 5′-TAM-CAACTTAGCTACAATGGCACCTCCGACCT-BHQ2-3′

[0044] Among them, TAM is a fluorescent reporter group, and BHQ2 is a fluorescent quencher group.

[0045] All were synthesized by Shanghai Yingwei Jieji B...

Embodiment 2

[0046] Embodiment 2: Sensitivity and specificity evaluation of pure Chlamydia flavinus real-time fluorescent PCR method

[0047] 1. Strain detection:

[0048]Using 2 pure standard strains of Chlamydomonas chrysalis (ATCC24474, ATCC24008), and Chlamydomonas snow-white (ATCC22260, ATCC18742), Neosartore fischeri (ATCC66640, ATCC66641), Paecilomyces wansonii (CICC4024, CICC4025), Aspergillus niger (ATCC16404), Aspergillus fumigatus (ATCC10894), Saccharomyces cerevisiae (ATCC26603), Shigella sonnei (ATCC25931), Staphylococcus aureus (ATCC6538), Vibrio parahaemolyticus (isolated and preserved in this laboratory), Escherichia coli The DNA was extracted from the suspension of non-pure chrysanthemums such as Sherella (ATCC25922), and then PCR amplification was carried out on the ABI7300 real-time fluorescent PCR instrument of ABI company in the United States with the upstream and downstream primers and probes for detection, and the specific detection results see figure 1 .

[0049...

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Abstract

The invention discloses a reagent for detecting byssochlamys fulva, a real-time fluorescent PCR kit and a detection method. The reagent comprises an upstream primer, a downstream primer and a fluorescent probe, wherein the upstream primer has the sequence of 5'-TTGGAAGGTCTATGAGATGAGGAAT-3', the downstream primer has the sequence of 5'-GCGGAGCGTCTTATTGCAAT-3', and the fluorescent probe has the sequence of 5'-TAM-CAACTTAGCTACAATGGCACCTCCGACCT-BHQ2-3'; the kit mainly comprises the upstream primer, the downstream primer, the fluorescent probe, a PCR buffer solution, dNTP, DNA polymerase and MgCl2; the detection method of the kit comprises the steps of genomic DNA extracting, real-time fluorescent PCR detection system establishing and real-time flurescent PCR detecting. The reagent, the kit and the detection method mainly have the advantages that the byssochlamys fulva can be accurately detected from a to-be-detected sample, the specificity and the sensitivity are high, and the PCR amplification process can be visually monitored in real time.

Description

technical field [0001] The invention relates to a reagent, a real-time fluorescent PCR kit and a detection method for detecting pure yellow silk mold, and belongs to the field of biotechnology. Background technique [0002] Pure yellow silk mold ( Byssochlamys fulva ) is one of the heat-resistant molds that are easily polluted in the fruit juice production process. Its heat resistance is much stronger than other molds, and it can survive after 30 minutes at 85°C or 10 minutes at 87.7°C. Pure yellow silk mold can grow in an environment with insufficient oxygen, and can also decompose fat, protein, carbohydrates, etc. in food. It has a strong effect on destroying pectin, softening and disintegrating the fruit, and producing carbon dioxide gas and poisonous Hazardous substances, causing product deterioration. Juice contaminated by heat-resistant molds is prone to spoilage, layered precipitation, and even bursting of fruit juices during storage, resulting in product quality pr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/645
CPCC12Q1/6895C12Q1/686C12Q2561/113C12Q2561/101
Inventor 张慧姜侃汪新陈小珍
Owner ZHEJIANG INST OF QUALITY INSPECTION SCI