Real-time fluorescence PCR (polymerase chain reaction) kit and detection method for detecting Byssochlamys nivea

A real-time fluorescence, silkworm mold technology, applied in biochemical equipment and methods, fluorescence/phosphorescence, microbial determination/inspection, etc., can solve problems such as cumbersome operation, fat cracking, product quality problems, etc., to reduce false positives efficiency, avoid cross-contamination, improve specificity

Inactive Publication Date: 2013-03-06
ZHEJIANG INST OF QUALITY INSPECTION SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Juices contaminated by heat-resistant molds are prone to discoloration, odor, layered precipitation and other phenomena during storage, and even fat cans burst, resulting in product quality problems
[0003] At present, C. snow white is mainly separated by culture method and identified by biochemical me

Method used

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  • Real-time fluorescence PCR (polymerase chain reaction) kit and detection method for detecting Byssochlamys nivea
  • Real-time fluorescence PCR (polymerase chain reaction) kit and detection method for detecting Byssochlamys nivea

Examples

Experimental program
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Effect test

Example Embodiment

[0029] Example 1: Specific primers, fluorescent probes

[0030] 1. Material:

[0031] PCR reaction buffer (10×) (Mg 2+ free), MgCl 2 , DNTP mixture, DNA polymerase were purchased from Takara Company;

[0032] ROX dye was purchased from Shanghai Weidian Biotechnology Co., Ltd.

[0033] The ABI7300 real-time fluorescent PCR instrument is a product of Applied Biosystems, USA.

[0034] 2. Design and synthesis of primers and probes:

[0035] Using the Beta tubulin gene sequence of S. snow white as template, using PrimerExpress TM (V3.0, American ABI company) software analyzes primer and TaqMan probe sites, and selects the best combination from them:

[0036] Upstream primer: 5'-GGTTGTTGTTGACTTCCCTGATG-3'

[0037] Downstream primer: 5′-CCATGGTACCAGGCTCAAGGT-3′

[0038] Fluorescent probe: 5'-FAM-ACGCTCCATATGCTGACCCTCCTCCTAG-BHQ1-3'

[0039] Among them, FAM is a fluorescent reporter group, and BHQ1 is a fluorescent quenching group.

[0040] They are all synthesized by Shanghai Yingweijieji Biotechn...

Example Embodiment

[0041] Example 2: Evaluation of the sensitivity and specificity of the real-time fluorescence PCR

[0042] 1. Strain detection:

[0043] Use 2 standard strains of Silkworm Snow White (ATCC22260, ATCC18742), and pure Silkworm (ATCC24474, ATCC24008), Paecilomyces variabilis (CICC4024, CICC4025), New Sartorius fischeri (ATCC66640, ATCC66641), Aspergillus fumigatus (ATCC10894, ATCC30367), Aspergillus niger (ATCC16404, ATCC10582), Saccharomyces cerevisiae (ATCC26603, ATCC26785), Staphylococcus aureus (ATCC6538), Shigella sonnei (ATCC25931), Escherichia coli (ATCC25922), Vibrio parahaemolyticus (isolated and preserved in our laboratory), Listeria monocytogenes (ATCC19114), Enterobacter sakazakii (ATCC51329), Shigella flexneri (ATCC12022), Streptococcus hemolyticus (CMCC32210), etc. Suspension of non-siliconia fungus, extract DNA, and then use upstream and downstream primers and probes for detection to perform PCR amplification on the ABI7300 real-time fluorescent PCR instrument from ABI...

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Abstract

The invention provides a real-time fluorescence PCR (polymerase chain reaction) kit and a detection method for detecting Byssochlamys nivea. The PCR kit mainly comprises specific primers, a fluorescence probe, a PCR buffer solution, dNTP (diethyl-nitrophenyl thiophosphate) compound (triphosphoric acid deoxyguanosine monophosphate, triphosphoric acid deoxyadenosine monophosphate, triphosphoric acid deoxythymidine monophosphate and triphosphoric acid deoxycytidine monophosphate are mixed according to the molar ratio of 1:1:1:1), MgCl2 and DNA (deoxyribonucleic acid) polymerase. The sequence of the specific primers and the fluorescence probe is as follows: an upstream primer 5'-GGTTGTTGTTGACTTCCCTGATG-3', a downstream primer: 5'-CCATGGTACCAGGCTCAAGGT-3', and the fluorescence probe: 5'-FAM-ACGCTCCATATGCTGACCCTCCTCCTAG-BHQ1-3', wherein the FAM is fluorescence reporter group, and the BHQ1 is fluorescence quenching group. The real-time fluorescence PCR kit and the detection method for detecting Byssochlamys nivea has the beneficial effects that the detection sensitivity is high, the specificity is good, the operation is simple, time and labor are saved, the results are reliable, and the detection is accurate and sensitive.

Description

technical field [0001] The invention relates to a real-time fluorescent PCR kit and a detection method for detecting the silky mold of snow white. Background technique [0002] Byssochlamys nivea is one of the heat-resistant molds that are easily polluted in the fruit juice production process. Mycotoxins can produce mycotoxins patulin / patulin and mycotic acid. Mycotoxins have toxic effects on many biological systems and can cause teratogenic and carcinogenic effects. Juices contaminated by heat-resistant molds are prone to discoloration, peculiar smell, layered precipitation and other phenomena during storage, and even the fruit juices may burst, resulting in product quality problems. [0003] At present, C. snow white is mainly separated by culture method and identified by biochemical method. The whole process is cumbersome to operate and the detection cycle is long, which is not conducive to product monitoring during production and emergency treatment of food safety incid...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04G01N21/64
Inventor 张慧姜侃陈小珍汪新
Owner ZHEJIANG INST OF QUALITY INSPECTION SCI
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