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Real-time fluorescence PCR ( Polymerase Chain Reaction) kit for detecting shigella and detection method thereof

A real-time fluorescence, kit technology, applied in microorganism-based methods, fluorescence/phosphorescence, biochemical equipment and methods, etc., can solve the problems of low positive rate, long time, complicated operation, etc. The effect of high positive rate and high detection sensitivity

Inactive Publication Date: 2011-11-16
ZHEJIANG CENT FOR DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] At present, Shigella is mainly separated by culture method and identified by biochemical method. The whole process is cumbersome, time-consuming, and the positive rate is low.

Method used

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  • Real-time fluorescence PCR ( Polymerase Chain Reaction) kit for detecting shigella and detection method thereof
  • Real-time fluorescence PCR ( Polymerase Chain Reaction) kit for detecting shigella and detection method thereof
  • Real-time fluorescence PCR ( Polymerase Chain Reaction) kit for detecting shigella and detection method thereof

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Experimental program
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Effect test

Embodiment 1

[0043] Example 1: Specific primers, fluorescent probes

[0044] 1. Materials:

[0045] Premix Ex Taq TM (2×) was purchased from TaKaRa Company (DRR039S);

[0046] MX3000P quantitative PCR instrument is a product of STRATAGNE, USA.

[0047] 2. Primer and probe design and synthesis:

[0048] Using the Shigella ipaH gene sequence (registration number DQ448042) as a template, use PrimerExpressTM (V2.0, American ABI Company) software analyzes TaqMan-MGB primers and probe sites, and selects the best combination:

[0049] Upstream primer: 5′-GGAAAACCCTCCTGGTCCAT-3′

[0050] Downstream primer: 5′-CGCCGGTATCATTATCGAAAA-3′

[0051] Fluorescent probe: 5′-FAM-AGGCATCAGAAGGC-MGB-3′

[0052] Wherein FAM is a fluorescent reporter group, and MGB is a fluorescent quencher group.

[0053] Both were synthesized by Dalian Bao Biological Engineering Co., Ltd.

Embodiment 2

[0054] Embodiment 2: Specificity evaluation of Shigella fluorescent quantitative PCR method

[0055] 1. Strain detection:

[0056] Three Shigella standard strains (Shigella dysenteriae CMCC 51570, Shigella sonnei CMCC51334, Shigella flexneri ATCC 12022), 30 strains of Shigella isolates, Escherichia coli, Salmonella, Citrobacter, Escherichia coli, Vibrio parahaemolyticus, Yersinia, Proteus, Staphylococcus epidermidis, Staphylococcus aureus, Streptococcus pyogenes, Alcaligenes faecalis, Lactobacillus lymannii, Bacillus cereus, Salmonella anativa, Streptococcus mirabilis Proteus, hemolytic streptococcus and other 91 strains of non-Shigella bacteria suspension were extracted by pyrolysis method, and then PCR amplification was carried out on the MX3000P quantitative PCR instrument of American STRATAGNE company with the upstream and downstream primers and probes for detection.

[0057] (1) Thermal cracking method: collect the strains in a 1.5mL tube, place in a boiling water bath f...

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Abstract

The invention provides a real-time fluorescence PCR ( Polymerase Chain Reaction) kit for detecting shigella and a detection method thereof. The kit mainly comprises specific primers, a fluorescent probe, PCR buffer liquid, deoxynucleotide triphosphate mixture and DNA polymerase. The sequences of the specific primers and fluorescent probe are as follows: an upstream primer has the sequence of 5'-GGAAAACCCTCCTGGTCCAT-3', a downstream primer has the sequence of 5'-CGCCGGTATCATTATCGAAAA-3', and the fluorescent probe has the sequence of 5'-FAM-AGGCATCAGAAGGC-MGB-3', wherein the FAM is a fluorescent report radical, and the MGB is a fluorescent quenching radical. The invention has the advantages that the detection sensitivity of target bacteria is high, the specificity is good, the false positive rate of regular PCR amplification is reduced, and the shigella can be rapidly, accurately and peculiarly detected.

Description

(1) Technical field [0001] The invention relates to a real-time fluorescent PCR kit for detecting Shigella and a detection method thereof. (2) Background technology [0002] As an invasive pathogen, Shigella can directly damage the intestinal mucosa, causing bacillary dysentery and food poisoning. There are 160 million Shigella patients worldwide each year, and about 1.1 million deaths. It is a common intestinal infectious disease in summer and autumn in my country, and it is also a Class C infectious disease pathogen stipulated by the Infectious Disease Prevention Law. It belongs to a class of food-borne pathogens that are extremely harmful to human and animal health. [0003] At present, Shigella is mainly separated by culture method and identified by biochemical method. The whole process is cumbersome, time-consuming, and the positive rate is low. TaqMan-MGB probe fluorescent PCR method is a new real-time quantitative in vitro nucleic acid amplification detection techno...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68G01N21/64C12R1/01
Inventor 梅玲玲朱敏张俊彦程苏云占利
Owner ZHEJIANG CENT FOR DISEASE CONTROL & PREVENTION
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