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Primers for RPA rapid detection of Shigella and tetracycline drug resistance genes

A drug-resistant gene, Shigella technology, applied in the determination/inspection of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of low accuracy, long detection time, and difficulty in adapting to rapid detection. , to achieve good interspecific specificity, reduced primer number, and good intraspecific conservation.

Active Publication Date: 2017-08-18
ZHEJIANG GONGSHANG UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the rapid detection methods of Shigella mainly include conventional detection method, rapid biochemical instrument detection method, molecular biology detection method, toxin detection method, loop-mediated constant temperature amplification technology and enzyme-linked immunoassay method, etc. Their operation procedures are complicated, the detection time is long, the cost is high, the accuracy is not high and the specificity is poor, it is difficult to meet the needs of modern food safety rapid detection

Method used

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  • Primers for RPA rapid detection of Shigella and tetracycline drug resistance genes
  • Primers for RPA rapid detection of Shigella and tetracycline drug resistance genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Step (1). Sample pretreatment

[0031] Take 25 g of pork samples from the vegetable market, add 225 mL of sterilized distilled water, and homogenize with a tissue homogenizer for 1 min to obtain a tissue homogenate sample.

[0032] Step (2). Sample DNA extraction

[0033] Take 50 mg of the tissue homogenate sample treated in step (1), add 200 μl of TE buffer solution with a pH value of 8.0, vortex and mix; add 400 μl of lysate, mix well, add 600 μl of phenol-chloroform-isoamyl alcohol mixed solvent, Shake vigorously, centrifuge at 12000g for 10min; take the supernatant, add a mixed solvent of chloroform-isoamyl alcohol equal to the volume of the supernatant, shake vigorously, and centrifuge at 12000g for 10min; take the supernatant, add the same volume of the supernatant Chloroform, shake vigorously, centrifuge at 12000g for 10min; take the supernatant, add isopropanol 0.8 times the volume of the supernatant, and centrifuge at 12000g for 10min; take the precipitate, wa...

Embodiment 2

[0045] Step (1). Sample pretreatment

[0046] Take 25g of fishmeal sample, add 225mL of sterilized distilled water, and homogenize with a tissue homogenizer for 1min to obtain a tissue homogenate sample.

[0047] Step (2). Sample DNA extraction

[0048] Take 50 mg of the tissue homogenate sample treated in step (1), add 200 μl TE buffer solution with a pH value of 8.0, and vortex mix; add 400 μl lysate, mix well, add 600 μl phenol-chloroform-isoamyl alcohol mixed solvent, Shake vigorously, centrifuge at 12000g for 10min; take the supernatant, add a mixed solvent of chloroform-isoamyl alcohol equal to the volume of the supernatant, shake vigorously, and centrifuge at 12000g for 10min; take the supernatant, add the same volume of the supernatant Chloroform, shake vigorously, centrifuge at 12000g for 10min; take the supernatant, add isopropanol 0.8 times the volume of the supernatant, centrifuge at 12000g for 10min; take the precipitate, wash once with ethanol with a volume cont...

Embodiment 3

[0060] Step (1). Sample pretreatment

[0061] Take 25 g of chicken sample from the vegetable market, add 225 mL of sterilized distilled water, and homogenize it with a tissue homogenizer for 1 min to obtain a tissue homogenate sample.

[0062] Step (2). Sample DNA extraction

[0063] Take 50 mg of the tissue homogenate sample treated in step (1), add 200 μl TE buffer solution with a pH value of 8.0, and vortex mix; add 400 μl lysate, mix well, add 600 μl phenol-chloroform-isoamyl alcohol mixed solvent, Shake vigorously, centrifuge at 12000g for 10min; take the supernatant, add a mixed solvent of chloroform-isoamyl alcohol equal to the volume of the supernatant, shake vigorously, and centrifuge at 12000g for 10min; take the supernatant, add the same volume of the supernatant Chloroform, shake vigorously, centrifuge at 12000g for 10min; take the supernatant, add isopropanol 0.8 times the volume of the supernatant, centrifuge at 12000g for 10min; take the precipitate, wash once ...

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Abstract

The invention discloses primers for RPA rapid detection of Shigella and tetracycline drug resistance genes. The designed primers have relatively good interspecies specificity and intraspecific conservative property. The reaction can specifically detect a target gene and can separately identify Shigella (Shigella flexneri and Shigella sonnei) and the tetracycline drug resistance gene (tetM) at the same time, so that false positive is avoided. The pair of specific primers is designed according to the tetracycline drug resistance gene (tetM) and the pair of primers is designed according to homology of the Shigella flexneri and Shigella sonnei, so that the two pairs of primers can amplify genes of three clauses. Compared with a conventional method, the quantity of the primers is greatly reduced (three pairs of primers are needed for amplifying the genes of three clauses in the conventional method), so that false positive is reduced. The lengths and sizes of fragments after amplification are different, and the fragments can be separately differentiated by electrophoresis.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a primer for rapid detection of Shigella and tetracycline-resistant gene recombinase polymerase amplification technology (Recombinase Polymerase Amplification, RPA) and its application, in particular to a primer that can be used in samples At the same time, the primers for RPA rapid detection of Shigella futiferii, Shigella sonnei, and tetracycline resistance gene (tetM) were detected. Background technique [0002] Shigellosis, also known as bacillary dysentery, is an acute intestinal infectious disease caused by Shigella spp. Infectious and seriously harmful, the main susceptible groups are children under 5 years old and people with immune disorders. Patients with dysentery infection often have abdominal pain, diarrhea, mucus pus and blood in the stool, and fever. Severe patients may also be accompanied by life-threatening symptoms such as kidney failure and neurotoxicity, which seri...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/10C12N15/11
CPCC12Q1/6844C12Q1/689C12Q2521/507C12Q2521/101Y02A50/30
Inventor 曲道峰韩剑众徐琳沈杨张恩宝
Owner ZHEJIANG GONGSHANG UNIVERSITY
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