Method for rapidly detecting shigella dysenteriae

A detection method, Shigella technology, applied in the direction of biochemical equipment and methods, microbiological determination/inspection, resistance to vector-borne diseases, etc., can solve the problems of high detection limit, specificity discount, and lack of specificity in target sequence selection Advanced problems to achieve the effect of overcoming tedious and time-consuming

Inactive Publication Date: 2009-12-16
陈福生 +1
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AI Technical Summary

Problems solved by technology

These methods can amplify the target nucleic acid to a similar order of magnitude and have higher detection limits, but they still have some defects that cannot be overcome
Some of them require sophisticated instruments to amplify, and some require sophisticated methods to detect the amplified products because of the low specificity of the selection of the target sequence
Despite its simplicity, the need for high-precision thermal cyclers keeps PCR fr

Method used

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Embodiment Construction

[0029] 1. Primer Design

[0030] Outer primer F3 (forward outer primer): 5′-GCCTTTCCGATACCGTCTCT; outer primer B3 (forwardouter primer): 5′-TGATGGACCAGGAGGGTT; inner primer FIP (backward inner primer): 5′-TCCGCAGAGGCACTGAGTTTTTCACGCAATACCTCCGGATTC; inner primer BIP (backwardinner primer): 5 '-TCGACAGCAGTCTTTCGCTGTTCCGGAGATTGTTC-CATGTGA; loop primer LB: 5'-TGCAGCGACCTGTTCACG; loop primer LF: 5'-CTGCTGATG-CCACTGAGAGC.

[0031] 2. Sample Processing

[0032] Samples were processed according to GB / T4789.4-2003. After enrichment culture, 1 mL of the culture was taken and centrifuged at 12000 r / min for 5 min; 0.8 mL of 1×TE (100 mmol / L Tris-Cl, 10 mmol / L EDTA, pH8.0 ), centrifuge at 12000r / min for 5min, add 100μL sterile water to the precipitate, mix well, place in boiling water bath at 100°C for 15min, immediately ice-bath for 5min, centrifuge at 12000r / min for 5min, and the supernatant is the extracted DNA template.

[0033] 3. LAMP reaction system

[0034] Add the following su...

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Abstract

The invention belongs to a rapid detection technology of food-borne pathogens, in particular to a loop-mediated isothermal amplification (LAMP) method for detecting shigella dysenteriae DNA. The method comprises the preparation of reagent comprising sample processing reagent, LAMP reactive reagent, a large fragment of Bst DNA polyase, agarose, bromophenol blue loading buffer solution and ethidium bromide solution and the procedures of the detection of amplification products and sediment generation. The method comprises the following steps: firstly, designing a specific primer; secondly, extracting sample DNA; and thirdly, preparing a reaction system. Compared with the currently adopted traditional bacterial culture method, an immunological detection method and other molecular biology methods, the method has the advantages of simple and rapid operation, no special instrument and reagent, sensitivity and specificity and can be applied to the rapid detection of shigella dysenteriae in foods and other samples.

Description

technical field [0001] The invention relates to the use of a loop-mediated isothermal DNA amplification technique (loop-mediated isothermal amplification, LAMP) in detecting Shigella. Background technique [0002] Shigella spp. is a class of Gram-negative bacilli, and is the most common pathogen of human bacillary dysentery, commonly known as Shigella. Shigellosis is often food-borne or water-borne. Shigella can spread rapidly in crowded and unsanitary conditions, and is often found in places where a large number of people gather, such as restaurants and canteens. The most common reasons for the prevalence of food-borne Shigella are bacillary dysentery in food processing workers or contamination of food by carriers, poor personal hygiene of people in contact with food, and storage of contaminated food at inappropriate temperatures. [0003] At present, there are mainly bacteriological examination and immunological methods to identify Shigella. The former is a national stan...

Claims

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Application Information

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IPC IPC(8): C12Q1/10C12Q1/68
CPCY02A50/30
Inventor 陈福生朱胜梅
Owner 陈福生
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