Primer for detection of shigella and detection method
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A Shigella, primer amplification technology, applied in microorganism-based methods, biochemical equipment and methods, and microbial assay/inspection, etc., can solve problems such as low specificity of target sequence amplification
Inactive Publication Date: 2010-09-29
SOUTH CHINA AGRI UNIV
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Although their detection sensitivity is greatly improved, their amplification specificity for the target sequence is...
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Embodiment 1
[0030]To detect Shigella using loop-mediated isothermal gene amplification, follow the steps below:
[0031] (1) Pretreatment of samples to be tested
[0032] Suspend the tested sample in sterile normal saline, centrifuge at 10,000r / min for 3min, remove the supernatant, then wash the cells twice with sterile normal saline, and air-dry;
[0033] (2) Preparation of template
[0034] Add 50 μL ddH to the cell sample obtained above 2 O. After fully mixing, place in a boiling water bath at 100°C for 10 minutes, in an ice bath for 5 minutes, and centrifuge at 12,000 r / min at 4°C for 5 minutes. The supernatant is the DNA template.
[0036] Take 2 μL of the above template, 1 μL of each primer, 1 μL (8U) of Bst DNA polymerase, 7.5-12.5 μL of the amplification reaction solution, add water to the reaction system of 25 μL, and react at a constant temperature of 65 °C for 1.5 h; then place it at 80 °C for 10 min, stop ...
Embodiment 2
[0042] To detect Shigella from food samples, proceed as follows:
[0043] (1) Pretreatment of samples to be tested
[0044] Take 25g of the sample and add it to 225mL of GN enrichment solution, and homogenize it in a tissue grinder to make a homogenate. The homogenate was diluted 10 times, and 1 mL was taken as a sample.
[0045] (2) Preparation of template
[0046] The above samples were placed in a boiling water bath at 100°C for 10 minutes, in an ice bath for 5 minutes, and centrifuged at 12,000 r / min at 4°C for 5 minutes, and the supernatant was the DNA template.
[0048] Take 2 μL of the above template, 1 μL of each primer, 1 μL of Bst DNA polymerase (8U), 7.5 μL of amplification reaction solution, add water to a 25 μL reaction system, and react at a constant temperature of 63 °C for 1 h; then place it at 80 °C for 2 min to terminate the reaction.
[0049] The primers used are shown in SEQ ID NO: 1-4...
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Abstract
The invention discloses a primer for detection of shigella and a detection method. The sequence of the primer for the detection of the shigella is expressed as SEQ ID NO: 1-4. The detection method for the shigella comprises the following steps of: (1) extracting DNA serving as a template; (2) adopting a loop-mediated isothermal amplification reaction system which comprises 2muL of template, 2muL of primer, 1muL of Bst DNA and 7.5 to 12.5muL of amplification reaction solution, adding water into the reaction system to obtain 25muL of reaction system, performing thermostatic reaction for 1 to 1.5 hours at the temperature of between 63 and 65 DEG C, then placing the reaction system at the temperature of 80 DEG C for 2 to 10 minutes, and terminating the reaction; and (3) observing the results, namely observing a detection tube by using naked eyes, wherein the result is positive shigella when turbid is produced, and the result is negative when no turbid is produced. The method for detecting the shigella by using the primer to perform the loop-mediated isothermal amplification reaction does not need special equipment, can quickly detect shigella dysenteriae genes in a detection sample, and has the advantages of specificity, high sensitivity and simple identification method.
Description
technical field [0001] The invention relates to a method for detecting food-borne pathogenic bacteria based on loop-mediated isothermal amplification, in particular to a detection primer and a detection method for Shigella. Background technique [0002] Shigella (Shigella) bacteria (commonly known as Shigella), is the pathogenic bacteria of bacillary dysentery. There are many pathogenic organisms that can cause dysentery clinically, including Shigella, Salmonella, Proteus, Escherichia coli, etc., as well as amoeba, whipworm, and viruses, which can cause human dysentery, among which Shigella bacillary dysentery is the most common. Humans have a high susceptibility to Shigella, which can cause acute toxic bacillary dysentery in young children, and the mortality rate is very high. [0003] At present, the routine inspection procedure of traditional Shigella is: sample→enrichment→isolation culture (and count the bacteria)→isolation and purification→Gram staining microscopic ex...
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