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Kit and method for staphylococcus aureus CRISPR locus detection

A detection kit and staphylococcus technology, applied in the field of microbial detection, can solve the problems of no specific detection, etc., achieve the effect of low detection cost, high sensitivity and specificity, and good promotion and application value

Inactive Publication Date: 2016-11-30
ZHENGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the research on CRISPR in Staphylococcus aureus mainly focuses on the functional research of CRISPR in Staphylococcus aureus. However, there is no relevant kit for specific detection of CRISPR loci in Staphylococcus aureus on the market.

Method used

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  • Kit and method for staphylococcus aureus CRISPR locus detection
  • Kit and method for staphylococcus aureus CRISPR locus detection
  • Kit and method for staphylococcus aureus CRISPR locus detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] There are three types of CRISPR locus gene sequences in Staphylococcus aureus, namely CRISPR-1, CRISPR-2 and CRISPR-3. Three primers for CRISPR loci were designed based on the published CRISPR-related gene sequences of Staphylococcus aureus in Genebank:

[0045] CRISPR-1 upstream primer 5’-GTAGTGATTTTTGGGAGTGG-3’,

[0046] Downstream primer 5'-GTGGTTAATGTGTAGGGACC-3',

[0047] CRISPR-2 upstream primer 5’-CAATGACTTGGAGTGTGA-3’,

[0048] Downstream primer 5'-GGTTAATGTGTAGGAACC-3',

[0049] CRISPR-3 upstream primer 5’-ACTCAATGGCATGGAGTGTGA-3’,

[0050] Downstream primer 5'-GGTGGTTAATGTGTAGGGACC-3'.

[0051] Staphylococcus aureus CRISPR locus detection kit includes primers designed for the CRISPR locus gene sequence of Staphylococcus aureus, 10×Tag Buffer, 0.2 mM dNTP, 2 mM MgCl 2 , 0.05U / μL Tag DNA polymerase, sterilized ultrapure water and DNA Marker 2000.

Embodiment 2

[0053] The detection method of Staphylococcus aureus CRISPR site, comprises the following steps:

[0054] (1) Genomic DNA of bacterial samples was extracted by boiling method.

[0055] (2) Using the genomic DNA of the bacterial sample as a template, PCR amplification was performed using primers of three kinds of CRISPR locus gene sequences respectively. The primer sequences are as shown in Example 1. The reaction system of the PCR amplification is: 10 μmol / L 1 μL of upstream primer, 1 μL of 10 μmol / L downstream primer, 2.5 μL of 10×Tag Buffer, 0.5 μL of 0.2 mM dNTP, 2 mM MgCl 2 2 μl, 0.125 μL of 0.05U / μL Taq DNA polymerase, 2 μL of bacterial sample genomic DNA, 15.875 μL of sterilized ultrapure water. The PCR amplification reaction procedures adopted for different CRISPR site gene sequences are as follows:

[0056] CRISPR-1 pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 30s, annealing at 51°C for 30s, extension at 72°C for 30s, a total of 35 cycles, and ex...

experiment example

[0062] 1. Sample collection

[0063] The bacterial sample used in the test was Staphylococcus aureus, which was isolated in a hospital in Zhengzhou, Henan in 2014.

[0064] 2. Experimental method

[0065] Using the detection kit of the present invention to detect the CRISPR site of the bacterial sample, the detection steps are as follows:

[0066] (1) Preparation of DNA template

[0067] Whole genomic DNA was extracted from bacterial samples using the boiling method. Take out the bacterial sample cryopreservation tube from the -80°C refrigerator, put it in a 4°C refrigerator for rewarming for 5 hours, use a sterile inoculation loop to quickly dip the bacterial solution in the ultra-clean bench, and inoculate it on Columbia blood agar culture by segmented streaking method On the plate, incubate for 20 hours in a 37°C incubator, pick a single colony on the blood plate, inoculate it in the brain heart infusion liquid medium, culture it with shaking at 37°C for 16 hours, take 1...

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Abstract

The invention discloses a kit and method for staphylococcus aureus CRISPR locus detection. The kit comprises three pairs of specific primers designed according to three CRISPR locus gene sequences of staphylococcus aureus. The invention further discloses the method for staphylococcus aureus CRISPR locus detection. The three pairs of specific primers are utilized to amplify corresponding CRISPR fragments from sample genome DNAs. The kit and the detection method are simple to operate, high in sensitivity and specificity and low in detection cost and have good popularization and application values.

Description

technical field [0001] The invention belongs to the technical field of microbial detection, in particular to a CRISPR site detection kit and detection method for Staphylococcus aureus. Background technique [0002] Staphylococcus aureus is an important pathogenic bacterium in humans, belonging to Gram-positive cocci, which can cause a variety of diseases, such as endocarditis, pneumonia, and toxic shock syndrome. In recent years, with the increasing rate and degree of drug resistance of Staphylococcus aureus, Staphylococcus aureus infection has become a serious health problem. Clustered regularly interspaced short palindromic repeats (CRISPR) is a bacterial immune system discovered in recent years against exogenous genes such as phages, which can effectively resist phages and various external genetic elements. Interference, thereby inhibiting horizontal gene transfer (HGT). CRISPR widely exists in bacteria and archaea, and is composed of a discontinuous repeat sequence (re...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/14C12R1/445
CPCC12Q1/6869C12Q1/689C12Q2600/156C12Q2565/125
Inventor 杨海燕刘静邵富叶
Owner ZHENGZHOU UNIV
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