Gene chip for serotype detection of shigella and its detection process and reagent kit

A technology for serotype detection and Shigella, which is applied in the field of gene chips and its detection methods and detection kits, can solve problems such as cross-contamination and false negatives, and achieve strong repeatability, high accuracy, detection and analysis The method is efficient and fast

Inactive Publication Date: 2007-06-27
TIANJIN BIOCHIP TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, if the PCR reaction conditions are not well controlled, false negatives and cross-...

Method used

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  • Gene chip for serotype detection of shigella and its detection process and reagent kit
  • Gene chip for serotype detection of shigella and its detection process and reagent kit
  • Gene chip for serotype detection of shigella and its detection process and reagent kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1: Design and preparation of probes

[0050] 1. Sequence acquisition: use Artemis software to organize the sequences of the oligosaccharide unit processing enzyme genes wzx and wzy in the O-antigen gene cluster of Shigella type 33, which is the most common in China and has great typing significance (Shigella Bowman's Bacteria O6 / O10 is ribosyltransferase gene orf8, Shigella baumannii O16 is ribosyltransferase gene orf5, Shigella dysenteriae O1 is wzy and rfpB gene) (its sequence source has been explained in the background technology, here No further details) and the Shigella 16S rRNA sequence downloaded from the GenBank public database.

[0051] 2. Probe design: Import the above sequence into OligoArray2.0 software, set the parameters as length 40bp±5bp, Tm80°C±2°C, and then run the program to design the probe online.

[0052] 3. Probe screening: Select a probe with a length of 40bp ± 5bp and a Tm of 80°C ± 2°C from the output results, and perform probe screeni...

Embodiment 2

[0060] Example 2: Design and preparation of primers

[0061] 1. Sequence acquisition: use Artemis software to sort out the wzx and wzy sequences of the most common and significant type 33 Shigella in China mentioned above (Shigella baumannii O6 / O10 is the ribosyltransferase gene orf8, Bao Shigella Shigella dysenteriae O16 is the ribosyltransferase gene orf5, and Shigella dysenteriae O1 is the wzy and rfpB genes).

[0062] 2. Design primers: Import the above sequence into the primer design software Primer Premier 5.0 software, set the parameters as Tm value 50°C±5°C, length 20bp±2bp, and then run the program.

[0063] 3. Primer selection: Select primers with a Tm value of 50°C±5°C, a length of 20bp±2bp, and the probe sequence used in the gene chip from the output results. Individual probes were manually adjusted, and primers were appropriately lengthened or shortened by a few bases to include probes, meet Tm values ​​of 50°C±5°C, and other parameters.

[0064] In a preferred ...

Embodiment 3

[0070] Example 3: Gene Chip Preparation - Chip Spotting

[0071] 1. Dissolving the probe: Dissolve the probe dry powder tube (synthesized by Aoke) synthesized in Example 1 above. The steps are as follows: centrifuge the above-mentioned probe dry powder tube at 12000 rpm for 5 minutes (be careful not to open the lid of the dry powder tube before centrifugation), then add 50% DMSO (adding 1OD to 14 μl), and mix well with a shaker After fast centrifugation, the liquid on the tube wall is separated. After standing at room temperature for 1 hour, take 1 μl and dilute 180 times with 50% DMSO to measure OD, measure the nucleic acid concentration (ng / μl) of the probe (single strand), and then use the following formula to dilute the probe to the final The concentration is 1 μg / μl.

[0072] Formula:

[0073] 2. Adding plates: add the above dissolved 113 probes to the corresponding positions in the 384-well plate, and add 10 μl of probes to each well.

[0074] 3. Spotting: Spot the...

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Abstract

The present invention relates to gene chip for serotype detection of Shigalla and its detection process and kit. The gene chip includes solid phase carrier and supported oligonucleotide probe, which includes the DNA segment selected from Shigalla 16S rRNA sequence and the DNA segment selected from oligose unit treating enzyme gene, glycosyl transferase gene or rfpB gene of one or several different serotype Shigalla O-antigen gene cluster. Through amplifying and marking the detected genome DNA with the designed primer and hybridization with the said gene chip, different serotype of Shigalla may be detected based on the hybridization signal. The present invention has simple operation, high accuracy, high repeatability and capacity of directing medicine administration.

Description

technical field [0001] The invention relates to a gene chip, a detection method thereof, and a detection kit, in particular to a gene chip, a detection method, and a detection kit for Shigella serotype detection. Background technique [0002] Shigella (Shigella) is a human pathogenic bacterium developed with human evolution, and it is the pathogenic bacterium that causes bacillary dysentery (hereinafter referred to as bacillary dysentery). Shigella is a Gram-negative bacillus, a facultative anaerobic bacterium that can decompose lactose, and generally produces acid but not gas. According to different antigen structures, it is divided into four groups: A, B, C, and D, that is, group A——Shigella dysenteriae (S.dysenteriae), group B——Shigella flexneri (S.flexneri), Group C - Shigella baumannii (S.boydii) and Group D - Shigella sonnei (S.sonnei). The proportion of dysentery bacilli varies with regions, ages, and years. Group D is the main group in Europe, America and Japan, a...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 王磊李雅玥刘丹冯露
Owner TIANJIN BIOCHIP TECH CO LTD
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