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Gene chip detecting marine pathogenic vibrios, and preparation method and detection method thereof

A gene chip and pathogenic vibrio technology, applied in the field of gene chip detection of marine pathogenic vibrio and its preparation, can solve the problems of tools that are difficult to monitor pathogenic bacteria, increase economic burden, easy misdiagnosis, etc., and achieve wide application , Simple operation, strong signal effect

Active Publication Date: 2015-12-09
浙江正合谷生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Early diagnosis and treatment are of great significance. Delayed diagnosis leads to blind application of antibacterial drugs, increases economic burden, and promotes the occurrence of bacterial resistance
The culture method is currently the gold standard for the diagnosis of bacterial infection, but it takes a long time and is often diagnosed based on biochemical reactions and other phenotypes. It is easy to misdiagnose and it is not easy to be used as a tool for monitoring pathogenic bacteria

Method used

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  • Gene chip detecting marine pathogenic vibrios, and preparation method and detection method thereof
  • Gene chip detecting marine pathogenic vibrios, and preparation method and detection method thereof
  • Gene chip detecting marine pathogenic vibrios, and preparation method and detection method thereof

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Embodiment 1

[0023] The preparation method of the gene chip of detecting marine pathogenic vibrio of the present invention is as follows:

[0024] (1) Design of primers and probes: 5 strains of Vibrio: Vibrio vulnificus ATCC27562, Vibrio splendidus ATCC33125, Vibrio harveyi ATCC33868, Vibrio parahaemolyticus ATCC17802, Vibrio anguillarum ATCC43308 were cultured in common beef extract Two-dimensional electrophoresis was used to find differential proteins on peptone and blood plates, specific amino acid and DNA sequences were obtained after mass spectrometry sequencing, and the gene sequences related to pathogenicity were determined after further online comparison. Use commonly used probe design software (such as ArrayDesigner4) to design according to the design requirements of primers and probes. Commercially synthesize the designed primers and probes (such as Shanghai Bioengineering Co., Ltd.). The primers are labeled with biotin, the probes do not need to be labeled.

[0025] The sequen...

Embodiment 2

[0040] The detection method of the gene chip of detecting marine pathogenic vibrio of the present invention is as follows:

[0041] (1) Preparation of the sample to be tested: After taking the water sample and enriching it with immunomagnetic beads, boil it at 95-100°C for 5-10 minutes, and take 1 μL as a PCR template;

[0042] (2) Biotin-labeled PCR amplification: 10×PCR buffer 2.5 μL, MgCl 2 (20mM) 2μL, Biotin-labeled upstream primer and downstream primer 1μL each (10 pairs in total, added at the same time), 2μL dNTP (dTTP is 0.25mmol / L), TaqDNA polymerase (5U / μL) 0.2μL, sample template to be tested 1μL , make up the volume to 25 μL with sterile ultrapure water. PCR amplification program: 94°C for 5min; 30 cycles of 94°C for 30s, 56°C for 30s, and 72°C for 30s; 72°C for 10min.

[0043] (3) The hybridization solution (5×Deharndt, 2×SSC, 50% deionized formamide, 0.2% SDS) used for hybridization, the PCR product obtained in step (2) was boiled in boiling water for 10 min, and...

Embodiment 3

[0047] Using Vibrio vulnificus (ATCC27562), Vibrio resplendent (ATCC33125), Vibrio harveii (ATCC33868), Vibrio parahaemolyticus (ATCC17802), Vibrio anguillarum (ATCC43308), Vibrio hardy (MCCC1H00104), Staphylococcus aureus (ATCC25923) and Enterobacter cloacae (ATCC13047) standard strains were tested as the samples to be tested.

[0048] The detection results of Vibrio vulnificus (ATCC27562) showed that the probes VV1 and VV2 were positive, and the other probes were negative.

[0049] Vibrio resplendent (ATCC33125) test results showed that probes VS1 and VS2 were positive, and other probes were negative.

[0050] The detection results of Vibrio harveyi (ATCC33868) showed that the probes VH1 and VH2 were positive, and the other probes were negative.

[0051] The detection results of Vibrio parahaemolyticus (ATCC17802) showed that the probes VP1 and VP2 were positive, and the other probes were negative.

[0052] The detection results of Vibrio anguillarum (ATCC43308) showed tha...

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Abstract

The invention discloses a gene chip detecting marine pathogenic vibrios, and a preparation method and a detection method thereof. The gene chip comprises a solid-phase carrier and detection probes fixedly disposed on the solid-phase carrier, and the detection probes comprise vibrio vulnificus gyrB gene probe, vibrio vulnificus virulence gene hemA probe, vibrio splendidus gyrB gene probe, vibrio splendidus virulence gene toxR probe, vibrio harveyi gyrB gene probe, vibrio harveyi virulence gene hemA probe, vibrio parahaemolyticus gyrB gene probe, vibrio parahaemolyticus virulence gene toxS probe, vibrio anguillarum gyrB gene probe and vibrio anguillarum virulence gene hem probe which are respectively shown as SEQ NO 1-10. The gene chip is firstly applied to detection on marine pathogenic vibrios. The gene chip is capable of rapidly sensitively detecting target bacterium infectio, also is good in repeatability and strong in signal, and does not easily cause a nonspecific signal.

Description

technical field [0001] The invention belongs to the technical field of gene chip detection, and in particular relates to a gene chip for detecting marine pathogenic vibrio, a preparation method and a detection method thereof. Background technique [0002] Pathogenic bacterial infection is a thorny problem faced by human beings, which often results in high mortality and disability rates. Early diagnosis and treatment are of great significance. Delayed diagnosis leads to blind application of antibacterial drugs, increases economic burden, and promotes the occurrence of bacterial drug resistance. The culture method is currently the gold standard for the diagnosis of bacterial infection, but it takes a long time and is often diagnosed based on biochemical reactions and other phenotypes. It is easy to misdiagnose and it is not easy to be used as a tool for monitoring pathogenic bacteria. [0003] Biochip technology is a technology that has developed rapidly in the field of biolo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B40/06C40B50/06C12Q1/68C12Q1/04
CPCY02A50/30
Inventor 周君
Owner 浙江正合谷生物科技有限公司
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