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Gene chip, kit and method for detecting common pathogenic bacteria of piglets

A gene chip and pathogenic bacteria technology, applied in the field of gene chip detection of common pathogenic bacteria in piglets, can solve the problems affecting the quality of growth and development, incomplete function of physiological system, low immunity, etc.

Active Publication Date: 2012-05-09
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Due to the low immunity and incomplete function of the physiological system, pigs in the piglet stage are often infected with pathogenic bacteria and suffer from various diseases, resulting in low survival rate or affecting the quality of subsequent growth and development, which brings great risks to farmers

Method used

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  • Gene chip, kit and method for detecting common pathogenic bacteria of piglets
  • Gene chip, kit and method for detecting common pathogenic bacteria of piglets
  • Gene chip, kit and method for detecting common pathogenic bacteria of piglets

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0105] Example 1. Design of gene chip-specific probes

[0106] Step 1: 23S rRNA sequence alignment analysis of target bacteria

[0107] Collected 108 23S rRNA complete or nearly complete gene sequences of 12 target pathogenic bacteria from Genbank (there are too few available sequences for individual bacteria, our laboratory sequenced 6 and submitted them to Genbank. A. suis has no choice but to submit them to us a sequence). The 108 sequences were constructed by species, compared with the clustalW program of DNAStar software, and 1 or 2 representative sequences of each bacterium were selected (14 in total), among which the representative sequence of Staphylococcus epidermidis (S.epi) was registered The number is CP000029, Haemophilus parasuis (Hps) is AB303974, Actinobacillus pleuropneumoniae (App) is NC009053, Pasteurella multocida (Pm) is NC_002663, Actinobacillus suis (A.suis) is EU333989, Bordetella bronchiseptica (Bb) is NC_002927, Clostridium perfringens (Clp) is NC_0...

Embodiment 2

[0127] Embodiment 2. Primer generality test

[0128] Step 1. Extract DNA

[0129] Take appropriate cultures of the 12 target bacteria in Table 1 (i.e., the reference strains with asterisks in Table 1), and use the Tiangen Bacterial Genome Kit to extract bacterial genomic DNA. The purity and concentration of DNA were measured by ultraviolet spectrophotometer, and 1ng / μl DNA was used for PCR.

[0130] Or obtain crude DNA by boiling: take the bacterial suspension, centrifuge at 12,000rpm for 2 minutes, pour off the supernatant or pick colonies, add 100-400μl double distilled water to shake and suspend, OD600 is 1.0-2.0, put it in a boiling water bath and boil Or heat in a metal bath at 95-100°C for 10-15 minutes, ice-bath for 10 minutes, centrifuge at 12,000 rpm for 2 minutes, and take 2 μl of the supernatant as a PCR template.

[0131] Use each pair of universal primers designed in Example 1 to perform conventional single-plex PCR on the DNA of 12 pathogenic bacteria respectiv...

Embodiment 3

[0133] Example 3. Optimizing the amplification system

[0134] The purpose of asymmetric PCR is to obtain a large number of single-stranded PCR products by adding more fluorescent-labeled downstream universal primers and less non-fluorescent upstream universal primers, and increase the hybridization efficiency of PCR products and single-stranded probes.

[0135] Each pair of universal primers was optimized three times using the genomic DNA of two pathogenic bacteria as templates. In the PCR reaction system, the final concentration of the downstream universal primer was fixed at 1 μM, and serially diluted upstream universal primers were added to make the final concentration decrease (1 μM, 0.2 μM, 0.1 μM, 0.05 μM, etc.), that is, the ratio of the upstream and downstream universal primers was 1:1, 1:5, 1:10...1:60.

[0136]The optimized results of two pairs of universal primers are

[0137] 23S-F1:23S-R1 is 1:10, 23S-F2:13S-R2 is 1:30. The ratio between two pairs of universal...

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Abstract

The invention provides a gene chip, a kit and a method for detecting common pathogenic bacteria of piglets, belonging to the field of medical diagnosis of animals. A 23S rDNA specific probe sequence with the pathogenic bacteria of the piglets is arranged on the gene chip, thereby being capable of detecting a plurality of pathogenic bacterial strains under a plurality of pathogenic bacterial species of the piglets. The invention further provides two pairs of general primers which are combined with the gene chip for forming the detection kit, the bacteria to be detected are amplified, on one hand, the template concentration can be expanded by multiple times and the chip detection sensitivity can be improved, on the other hand, and the specificity of combining with a chip probe of a fluorescent labeled fragment obtained by the amplification process of the universal primers is significantly higher than chip direct hybridization of DNA of the bacteria to be detected. The invention further provides an application method of the universal primers and the chip. The gene chip, the kit and the detection method provide a practical and convenient tool for detecting the types of the pathogenic bacteria and preventing the spread of epidemic diseases of the piglets.

Description

technical field [0001] The invention relates to the field of veterinary medical diagnosis, in particular to a gene chip, kit and method for detecting common pathogenic bacteria in piglets Background technique [0002] The problem of infectious diseases in my country's livestock and poultry production is serious, and multiple pathogenic infections often occur. Bacterial infection is the most common secondary and multiple infection source. For a long time, the isolation and identification of bacteria has always been a difficult problem in laboratory testing. The traditional methods of bacterial identification mainly include bacterial isolation and culture, biochemical tests, serological tests, etc. The procedures are cumbersome, and it usually takes at least 4-7 days for a specialized authoritative inspection department to obtain the results. There are also bacteria that are difficult to culture in vitro, cannot be cultured so far, or are newly discovered bacteria, which brin...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C40B40/06C12R1/37C12R1/22C12R1/385C12R1/35C12R1/145C12R1/46C12R1/42C12R1/19C12R1/45C12R1/21C12R1/01
Inventor 陈小玲伍诚意曹峰子梁之昶杨兵章振华周宏专季海峰
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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