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Visualized loop-mediated isothermal amplification kit for detecting Haemophilus parasuis

A ring-mediated isotherm, Haemophilus suis technology, applied in the direction of microbe-based methods, microbiological measurement/testing, biochemical equipment and methods, etc., can solve problems such as limiting practical applications, achieve high specificity and simple operation Convenience and good sensitivity

Inactive Publication Date: 2012-11-14
INST OF ANIMAL HUSBANDRY & VETERINARY FUJIAN ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods have certain defects in cost, sensitivity, and convenience of operation, which limit their practical application in production.

Method used

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  • Visualized loop-mediated isothermal amplification kit for detecting Haemophilus parasuis
  • Visualized loop-mediated isothermal amplification kit for detecting Haemophilus parasuis
  • Visualized loop-mediated isothermal amplification kit for detecting Haemophilus parasuis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Preparation of Visualized Loop-Mediated Isothermal Amplification Kit for Detection of Haemophilus parasuis

[0056] 1. Synthesis of Primers

[0057] Artificially synthesize the following 3 pairs of primers:

[0058]

[0059] 2. Preparation of loop-mediated isothermal amplification reaction solution

[0060] Each 23 μL ring-mediated isothermal amplification reaction solution contains the following components: Tris-HCl (1.0 mol / L) 0.5 μL, KCl (0.5 mol / L) 0.5 μL, (NH 4 ) 2 SO 4 (0.5 mol / L) 0.5 μL, MgSO 4 (20 mmol / L) 5 μL, 0.1% Triton X-100, betaine (18 mmol / L) 3 μL, dNTP (2.5 mmol / L) 2.5 μL, Bst DNA large fragment polymerase (8U / μL) 1 μL , FIP and BIP (25 μmol / L) each 1.6 μL, F3 and B3 (25 μmol / L) each 0.4 μL, LF and LB (25 μmol / L) each 0.8 μL.

[0061] 3. Assembly of the Kit

[0062] The kit consists of the following materials: the ring-mediated isothermal amplification reaction solution prepared in step 2, Haemophilus parasuis DNA (positive control) (preserved ...

Embodiment 2

[0064] PCR sensitivity test of palA gene:

[0065] The prepared Haemophilus parasuis genomic DNA was diluted in 10-fold increments, and the DNA concentration of each gradient after dilution was measured with a protein and nucleic acid quantifier, which were 4ng, 0.4ng, 40pg, 4pg, and 0.4pg, respectively. The DNA of each concentration was amplified by PCR using the established PCR method for detecting palA. 1% agarose gel electrophoresis analysis. The sensitivity test results of PCR were as follows: figure 1 As shown, it shows that the PCR method has a minimum detection limit of 4pg of Haemophilus parasuis genomic DNA.

[0066] Sensitivity test of the kit:

[0067] Use Haemophilus parasuis genomic DNA at a concentration of 4pg, 0.4pg, 0.04pg, 4fg, and 0.4fg as the template DNA to be tested; add 23 μL of the loop-mediated isothermal amplification reaction solution into the reaction tube, and 2 μL Mix the template DNA; place the reaction tube in a water bath at 55°C for 30 mi...

Embodiment 3

[0069] Specificity test of the kit:

[0070] Use the genomic DNA of Haemophilus parasuis, Pasteurella multocida, Actinobacillus pleuropneumoniae, and Streptococcus suis as the template DNA to be tested; add the ring-mediated isothermal amplification reaction solution 23 into the reaction tube μL, 2 μL template DNA, mix well; place the reaction tube in a water bath at 55°C for 40 min; react in a water bath at 80°C for another 5 minutes; analyze the results by 2% agarose gel electrophoresis. The result is as image 3 As shown, only the genomic DNA samples of Haemophilus parasuis were positive, while the genomic DNAs of Pasteurella multocida, Actinobacillus pleuropneumoniae, and Streptococcus suis had no characteristic ladder-shaped bands.

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Abstract

The invention discloses a visualized loop-mediated isothermal amplification kit for detecting Haemophilus parasuis. The kit provided by the invention comprises loop-mediated isothermal amplification reaction solution, fluorescent color-developing agent, positive control and negative control, wherein the positive control is Haemophilus parasuis DNA (deoxyribonucleic acid) and the negative control is distilled water. The loop-mediated isothermal amplification reaction solution comprises three pairs of primers, i.e. an inner primer pair, an outer primer pair and a loop primer pair are designed by reference to a conserved region of a Haemophilus parasuis peptidoglycan-associated lipoprotein gene (palA). The kit provided by the invention has the advantages that the detection sensitivity is high, the lowest detection limit of genome DNA is 0.04pg, the kit is simple and convenient to operate, and the kit is particularly suitable for detection of pathogens in fields at a basic level and detection of Haemophilus parasuis in possibly polluted animal food.

Description

technical field [0001] The invention relates to a visualization loop-mediated isothermal amplification kit for detecting Haemophilus parasuis. Background technique [0002] Haemophilus parasuis (Hps) is the pathogenic bacterium of Galasser's disease. It is a common bacterium in the upper respiratory tract of healthy pigs. Fibrinous polyserositis in young pigs. In recent years, immunosuppressive diseases such as porcine circovirus disease and porcine reproductive and respiratory syndrome are often secondary to Hps infection, which increases the morbidity and mortality, and causes serious economic losses to the pig industry. [0003] There are many serotypes of Haemophilus parasuis, with 15 serotypes and more than 20% untyped strains, among which serotypes 1, 5, 10, 12, 13 and 14 are highly virulent serotypes, and serotypes 2, 4, Type 15 is a moderately virulent serotype, and serotypes 3, 6, 7, 8, 9 and 11 are avirulent serotypes. Moreover, the cross-protection between the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12R1/21
Inventor 车勇良周伦江陈如敬王隆柏庄向生江斌吴学敏刘玉涛
Owner INST OF ANIMAL HUSBANDRY & VETERINARY FUJIAN ACADEMY OF AGRI SCI
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