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A kit and detection method for detecting bacteria

A kit, the technology of Erysipelas swine, applied in the field of microbial detection, can solve problems such as easy decolorization, and achieve the effects of low false positive rate, good specificity and efficient operation

Inactive Publication Date: 2017-10-27
WUHAN BAIYUAN TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This bacterium has no motility, does not produce flagella, is easily stained by common dyes, but is easy to decolorize

Method used

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  • A kit and detection method for detecting bacteria
  • A kit and detection method for detecting bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] The preparation of embodiment 1 kit of the present invention

[0027] 1.1 Reagents

[0028] Primers were synthesized by Tiangen Biochemical Technology (Beijing) Co., Ltd.; Bst DNA polymerase and 10×ThermoPol reaction buffer were purchased from Takara; SYBR Green I was purchased from Invitrogen; other PCR reagents and reagents required for preparing CTAB extraction buffer were purchased from From Sigma.

[0029] 1.2 Preparation of the kit:

[0030] The test kit of the present invention includes following reagents:

[0031] CTAB extraction buffer: prepared according to the following formula: 100mM Tris-HCl pH 8.0, 50mM EDTA, 1M NaCl, 1% (v / v) β-mercaptoethanol, 2% CTAB, adjust the pH value to 7.2;

[0032] Reaction buffer: prepared according to the following formula: 2mM dNTP, 10×ThermoPol reaction buffer, 6mMMgSO 4 ;

[0033] Primer: outer primer F3, its nucleotide sequence is as shown in SEQ ID NO:1; Outer primer B3, its nucleotide sequence is as shown in SEQ ID NO...

Embodiment 2

[0038] Example 2 Erysipelas suis specificity detection

[0039] 2.1 LAMP-specific detection

[0040] 2.1.1 Samples to be tested

[0041] The samples to be tested were collected from the Changxing Breeding Service Professional Cooperative in Renqiu City, Hebei Province, including the disease materials of a suspected porcine erysipelas outbreak in March 2016, including 6 heart, liver, and spleen tissues. The type strains Erysipelas suis CVCC134, Actinobacillus pleuropneumoniae CVCC259, and Streptococcus pyogenes CVCC594 were purchased from China Center for Veterinary Microbiology Culture Collection.

[0042] 2.1.2 Sample pretreatment:

[0043] Cut a little diseased tissue into small pieces with sterilized scissors, put it into a homogenizer, centrifuge at 700×g for 5 minutes, then discard the precipitate, take a part of the supernatant and inoculate it on a 5% goat blood nutrient agar plate, at 30°C Cultivate and observe the bacterial growth after about 48 hours. The dominan...

Embodiment 3

[0053] Example 3 Erysipelas suis sensitivity detection

[0054] 3.1 LAMP sensitivity test

[0055] According to the step (1) of embodiment 2, extract the DNA of model bacterial strain Erysipelas suis CVCC134, measure its OD value with ultraviolet spectrophotometer, and be diluted into 10ng, 1ng, 100pg, 10pg, 1pg, 100fg total with 10 times concentration serial dilution method 6 gradients.

[0056] LAMP detection was performed according to steps (2)-(4) in Section 2.1.3 of Example 2.

[0057] 3.2 Test results

[0058] The PCR tubes with DNA concentrations of 10ng, 1ng, 100pg, 10pg, and 1pg of Erysipelas suis all showed green, indicating that the detection method of the present invention has a minimum detection limit of 1pg DNA and high sensitivity.

[0059]

[0060]

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Abstract

The invention provides an LAMP kit for detection of bacillus erysipelatos-suis.The LAMP kit comprises a primer, a Bst DNA polymerase, a reaction buffer solution and nucleic acid dye.The invention further provides an LAMP method for detection of bacillus erysipelatos-suis.Through the LAMP kit or method, bacillus erysipelatos-suis can be authenticated and detected quickly and accurately, and the LAMP kit or method has the advantages of high specificity, short consumption time, high sensitivity, easy and convenient authentication and the like and is suitable for a laboratory or field tests.

Description

technical field [0001] The invention relates to the field of microorganism detection, in particular to a LAMP kit and a detection method for detecting Erysipelas suis. Background technique [0002] Erysipelothrix rhusiopathiae, commonly known as Erysipelothrix rhusiopathiae or Erysipelothrix, belongs to the genus Erysipelothrix, is a slender small bacillus with positive Gram staining. It can exist in the environment widely and for a long time, and it can co-exist with a wide variety of wild and domestic animals, birds and fish to cause disease. The morphology of Erysipelothrix suis is variable, slender, straight or slightly curved rod-shaped, existing in single or chain, sometimes spherical or rod-shaped, but commonly palisade and pointed, with formation Tendency to be filamentous. The bacterium has no motility, does not produce flagella, and is easy to be colored by common dyes, but it is easy to decolorize. Filaments begin to form after being uploaded for several genera...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04C12R1/01
CPCC12Q1/6844C12Q1/689C12Q2531/119C12Q2545/113
Inventor 李则轩周静郭田田
Owner WUHAN BAIYUAN TECH CO LTD
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