Brucella detection kit and using method thereof

A detection kit and technology for Brucella, which is applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve problems such as long detection cycle, high cost of fluorescent probes, and complicated procedures

Active Publication Date: 2011-02-23
GUANGZHOU HUAFENG BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The traditional Brucella detection method is far from meeting the requirements of modern detection due to its shortcomings such as long detection cycle, complicated procedures, and various reagents required.
The pathogenic nucleic acid detection technology represented by polymerase chain reaction (PCR) technology has some problems in practical application, such as ordinary polymerase chain reaction (PCR) technology requires special equipment, and there are easy cross-contamination and cumbersome operation process Shortcomings
Although the fluorescent real-time quantitative polymerase chain reaction (real time PCR) techno

Method used

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  • Brucella detection kit and using method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0119] The preparation of embodiment 1 kit

[0120](1) Synthesize oligodeoxynucleic acid primers by DNA synthesizer according to the following sequence:

[0121] Outer primer F3(1):

[0122] CCTTTGCTGGCTGGAACT

[0123] Outer primer B3(1):

[0124] GCAATACCAGCCGTGAGG

[0125] Internal primer FIP(1):

[0126] GTCCTTGGACTTCTTGGCCCAGttttTCCAGCAGGACCAGATCG

[0127] Internal primer BIP(1):

[0128] GGCCTGGAAGTCAAGCAGGGttttACGGCATAACCGGGTTCA.

[0129] (2) Purchase DNA polymerase: Bst DNA polymerase is placed in the container;

[0130] (3) Preparation of reaction solution and primers: the reaction solution contains 2mmol / LdNTP, 25mmol / L Tris-Cl, 12.5mmol / L KCl, 12.5mmol / L (NH 4 ) 2 SO 4 , 10mmol / L MgSO 4 , 0.125% by volume TritonX-100, 1mol / L betaine, 1.2 μmol / L each of the inner primer FIP / BIP and 0.25 μmol / L each of the outer primer F3 / B3, placed in the container;

[0131] (4) Preparation of sample pretreatment solution: the sample pretreatment solution contains 20mmol / L...

Embodiment 2

[0151] The preparation of embodiment 2 kit

[0152] (1) Synthesize oligodeoxynucleic acid primers by DNA synthesizer according to the following sequence:

[0153] Outer primer F3(2):

[0154] CAAGACCAGCACCGTTGG

[0155] Outer primer B3(2):

[0156] GGTTCAGGTCGTAGCCGA

[0157] Internal primer FIP(2):

[0158] GGTCCTGCTGGAAGTTCCAGCttttAGCATCAAGCCTGACGATTG

[0159] Internal primer BIP(2):

[0160] CGGTGTTGAAGGTGATGCAGGTttttTTCAAAGCCCTGCTTGACTT

[0161] (2) Purchase DNA polymerase: Bst DNA polymerase is placed in the container;

[0162] (3) Preparation of reaction solution and primers: the reaction solution contains 1.6mmol / LdNTP, 20mmol / L Tris-Cl, 10mmol / L KCl, 10mmol / L (NH 4 ) 2 SO 4 , 8mmol / L MgSO 4 , 0.1 volume % TritonX-100, 0.8 mol / L betaine, 2.0 μmol / L each of the inner primer FIP / BIP and 0.2 μmol / L each of the outer primer F3 / B3, placed in the container;

[0163] (4) Preparation of sample pretreatment solution: the sample pretreatment solution contains 10mmol / L...

Embodiment 3

[0178] The application of embodiment 3 Brucella detection kits

[0179] 1 Materials and methods

[0180] 1.1 Materials

[0181] 1.1.1 Strains

[0182] There are 28 bacterial strains used in the present invention, which are mainly derived from the Guangzhou Center for Disease Control and Prevention, clinical isolated bacterial strains and environmental isolated bacterial strains. See Table 1 for details.

[0183] Table 1 strain name and source

[0184]

[0185] 1.1.2 Main instruments and reagents

[0186] 1.2 Identification of isolated strains

[0187] 1.2.1 Cultivation of Brucella Puncture-cultured strains of Brucella were resuscitated with Brucella broth, cultured at 36±1°C for 24±2 hours.

[0188] 1.2.2 Isolation and identification of clinical isolates. Smear the bacterial solution that is slightly turbid, precipitated and does not form a bacterial film directly on blood agar, liver infusion agar or Brooke's agar plate, respectively, in microaerobic (10% carbon diox...

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Abstract

The invention provides a brucella detection kit. The brucella detection kit comprises two pairs of primers, namely internal primers FIP/BIP and external primers F3/B3 which take the OMP25 gene of brucella as a target gene and are designed based on loop-mediated isothermal amplification technology. The brucella detection kit has more comprehensive detection effect and low undetected rate.

Description

technical field [0001] The invention relates to a biological detection reagent, in particular to a Brucella detection kit and a use method thereof. Background technique [0002] Brucella is a Gram-negative, non-motile bacterium that can survive as an intracellular parasite in many species of livestock. The principal cause of infection in humans is Brucella malta ("Maltese fever"), abortive cloth Bacillus, Brucella bovine, sheep, Brucella suis, and Brucella canis. After a person is infected, the first symptom is fever, the body temperature can reach 38-40 degrees, and it lasts for a long time, and it is in a long-term low-grade state; , Repeated many times, so brucellosis is also called undulant fever. Therefore, it is of great significance to detect Brucella accurately and rapidly. [0003] Traditional Brucella detection methods are far from meeting the requirements of modern detection due to their shortcomings such as long detection cycle, complicated procedures, and var...

Claims

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Application Information

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IPC IPC(8): C12Q1/04C12Q1/68
Inventor 曹以诚陈守义杜正平宋榴艳冯雪梅邓志爱陈洵杨智聪
Owner GUANGZHOU HUAFENG BIOTECH
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